Samant, R.S.
Batista, S.
Larance, M.
Ozer, B.
Milton, C.I.
Bludau, I.
Wu, E.
Biggins, L.
Andrews, S.
Hervieu, A.
Johnston, H.E.
Al-Lazikhani, B.
Lamond, A.I.
Clarke, P.A.
Workman, P.
(2023). Native Size-Exclusion Chromatography-Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes. Mol cell proteomics,
Vol.22
(2),
p. 100485.
show abstract
full text
The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8Â h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29Â cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459..
Workman, P.
(2023). The NCI-60 Human Tumor Cell Line Screen: A Catalyst for Progressive Evolution of Models for Discovery and Development of Cancer Drugs. Cancer res,
Vol.83
(19),
pp. 3170-3173.
show abstract
Following three decades of systematic primary empirical screening against mice bearing two transplantable murine leukemias, the NCI took the bold step of switching to a radically different approach-initial screening of 10,000 diverse compounds/year against a panel of 60 human tumor cell lines in vitro. The establishment of the "NCI-60" screen was announced in the landmark Cancer Research article by Alley and colleagues, published in 1988, which exemplified the technological basis for the new microculture screen, operating at unprecedented scale. The underlying concept was that NCI-60 might expedite the discovery of innovative cancer drugs, especially those with predicted activity against particular solid cancers-not then possible. We discuss how NCI-60 provided a major technological advance and delivered a successful legacy for cancer research and development. While not immediately cracking the thorny problem of model-to-human tumor type prediction, NCI-60 nevertheless provided the conceptual and methodologic foundation for subsequent, much larger-scale human cancer cell panel screens with detailed molecular annotation and sophisticated informatics. Now used in modern molecular target-based drug discovery, these panels help enable the implementation of contemporary biomarker-led precision oncology. See related article by Alley and colleagues, Cancer Res 1988;48:589-601..
Mader, M.M.
Rudolph, J.
Hartung, I.V.
Uehling, D.
Workman, P.
Zuercher, W.
(2023). Which Small Molecule? Selecting Chemical Probes for Use in Cancer Research and Target Validation. Cancer discov,
Vol.13
(10),
pp. 2150-2165.
show abstract
UNLABELLED: Small-molecule chemical "probes" complement the use of molecular biology techniques to explore, validate, and generate hypotheses on the function of proteins in diseases such as cancer. Unfortunately, the poor selection and use of small-molecule reagents can lead to incorrect conclusions. Here, we illustrate examples of poor chemical tools and suggest best practices for the selection, validation, and use of high-quality chemical probes in cancer research. We also note the complexity associated with tools for novel drug modalities, exemplified by protein degraders, and provide advice and resources to facilitate the independent identification of appropriate small-molecule probes by researchers. SIGNIFICANCE: Validation of biological targets and pathways will be aided by a shared understanding of the criteria of potency, selectivity, and target engagement associated with small-molecule reagents ("chemical probes") that enable that work. Interdisciplinary collaboration between cancer biologists, medicinal chemists, and chemical biologists and the awareness of available resources will reduce misleading data generation and interpretation, strengthen data robustness, and improve productivity in academic and industrial research..
Antolin, A.A.
Sanfelice, D.
Crisp, A.
Villasclaras Fernandez, E.
Mica, I.L.
Chen, Y.
Collins, I.
Edwards, A.
Müller, S.
Al-Lazikani, B.
Workman, P.
(2023). The Chemical Probes Portal: an expert review-based public resource to empower chemical probe assessment, selection and use. Nucleic acids res,
Vol.51
(D1),
pp. D1492-D1502.
show abstract
full text
We describe the Chemical Probes Portal (https://www.chemicalprobes.org/), an expert review-based public resource to empower chemical probe assessment, selection and use. Chemical probes are high-quality small-molecule reagents, often inhibitors, that are important for exploring protein function and biological mechanisms, and for validating targets for drug discovery. The publication, dissemination and use of chemical probes provide an important means to accelerate the functional annotation of proteins, the study of proteins in cell biology, physiology, and disease pathology, and to inform and enable subsequent pioneering drug discovery and development efforts. However, the widespread use of small-molecule compounds that are claimed as chemical probes but are lacking sufficient quality, especially being inadequately selective for the desired target or even broadly promiscuous in behaviour, has resulted in many erroneous conclusions in the biomedical literature. The Chemical Probes Portal was established as a public resource to aid the selection and best-practice use of chemical probes in basic and translational biomedical research. We describe the background, principles and content of the Portal and its technical development, as well as examples of its applications and use. The Chemical Probes Portal is a community resource and we therefore describe how researchers can be involved in its content and development..
di Micco, P.
Antolin, A.A.
Mitsopoulos, C.
Villasclaras-Fernandez, E.
Sanfelice, D.
Dolciami, D.
Ramagiri, P.
Mica, I.L.
Tym, J.E.
Gingrich, P.W.
Hu, H.
Workman, P.
Al-Lazikani, B.
(2023). canSAR: update to the cancer translational research and drug discovery knowledgebase. Nucleic acids res,
Vol.51
(D1),
pp. D1212-D1219.
show abstract
full text
canSAR (https://cansar.ai) is the largest public cancer drug discovery and translational research knowledgebase. Now hosted in its new home at MD Anderson Cancer Center, canSAR integrates billions of experimental measurements from across molecular profiling, pharmacology, chemistry, structural and systems biology. Moreover, canSAR applies a unique suite of machine learning algorithms designed to inform drug discovery. Here, we describe the latest updates to the knowledgebase, including a focus on significant novel data. These include canSAR's ligandability assessment of AlphaFold; mapping of fragment-based screening data; and new chemical bioactivity data for novel targets. We also describe enhancements to the data and interface..
Pasqua, A.E.
Sharp, S.Y.
Chessum, N.E.
Hayes, A.
Pellegrino, L.
Tucker, M.J.
Miah, A.
Wilding, B.
Evans, L.E.
Rye, C.S.
Mok, N.Y.
Liu, M.
Henley, A.T.
Gowan, S.
De Billy, E.
Te Poele, R.
Powers, M.
Eccles, S.A.
Clarke, P.A.
Raynaud, F.I.
Workman, P.
Jones, K.
Cheeseman, M.D.
(2023). HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies. J med chem,
Vol.66
(8),
pp. 5907-5936.
show abstract
full text
CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies..
Hartung, I.V.
Rudolph, J.
Mader, M.M.
Mulder, M.P.
Workman, P.
(2023). Expanding Chemical Probe Space: Quality Criteria for Covalent and Degrader Probes. J med chem,
Vol.66
(14),
pp. 9297-9312.
show abstract
full text
Within druggable target space, new small-molecule modalities, particularly covalent inhibitors and targeted degraders, have expanded the repertoire of medicinal chemists. Molecules with such modes of action have a large potential not only as drugs but also as chemical probes. Criteria have previously been established to describe the potency, selectivity, and properties of small-molecule probes that are qualified to enable the interrogation and validation of drug targets. These definitions have been tailored to reversibly acting modulators but fall short in their applicability to other modalities. While initial guidelines have been proposed, we delineate here a full set of criteria for the characterization of covalent, irreversible inhibitors as well as heterobifunctional degraders ("proteolysis-targeting chimeras", or PROTACs) and molecular glue degraders. We propose modified potency and selectivity criteria compared to those for reversible inhibitors. We discuss their relevance and highlight examples of suitable probe and pathfinder compounds..
Licciardello, M.P.
Workman, P.
(2022). The era of high-quality chemical probes. Rsc med chem,
Vol.13
(12),
pp. 1446-1459.
show abstract
full text
Small-molecule chemical probes are among the most important tools to study the function of proteins in cells and organisms. Regrettably, the use of weak and non-selective small molecules has generated an abundance of erroneous conclusions in the scientific literature. More recently, minimal criteria have been outlined for investigational compounds, encouraging the selection and use of high-quality chemical probes. Here, we briefly recall the milestones and key initiatives that have paved the way to this new era, illustrate examples of recent high-quality chemical probes and provide our perspective on future challenges and developments..
Antolin, A.A.
Workman, P.
Al-Lazikani, B.
(2021). Public resources for chemical probes: the journey so far and the road ahead. Future med chem,
Vol.13
(8),
pp. 731-747.
show abstract
full text
High-quality small molecule chemical probes are extremely valuable for biological research and target validation. However, frequent use of flawed small-molecule inhibitors produces misleading results and diminishes the robustness of biomedical research. Several public resources are available to facilitate assessment and selection of better chemical probes for specific protein targets. Here, we review chemical probe resources, discuss their current strengths and limitations, and make recommendations for further improvements. Expert review resources provide in-depth analysis but currently cover only a limited portion of the liganded proteome. Computational resources encompass more proteins and are regularly updated, but have limitations in data availability and curation. We show how biomedical scientists may use these resources to choose the best available chemical probes for their research..
Pal, A.
Shinde, R.
Miralles, M.S.
Workman, P.
de Bono, J.
(2021). Applications of liquid biopsy in the Pharmacological Audit Trail for anticancer drug development. Nat rev clin oncol,
Vol.18
(7),
pp. 454-467.
show abstract
Anticancer drug development is a costly and protracted activity, and failure at late phases of clinical testing is common. We have previously proposed the Pharmacological Audit Trail (PhAT) intended to improve the efficiency of drug development, with a focus on the use of tumour tissue-based biomarkers. Blood-based 'liquid biopsy' approaches, such as targeted or whole-genome sequencing studies of plasma circulating cell-free tumour DNA (ctDNA) and circulating tumour cells (CTCs), are of increasing relevance to this drug development paradigm. Liquid biopsy assays can provide quantitative and qualitative data on prognostic, predictive, pharmacodynamic and clinical response biomarkers, and can also enable the characterization of disease evolution and resistance mechanisms. In this Perspective, we examine the promise of integrating liquid biopsy analyses into the PhAT, focusing on the current evidence, advances, limitations and challenges. We emphasize the continued importance of analytical validation and clinical qualification of circulating tumour biomarkers through prospective clinical trials..
Mitsopoulos, C.
Di Micco, P.
Fernandez, E.V.
Dolciami, D.
Holt, E.
Mica, I.L.
Coker, E.A.
Tym, J.E.
Campbell, J.
Che, K.H.
Ozer, B.
Kannas, C.
Antolin, A.A.
Workman, P.
Al-Lazikani, B.
(2021). canSAR: update to the cancer translational research and drug discovery knowledgebase. Nucleic acids res,
Vol.49
(D1),
pp. D1074-D1082.
show abstract
full text
canSAR (http://cansar.icr.ac.uk) is the largest, public, freely available, integrative translational research and drug discovery knowledgebase for oncology. canSAR integrates vast multidisciplinary data from across genomic, protein, pharmacological, drug and chemical data with structural biology, protein networks and more. It also provides unique data, curation and annotation and crucially, AI-informed target assessment for drug discovery. canSAR is widely used internationally by academia and industry. Here we describe significant developments and enhancements to the data, web interface and infrastructure of canSAR in the form of the new implementation of the system: canSARblack. We demonstrate new functionality in aiding translation hypothesis generation and experimental design, and show how canSAR can be adapted and utilised outside oncology..
Gil, V.
Miranda, S.
Riisnaes, R.
Gurel, B.
D'Ambrosio, M.
Vasciaveo, A.
Crespo, M.
Ferreira, A.
Brina, D.
Troiani, M.
Sharp, A.
Sheehan, B.
Christova, R.
Seed, G.
Figueiredo, I.
Lambros, M.
Dolling, D.
Rekowski, J.
Alajati, A.
Clarke, M.
Pereira, R.
Flohr, P.
Fowler, G.
Boysen, G.
Sumanasuriya, S.
Bianchini, D.
Rescigno, P.
Aversa, C.
Tunariu, N.
Guo, C.
Paschalis, A.
Bertan, C.
Buroni, L.
Ning, J.
Carreira, S.
Workman, P.
Swain, A.
Califano, A.
Shen, M.M.
Alimonti, A.
Neeb, A.
Welti, J.
Yuan, W.
de Bono, J.
PCF/SU2C International Prostate Cancer Dream Team,
(2021). HER3 Is an Actionable Target in Advanced Prostate Cancer. Cancer res,
Vol.81
(24),
pp. 6207-6218.
show abstract
full text
It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials..
Carotenuto, P.
Amato, F.
Lampis, A.
Rae, C.
Hedayat, S.
Previdi, M.C.
Zito, D.
Raj, M.
Guzzardo, V.
Sclafani, F.
Lanese, A.
Parisi, C.
Vicentini, C.
Said-Huntingford, I.
Hahne, J.C.
Hallsworth, A.
Kirkin, V.
Young, K.
Begum, R.
Wotherspoon, A.
Kouvelakis, K.
Azevedo, S.X.
Michalarea, V.
Upstill-Goddard, R.
Rao, S.
Watkins, D.
Starling, N.
Sadanandam, A.
Chang, D.K.
Biankin, A.V.
Jamieson, N.B.
Scarpa, A.
Cunningham, D.
Chau, I.
Workman, P.
Fassan, M.
Valeri, N.
Braconi, C.
(2021). Modulation of pancreatic cancer cell sensitivity to FOLFIRINOX through microRNA-mediated regulation of DNA damage. Nat commun,
Vol.12
(1),
p. 6738.
show abstract
full text
FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome..
Antolin, A.A.
Clarke, P.A.
Collins, I.
Workman, P.
Al-Lazikani, B.
(2021). Evolution of kinase polypharmacology across HSP90 drug discovery. Cell chem biol,
Vol.28
(10),
pp. 1433-1445.e3.
show abstract
full text
Most small molecules interact with several target proteins but this polypharmacology is seldom comprehensively investigated or explicitly exploited during drug discovery. Here, we use computational and experimental methods to identify and systematically characterize the kinase cross-pharmacology of representative HSP90 inhibitors. We demonstrate that the resorcinol clinical candidates ganetespib and, to a lesser extent, luminespib, display unique off-target kinase pharmacology as compared with other HSP90 inhibitors. We also demonstrate that polypharmacology evolved during the optimization to discover luminespib and that the hit, leads, and clinical candidate all have different polypharmacological profiles. We therefore recommend the computational and experimental characterization of polypharmacology earlier in drug discovery projects to unlock new multi-target drug design opportunities..
Poon, E.
Liang, T.
Jamin, Y.
Walz, S.
Kwok, C.
Hakkert, A.
Barker, K.
Urban, Z.
Thway, K.
Zeid, R.
Hallsworth, A.
Box, G.
Ebus, M.E.
Licciardello, M.P.
Sbirkov, Y.
Lazaro, G.
Calton, E.
Costa, B.M.
Valenti, M.
De Haven Brandon, A.
Webber, H.
Tardif, N.
Almeida, G.S.
Christova, R.
Boysen, G.
Richards, M.W.
Barone, G.
Ford, A.
Bayliss, R.
Clarke, P.A.
De Bono, J.
Gray, N.S.
Blagg, J.
Robinson, S.P.
Eccles, S.A.
Zheleva, D.
Bradner, J.E.
Molenaar, J.
Vivanco, I.
Eilers, M.
Workman, P.
Lin, C.Y.
Chesler, L.
(2020). Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma. J clin invest,
Vol.130
(11),
pp. 5875-5892.
show abstract
full text
The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma..
Carotenuto, P.
Hedayat, S.
Fassan, M.
Cardinale, V.
Lampis, A.
Guzzardo, V.
Vicentini, C.
Scarpa, A.
Cascione, L.
Costantini, D.
Carpino, G.
Alvaro, D.
Ghidini, M.
Trevisani, F.
Te Poele, R.
Salati, M.
Ventura, S.
Vlachogiannis, G.
Hahne, J.C.
Boulter, L.
Forbes, S.J.
Guest, R.V.
Cillo, U.
Said-Huntingford, I.
Begum, R.
Smyth, E.
Michalarea, V.
Cunningham, D.
Rimassa, L.
Santoro, A.
Roncalli, M.
Kirkin, V.
Clarke, P.
Workman, P.
Valeri, N.
Braconi, C.
(2020). Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells. Hepatology,
Vol.72
(3),
pp. 982-996.
show abstract
full text
BACKGROUND AND AIMS: Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy. APPROACH AND RESULTS: High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment. CONCLUSIONS: MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics..
Sharpe, E.
Hoey, R.
Yap, C.
Workman, P.
(2020). From patent to patient: analysing access to innovative cancer drugs. Drug discov today,
Vol.25
(9),
pp. 1561-1568.
show abstract
full text
Analysis of cancer drugs licensed through the European Medicines Agency (EMA) in 2000-2016 shows that the number of authorisations increased over that timeframe. The median number of licensed drugs each year rose from six for 2000-2008 to 13.5 for 2009-2016. Over 2000-2016, there were 64 drug authorisations for haematological, 15 for breast, and 12 for skin cancer, but none for oesophageal, brain, bladder, or uterine cancer. Only 6% of authorisations included a paediatric indication. The average time for a drug to progress from patent priority date to availability on the National Health Service (NHS) increased from 12.8 years for drugs first licensed in 2000-2008 to 14.0 years for those licensed in 2009-2016. There was evidence that the most innovative drugs were not being prioritised for EMA licensing and NICE approval..
Rogers, R.F.
Walton, M.I.
Cherry, D.L.
Collins, I.
Clarke, P.A.
Garrett, M.D.
Workman, P.
(2020). CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells. Cancer res,
Vol.80
(8),
pp. 1735-1747.
show abstract
full text
Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies..
Antolin, A.A.
Ameratunga, M.
Banerji, U.
Clarke, P.A.
Workman, P.
Al-Lazikani, B.
(2020). The kinase polypharmacology landscape of clinical PARP inhibitors. Sci rep,
Vol.10
(1),
p. 2585.
show abstract
full text
Polypharmacology plays an important role in defining response and adverse effects of drugs. For some mechanisms, experimentally mapping polypharmacology is commonplace, although this is typically done within the same protein class. Four PARP inhibitors have been approved by the FDA as cancer therapeutics, yet a precise mechanistic rationale to guide clinicians on which to choose for a particular patient is lacking. The four drugs have largely similar PARP family inhibition profiles, but several differences at the molecular and clinical level have been reported that remain poorly understood. Here, we report the first comprehensive characterization of the off-target kinase landscape of four FDA-approved PARP drugs. We demonstrate that all four PARP inhibitors have a unique polypharmacological profile across the kinome. Niraparib and rucaparib inhibit DYRK1s, CDK16 and PIM3 at clinically achievable, submicromolar concentrations. These kinases represent the most potently inhibited off-targets of PARP inhibitors identified to date and should be investigated further to clarify their potential implications for efficacy and safety in the clinic. Moreover, broad kinome profiling is recommended for the development of PARP inhibitors as PARP-kinase polypharmacology could potentially be exploited to modulate efficacy and side-effect profiles..
Darby, J.F.
Vidler, L.R.
Simpson, P.J.
Al-Lazikani, B.
Matthews, S.J.
Sharp, S.Y.
Pearl, L.H.
Hoelder, S.
Workman, P.
(2020). Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches. Sci rep,
Vol.10
(1),
p. 16000.
show abstract
full text
Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in tumour biology by promoting the stabilisation and activity of oncogenic 'client' proteins. Inhibition of Hsp90 by small-molecule drugs, acting via its ATP hydrolysis site, has shown promise as a molecularly targeted cancer therapy. Owing to the importance of Hop and other tetratricopeptide repeat (TPR)-containing cochaperones in regulating Hsp90 activity, the Hsp90-TPR domain interface is an alternative site for inhibitors, which could result in effects distinct from ATP site binders. The TPR binding site of Hsp90 cochaperones includes a shallow, positively charged groove that poses a significant challenge for druggability. Herein, we report the apo, solution-state structure of Hop TPR2A which enables this target for NMR-based screening approaches. We have designed prototype TPR ligands that mimic key native 'carboxylate clamp' interactions between Hsp90 and its TPR cochaperones and show that they block binding between Hop TPR2A and the Hsp90 C-terminal MEEVD peptide. We confirm direct TPR-binding of these ligands by mapping 1H-15N HSQC chemical shift perturbations to our new NMR structure. Our work provides a novel structure, a thorough assessment of druggability and robust screening approaches that may offer a potential route, albeit difficult, to address the chemically challenging nature of the Hop TPR2A target, with relevance to other TPR domain interactors..
Slovin, S.
Hussain, S.
Saad, F.
Garcia, J.
Picus, J.
Ferraldeschi, R.
Crespo, M.
Flohr, P.
Riisnaes, R.
Lin, C.
Keer, H.
Oganesian, A.
Workman, P.
de Bono, J.
(2019). Pharmacodynamic and Clinical Results from a Phase I/II Study of the HSP90 Inhibitor Onalespib in Combination with Abiraterone Acetate in Prostate Cancer. Clin cancer res,
Vol.25
(15),
pp. 4624-4633.
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PURPOSE: Onalespib is a potent, fragment-derived second-generation HSP90 inhibitor with preclinical activity in castration-resistant prostate cancer (CPRC) models. This phase I/II trial evaluated onalespib in combination with abiraterone acetate (AA) and either prednisone or prednisolone (P) in men with CRPC progressing on AA/P. PATIENTS AND METHODS: Patients with progressing CRPC were randomly assigned to receive 1 of 2 regimens of onalespib combined with AA/P. Onalespib was administered as intravenous infusion starting at 220 mg/m2 once weekly for 3 of 4 weeks (regimen 1); or at 120 mg/m2 on day 1 and day 2 weekly for 3 of 4 weeks (regimen 2). Primary endpoints were response rate and safety. Secondary endpoints included evaluation of androgen receptor (AR) depletion in circulating tumor cells (CTC) and in fresh tumor tissue biopsies. RESULTS: Forty-eight patients were treated with onalespib in combination with AA/P. The most common ≥grade 3 toxicities related to onalespib included diarrhea (21%) and fatigue (13%). Diarrhea was dose limiting at 260 and 160 mg/m2 for regimens 1 and 2, respectively. Transient decreases in CTC counts and AR expression in CTC were observed in both regimens. HSP72 was significantly upregulated following onalespib treatment, but only a modest decrease in AR and GR was shown in paired pre- and posttreatment tumor biopsy samples. No patients showed an objective or PSA response. CONCLUSIONS: Onalespib in combination with AA/P showed mild evidence of some biological effect; however, this effect did not translate into clinical activity, hence further exploration of this combination was not justified..
Clarke, P.A.
Roe, T.
Swabey, K.
Hobbs, S.M.
McAndrew, C.
Tomlin, K.
Westwood, I.
Burke, R.
van Montfort, R.
Workman, P.
(2019). Dissecting mechanisms of resistance to targeted drug combination therapy in human colorectal cancer. Oncogene,
Vol.38
(25),
pp. 5076-5090.
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Genomic alterations in cancer cells result in vulnerabilities that clinicians can exploit using molecularly targeted drugs, guided by knowledge of the tumour genotype. However, the selective activity of these drugs exerts an evolutionary pressure on cancers that can result in the outgrowth of resistant clones. Use of rational drug combinations can overcome resistance to targeted drugs, but resistance may eventually develop to combinatorial therapies. We selected MAPK- and PI3K-pathway inhibition in colorectal cancer as a model system to dissect out mechanisms of resistance. We focused on these signalling pathways because they are frequently activated in colorectal tumours, have well-characterised mutations and are clinically relevant. By treating a panel of 47 human colorectal cancer cell lines with a combination of MEK- and PI3K-inhibitors, we observe a synergistic inhibition of growth in almost all cell lines. Cells with KRAS mutations are less sensitive to PI3K inhibition, but are particularly sensitive to the combined treatment. Colorectal cancer cell lines with inherent or acquired resistance to monotherapy do not show a synergistic response to the combination treatment. Cells that acquire resistance to an MEK-PI3K inhibitor combination treatment still respond to an ERK-PI3K inhibitor regimen, but subsequently also acquire resistance to this combination treatment. Importantly, the mechanisms of resistance to MEK and PI3K inhibitors observed, MEK1/2 mutation or loss of PTEN, are similar to those detected in the clinic. ERK inhibitors may have clinical utility in overcoming resistance to MEK inhibitor regimes; however, we find a recurrent active site mutation of ERK2 that drives resistance to ERK inhibitors in mono- or combined regimens, suggesting that resistance will remain a hurdle. Importantly, we find that the addition of low concentrations of the BCL2-family inhibitor navitoclax to the MEK-PI3K inhibitor regimen improves the synergistic interaction and blocks the acquisition of resistance..
Zhang, C.
Stockwell, S.R.
Elbanna, M.
Ketteler, R.
Freeman, J.
Al-Lazikani, B.
Eccles, S.
De Haven Brandon, A.
Raynaud, F.
Hayes, A.
Clarke, P.A.
Workman, P.
Mittnacht, S.
(2019). Signalling involving MET and FAK supports cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases. Oncogene,
Vol.38
(30),
pp. 5905-5920.
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Deregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer; yet, inhibitors against these kinases are currently used only in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and the mechanisms that may undermine such dependency are poorly understood. Here, we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2 activation, involving as critical mechanistic events loss of the CDKI p21CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates the proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic..
Aldape, K.
Brindle, K.M.
Chesler, L.
Chopra, R.
Gajjar, A.
Gilbert, M.R.
Gottardo, N.
Gutmann, D.H.
Hargrave, D.
Holland, E.C.
Jones, D.T.
Joyce, J.A.
Kearns, P.
Kieran, M.W.
Mellinghoff, I.K.
Merchant, M.
Pfister, S.M.
Pollard, S.M.
Ramaswamy, V.
Rich, J.N.
Robinson, G.W.
Rowitch, D.H.
Sampson, J.H.
Taylor, M.D.
Workman, P.
Gilbertson, R.J.
(2019). Challenges to curing primary brain tumours. Nat rev clin oncol,
Vol.16
(8),
pp. 509-520.
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Despite decades of research, brain tumours remain among the deadliest of all forms of cancer. The ability of these tumours to resist almost all conventional and novel treatments relates, in part, to the unique cell-intrinsic and microenvironmental properties of neural tissues. In an attempt to encourage progress in our understanding and ability to successfully treat patients with brain tumours, Cancer Research UK convened an international panel of clinicians and laboratory-based scientists to identify challenges that must be overcome if we are to cure all patients with a brain tumour. The seven key challenges summarized in this Position Paper are intended to serve as foci for future research and investment..
Coker, E.A.
Mitsopoulos, C.
Tym, J.E.
Komianou, A.
Kannas, C.
Di Micco, P.
Villasclaras Fernandez, E.
Ozer, B.
Antolin, A.A.
Workman, P.
Al-Lazikani, B.
(2019). canSAR: update to the cancer translational research and drug discovery knowledgebase. Nucleic acids res,
Vol.47
(D1),
pp. D917-D922.
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canSAR (http://cansar.icr.ac.uk) is a public, freely available, integrative translational research and drug discovery knowlegebase. canSAR informs researchers to help solve key bottlenecks in cancer translation and drug discovery. It integrates genomic, protein, pharmacological, drug and chemical data with structural biology, protein networks and unique, comprehensive and orthogonal 'druggability' assessments. canSAR is widely used internationally by academia and industry. Here we describe major enhancements to canSAR including new and expanded data. We also describe the first components of canSARblack-an advanced, responsive, multi-device compatible redesign of canSAR with a question-led interface..
Felisberto-Rodrigues, C.
Thomas, J.C.
McAndrew, C.
Le Bihan, Y.-.
Burke, R.
Workman, P.
van Montfort, R.L.
(2019). Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release. Biochem j,
Vol.476
(18),
pp. 2521-2543.
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DHX8 is a crucial DEAH-box RNA helicase involved in splicing and required for the release of mature mRNA from the spliceosome. Here, we report the biochemical characterisation of full-length human DHX8 and the catalytically active helicase core DHX8Δ547, alongside crystal structures of DHX8Δ547 bound to ADP and a structure of DHX8Δ547 bound to poly(A)6 single-strand RNA. Our results reveal that DHX8 has an in vitro binding preference for adenine-rich RNA and that RNA binding triggers the release of ADP through significant conformational flexibility in the conserved DEAH-, P-loop and hook-turn motifs. We demonstrate the importance of R620 and both the hook-turn and hook-loop regions for DHX8 helicase activity and propose that the hook-turn acts as a gatekeeper to regulate the directional movement of the 3' end of RNA through the RNA-binding channel. This study provides an in-depth understanding of the activity of DHX8 and contributes insights into the RNA-unwinding mechanisms of the DEAH-box helicase family..
Whittaker, S.R.
Barlow, C.
Martin, M.P.
Mancusi, C.
Wagner, S.
Self, A.
Barrie, E.
Te Poele, R.
Sharp, S.
Brown, N.
Wilson, S.
Jackson, W.
Fischer, P.M.
Clarke, P.A.
Walton, M.I.
McDonald, E.
Blagg, J.
Noble, M.
Garrett, M.D.
Workman, P.
(2018). Molecular profiling and combinatorial activity of CCT068127: a potent CDK2 and CDK9 inhibitor. Mol oncol,
Vol.12
(3),
pp. 287-304.
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Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small-molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer..
Lampis, A.
Carotenuto, P.
Vlachogiannis, G.
Cascione, L.
Hedayat, S.
Burke, R.
Clarke, P.
Bosma, E.
Simbolo, M.
Scarpa, A.
Yu, S.
Cole, R.
Smyth, E.
Mateos, J.F.
Begum, R.
Hezelova, B.
Eltahir, Z.
Wotherspoon, A.
Fotiadis, N.
Bali, M.A.
Nepal, C.
Khan, K.
Stubbs, M.
Hahne, J.C.
Gasparini, P.
Guzzardo, V.
Croce, C.M.
Eccles, S.
Fassan, M.
Cunningham, D.
Andersen, J.B.
Workman, P.
Valeri, N.
Braconi, C.
(2018). MIR21 Drives Resistance to Heat Shock Protein 90 Inhibition in Cholangiocarcinoma. Gastroenterology,
Vol.154
(4),
pp. 1066-1079.e5.
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BACKGROUND & AIMS: Cholangiocarcinomas (CCA) are resistant to chemotherapy, so new therapeutic agents are needed. We performed a screen to identify small-molecule compounds that are active against CCAs. Levels of microRNA 21 (MIR21 or miRNA21) are increased in CCAs. We investigated whether miRNA21 mediates resistance of CCA cells and organoids to HSP90 inhibitors. METHODS: We performed a high-throughput screen of 484 small-molecule compounds to identify those that reduced viability of 6 human CCA cell lines. We tested the effects of HSP90 inhibitors on cells with disruption of the MIR21 gene, cells incubated with MIR21 inhibitors, and stable cell lines with inducible expression of MIR21. We obtained CCA biopsies from patients, cultured them as organoids (patient-derived organoids). We assessed their architecture, mutation and gene expression patterns, response to compounds in culture, and when grown as subcutaneous xenograft tumors in mice. RESULTS: Cells with IDH1 and PBRM1 mutations had the highest level of sensitivity to histone deacetylase inhibitors. HSP90 inhibitors were effective in all cell lines, irrespective of mutations. Sensitivity of cells to HSP90 inhibitors correlated inversely with baseline level of MIR21. Disruption of MIR21 increased cell sensitivity to HSP90 inhibitors. CCA cells that expressed transgenic MIR21 were more resistant to HSP90 inhibitors than cells transfected with control vectors; inactivation of MIR21 in these cells restored sensitivity to these agents. MIR21 was shown to target the DnaJ heat shock protein family (Hsp40) member B5 (DNAJB5). Transgenic expression of DNAJB5 in CCA cells that overexpressed MIR21 re-sensitized them to HSP90 inhibitors. Sensitivity of patient-derived organoids to HSP90 inhibitors, in culture and when grown as xenograft tumors in mice, depended on expression of miRNA21. CONCLUSIONS: miRNA21 appears to mediate resistance of CCA cells to HSP90 inhibitors by reducing levels of DNAJB5. HSP90 inhibitors might be developed for the treatment of CCA and miRNA21 might be a marker of sensitivity to these agents..
Bright, M.D.
Clarke, P.A.
Workman, P.
Davies, F.E.
(2018). Oncogenic RAC1 and NRAS drive resistance to endoplasmic reticulum stress through MEK/ERK signalling. Cell signal,
Vol.44,
pp. 127-137.
show abstract
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Cancer cells are able to survive under conditions that cause endoplasmic reticulum stress (ER-stress), and can adapt to this stress by upregulating cell-survival signalling pathways and down-regulating apoptotic pathways. The cellular response to ER-stress is controlled by the unfolded protein response (UPR). Small Rho family GTPases are linked to many cell responses including cell growth and apoptosis. In this study, we investigate the function of small GTPases in cell survival under ER-stress. Using siRNA screening we identify that RAC1 promotes cell survival under ER-stress in cells with an oncogenic N92I RAC1 mutation. We uncover a novel connection between the UPR and N92I RAC1, whereby RAC1 attenuates phosphorylation of EIF2S1 under ER-stress and drives over-expression of ATF4 in basal conditions. Interestingly, the UPR connection does not drive resistance to ER-stress, as knockdown of ATF4 did not affect this. We further investigate cancer-associated kinase signalling pathways and show that RAC1 knockdown reduces the activity of AKT and ERK, and using a panel of clinically important kinase inhibitors, we uncover a role for MEK/ERK, but not AKT, in cell viability under ER-stress. A known major activator of ERK phosphorylation in cancer is oncogenic NRAS and we show that knockdown of NRAS in cells, which bear a Q61 NRAS mutation, sensitises to ER-stress. These findings highlight a novel mechanism for resistance to ER-stress through oncogenic activation of MEK/ERK signalling by small GTPases..
Wedge, D.C.
Gundem, G.
Mitchell, T.
Woodcock, D.J.
Martincorena, I.
Ghori, M.
Zamora, J.
Butler, A.
Whitaker, H.
Kote-Jarai, Z.
Alexandrov, L.B.
Van Loo, P.
Massie, C.E.
Dentro, S.
Warren, A.Y.
Verrill, C.
Berney, D.M.
Dennis, N.
Merson, S.
Hawkins, S.
Howat, W.
Lu, Y.-.
Lambert, A.
Kay, J.
Kremeyer, B.
Karaszi, K.
Luxton, H.
Camacho, N.
Marsden, L.
Edwards, S.
Matthews, L.
Bo, V.
Leongamornlert, D.
McLaren, S.
Ng, A.
Yu, Y.
Zhang, H.
Dadaev, T.
Thomas, S.
Easton, D.F.
Ahmed, M.
Bancroft, E.
Fisher, C.
Livni, N.
Nicol, D.
Tavaré, S.
Gill, P.
Greenman, C.
Khoo, V.
Van As, N.
Kumar, P.
Ogden, C.
Cahill, D.
Thompson, A.
Mayer, E.
Rowe, E.
Dudderidge, T.
Gnanapragasam, V.
Shah, N.C.
Raine, K.
Jones, D.
Menzies, A.
Stebbings, L.
Teague, J.
Hazell, S.
Corbishley, C.
CAMCAP Study Group,
de Bono, J.
Attard, G.
Isaacs, W.
Visakorpi, T.
Fraser, M.
Boutros, P.C.
Bristow, R.G.
Workman, P.
Sander, C.
TCGA Consortium,
Hamdy, F.C.
Futreal, A.
McDermott, U.
Al-Lazikani, B.
Lynch, A.G.
Bova, G.S.
Foster, C.S.
Brewer, D.S.
Neal, D.E.
Cooper, C.S.
Eeles, R.A.
(2018). Sequencing of prostate cancers identifies new cancer genes, routes of progression and drug targets. Nat genet,
Vol.50
(5),
pp. 682-692.
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full text
Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62Â targets of compounds that may be active and should be considered candidates for future clinical trials..
Chessum, N.E.
Sharp, S.Y.
Caldwell, J.J.
Pasqua, A.E.
Wilding, B.
Colombano, G.
Collins, I.
Ozer, B.
Richards, M.
Rowlands, M.
Stubbs, M.
Burke, R.
McAndrew, P.C.
Clarke, P.A.
Workman, P.
Cheeseman, M.D.
Jones, K.
(2018). Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766). J med chem,
Vol.61
(3),
pp. 918-933.
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full text
Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets..
Meyers, J.
Chessum, N.E.
Ali, S.
Mok, N.Y.
Wilding, B.
Pasqua, A.E.
Rowlands, M.
Tucker, M.J.
Evans, L.E.
Rye, C.S.
O'Fee, L.
Le Bihan, Y.-.
Burke, R.
Carter, M.
Workman, P.
Blagg, J.
Brown, N.
van Montfort, R.L.
Jones, K.
Cheeseman, M.D.
(2018). Privileged Structures and Polypharmacology within and between Protein Families. Acs med chem lett,
Vol.9
(12),
pp. 1199-1204.
show abstract
full text
Polypharmacology is often a key contributor to the efficacy of a drug, but is also a potential risk. We investigated two hits discovered via a cell-based phenotypic screen, the CDK9 inhibitor CCT250006 (1) and the pirin ligand CCT245232 (2), to establish methodology to elucidate their secondary protein targets. Using computational pocket-based analysis, we discovered intrafamily polypharmacology for our kinase inhibitor, despite little overall sequence identity. The interfamily polypharmacology of 2 with B-Raf was used to discover a novel pirin ligand from a very small but privileged compound library despite no apparent ligand or binding site similarity. Our data demonstrates that in areas of drug discovery where intrafamily polypharmacology is often an issue, ligand dissimilarity cannot necessarily be used to assume different off-target profiles and that understanding interfamily polypharmacology will be important in the future to reduce the risk of idiopathic toxicity and in the design of screening libraries..
Antolin, A.A.
Tym, J.E.
Komianou, A.
Collins, I.
Workman, P.
Al-Lazikani, B.
(2018). Objective, Quantitative, Data-Driven Assessment of Chemical Probes. Cell chem biol,
Vol.25
(2),
pp. 194-205.e5.
show abstract
full text
Chemical probes are essential tools for understanding biological systems and for target validation, yet selecting probes for biomedical research is rarely based on objective assessment of all potential compounds. Here, we describe the Probe Miner: Chemical Probes Objective Assessment resource, capitalizing on the plethora of public medicinal chemistry data to empower quantitative, objective, data-driven evaluation of chemical probes. We assess >1.8 million compounds for their suitability as chemical tools against 2,220 human targets and dissect the biases and limitations encountered. Probe Miner represents a valuable resource to aid the identification of potential chemical probes, particularly when used alongside expert curation..
Fok, J.H.
Hedayat, S.
Zhang, L.
Aronson, L.I.
Mirabella, F.
Pawlyn, C.
Bright, M.D.
Wardell, C.P.
Keats, J.J.
De Billy, E.
Rye, C.S.
Chessum, N.E.
Jones, K.
Morgan, G.J.
Eccles, S.A.
Workman, P.
Davies, F.E.
(2018). HSF1 Is Essential for Myeloma Cell Survival and A Promising Therapeutic Target. Clin cancer res,
Vol.24
(10),
pp. 2395-2407.
show abstract
full text
Purpose: Myeloma is a plasma cell malignancy characterized by the overproduction of immunoglobulin, and is therefore susceptible to therapies targeting protein homeostasis. We hypothesized that heat shock factor 1 (HSF1) was an attractive therapeutic target for myeloma due to its direct regulation of transcriptional programs implicated in both protein homeostasis and the oncogenic phenotype. Here, we interrogate HSF1 as a therapeutic target in myeloma using bioinformatic, genetic, and pharmacologic means.Experimental Design: To assess the clinical relevance of HSF1, we analyzed publicly available patient myeloma gene expression datasets. Validation of this novel target was conducted in in vitro experiments using shRNA or inhibitors of the HSF1 pathway in human myeloma cell lines and primary cells as well as in in vivo human myeloma xenograft models.Results: Expression of HSF1 and its target genes were associated with poorer myeloma patient survival. ShRNA-mediated knockdown or pharmacologic inhibition of the HSF1 pathway with a novel chemical probe, CCT251236, or with KRIBB11, led to caspase-mediated cell death that was associated with an increase in EIF2α phosphorylation, CHOP expression and a decrease in overall protein synthesis. Importantly, both CCT251236 and KRIBB11 induced cytotoxicity in human myeloma cell lines and patient-derived primary myeloma cells with a therapeutic window over normal cells. Pharmacologic inhibition induced tumor growth inhibition and was well-tolerated in a human myeloma xenograft murine model with evidence of pharmacodynamic biomarker modulation.Conclusions: Taken together, our studies demonstrate the dependence of myeloma cells on HSF1 for survival and support the clinical evaluation of pharmacologic inhibitors of the HSF1 pathway in myeloma. Clin Cancer Res; 24(10); 2395-407. ©2018 AACRSee related commentary by Parekh, p. 2237..
Vlachogiannis, G.
Hedayat, S.
Vatsiou, A.
Jamin, Y.
Fernández-Mateos, J.
Khan, K.
Lampis, A.
Eason, K.
Huntingford, I.
Burke, R.
Rata, M.
Koh, D.-.
Tunariu, N.
Collins, D.
Hulkki-Wilson, S.
Ragulan, C.
Spiteri, I.
Moorcraft, S.Y.
Chau, I.
Rao, S.
Watkins, D.
Fotiadis, N.
Bali, M.
Darvish-Damavandi, M.
Lote, H.
Eltahir, Z.
Smyth, E.C.
Begum, R.
Clarke, P.A.
Hahne, J.C.
Dowsett, M.
de Bono, J.
Workman, P.
Sadanandam, A.
Fassan, M.
Sansom, O.J.
Eccles, S.
Starling, N.
Braconi, C.
Sottoriva, A.
Robinson, S.P.
Cunningham, D.
Valeri, N.
(2018). Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science,
Vol.359
(6378),
pp. 920-926.
show abstract
full text
Patient-derived organoids (PDOs) have recently emerged as robust preclinical models; however, their potential to predict clinical outcomes in patients has remained unclear. We report on a living biobank of PDOs from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients recruited in phase 1/2 clinical trials. Phenotypic and genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. Molecular profiling of tumor organoids was matched to drug-screening results, suggesting that PDOs could complement existing approaches in defining cancer vulnerabilities and improving treatment responses. We compared responses to anticancer agents ex vivo in organoids and PDO-based orthotopic mouse tumor xenograft models with the responses of the patients in clinical trials. Our data suggest that PDOs can recapitulate patient responses in the clinic and could be implemented in personalized medicine programs..
O'Connor, J.P.
Aboagye, E.O.
Adams, J.E.
Aerts, H.J.
Barrington, S.F.
Beer, A.J.
Boellaard, R.
Bohndiek, S.E.
Brady, M.
Brown, G.
Buckley, D.L.
Chenevert, T.L.
Clarke, L.P.
Collette, S.
Cook, G.J.
deSouza, N.M.
Dickson, J.C.
Dive, C.
Evelhoch, J.L.
Faivre-Finn, C.
Gallagher, F.A.
Gilbert, F.J.
Gillies, R.J.
Goh, V.
Griffiths, J.R.
Groves, A.M.
Halligan, S.
Harris, A.L.
Hawkes, D.J.
Hoekstra, O.S.
Huang, E.P.
Hutton, B.F.
Jackson, E.F.
Jayson, G.C.
Jones, A.
Koh, D.-.
Lacombe, D.
Lambin, P.
Lassau, N.
Leach, M.O.
Lee, T.-.
Leen, E.L.
Lewis, J.S.
Liu, Y.
Lythgoe, M.F.
Manoharan, P.
Maxwell, R.J.
Miles, K.A.
Morgan, B.
Morris, S.
Ng, T.
Padhani, A.R.
Parker, G.J.
Partridge, M.
Pathak, A.P.
Peet, A.C.
Punwani, S.
Reynolds, A.R.
Robinson, S.P.
Shankar, L.K.
Sharma, R.A.
Soloviev, D.
Stroobants, S.
Sullivan, D.C.
Taylor, S.A.
Tofts, P.S.
Tozer, G.M.
van Herk, M.
Walker-Samuel, S.
Wason, J.
Williams, K.J.
Workman, P.
Yankeelov, T.E.
Brindle, K.M.
McShane, L.M.
Jackson, A.
Waterton, J.C.
(2017). Imaging biomarker roadmap for cancer studies. Nat rev clin oncol,
Vol.14
(3),
pp. 169-186.
show abstract
full text
Imaging biomarkers (IBs) are integral to the routine management of patients with cancer. IBs used daily in oncology include clinical TNM stage, objective response and left ventricular ejection fraction. Other CT, MRI, PET and ultrasonography biomarkers are used extensively in cancer research and drug development. New IBs need to be established either as useful tools for testing research hypotheses in clinical trials and research studies, or as clinical decision-making tools for use in healthcare, by crossing 'translational gaps' through validation and qualification. Important differences exist between IBs and biospecimen-derived biomarkers and, therefore, the development of IBs requires a tailored 'roadmap'. Recognizing this need, Cancer Research UK (CRUK) and the European Organisation for Research and Treatment of Cancer (EORTC) assembled experts to review, debate and summarize the challenges of IB validation and qualification. This consensus group has produced 14 key recommendations for accelerating the clinical translation of IBs, which highlight the role of parallel (rather than sequential) tracks of technical (assay) validation, biological/clinical validation and assessment of cost-effectiveness; the need for IB standardization and accreditation systems; the need to continually revisit IB precision; an alternative framework for biological/clinical validation of IBs; and the essential requirements for multicentre studies to qualify IBs for clinical use..
Whittaker, S.R.
Mallinger, A.
Workman, P.
Clarke, P.A.
(2017). Inhibitors of cyclin-dependent kinases as cancer therapeutics. Pharmacol ther,
Vol.173,
pp. 83-105.
show abstract
full text
Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin-dependent kinases (CDKs). This attention initially stemmed from observations that different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non-selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform-selective inhibitors with accompanying biomarkers has re-ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER+/HER2- advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies..
Carotenuto, P.
Fassan, M.
Pandolfo, R.
Lampis, A.
Vicentini, C.
Cascione, L.
Paulus-Hock, V.
Boulter, L.
Guest, R.
Quagliata, L.
Hahne, J.C.
Ridgway, R.
Jamieson, T.
Athineos, D.
Veronese, A.
Visone, R.
Murgia, C.
Ferrari, G.
Guzzardo, V.
Evans, T.R.
MacLeod, M.
Feng, G.J.
Dale, T.
Negrini, M.
Forbes, S.J.
Terracciano, L.
Scarpa, A.
Patel, T.
Valeri, N.
Workman, P.
Sansom, O.
Braconi, C.
(2017). Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers. Gut,
Vol.66
(7),
pp. 1268-1277.
show abstract
full text
OBJECTIVE: Transcribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/β-catenin pathway in liver cancer. DESIGN: Hypomorphic Apc mice (Apcfl/fl) and thiocetamide (TAA)-treated rats developed Wnt/β-catenin dependent hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), respectively. T-UCR expression was assessed by microarray, real-time PCR and in situ hybridisation. RESULTS: Overexpression of the T-UCR uc.158- could differentiate Wnt/β-catenin dependent HCC from normal liver and from β-catenin negative diethylnitrosamine (DEN)-induced HCC. uc.158- was overexpressed in human HepG2 versus Huh7 cells in line with activation of the Wnt pathway. In vitro modulation of β-catenin altered uc.158- expression in human malignant hepatocytes. uc.158- expression was increased in CTNNB1-mutated human HCCs compared with non-mutated human HCCs, and in human HCC with nuclear localisation of β-catenin. uc.158- was increased in TAA rat CCA and reduced after treatment with Wnt/β-catenin inhibitors. uc.158- expression was negative in human normal liver and biliary epithelia, while it was increased in human CCA in two different cohorts. Locked nucleic acid-mediated inhibition of uc.158- reduced anchorage cell growth, 3D-spheroid formation and spheroid-based cell migration, and increased apoptosis in HepG2 and SW1 cells. miR-193b was predicted to have binding sites within the uc.158- sequence. Modulation of uc.158- changed miR-193b expression in human malignant hepatocytes. Co-transfection of uc.158- inhibitor and anti-miR-193b rescued the effect of uc.158- inhibition on cell viability. CONCLUSIONS: We showed that uc.158- is activated by the Wnt pathway in liver cancers and drives their growth. Thus, it may represent a promising target for the development of novel therapeutics..
van Montfort, R.L.
Workman, P.
(2017). Structure-based drug design: aiming for a perfect fit. Essays biochem,
Vol.61
(5),
pp. 431-437.
show abstract
full text
Knowledge of the three-dimensional structure of therapeutically relevant targets has informed drug discovery since the first protein structures were determined using X-ray crystallography in the 1950s and 1960s. In this editorial we provide a brief overview of the powerful impact of structure-based drug design (SBDD), which has its roots in computational and structural biology, with major contributions from both academia and industry. We describe advances in the application of SBDD for integral membrane protein targets that have traditionally proved very challenging. We emphasize the major progress made in fragment-based approaches for which success has been exemplified by over 30 clinical drug candidates and importantly three FDA-approved drugs in oncology. We summarize the articles in this issue that provide an excellent snapshot of the current state of the field of SBDD and fragment-based drug design and which offer key insights into exciting new developments, such as the X-ray free-electron laser technology, cryo-electron microscopy, open science approaches and targeted protein degradation. We stress the value of SBDD in the design of high-quality chemical tools that are used to interrogate biology and disease pathology, and to inform target validation. We emphasize the need to maintain the scientific rigour that has been traditionally associated with structural biology and extend this to other methods used in drug discovery. This is particularly important because the quality and robustness of any form of contributory data determines its usefulness in accelerating drug design, and therefore ultimately in providing patient benefit..
Ang, J.E.
Pal, A.
Asad, Y.J.
Henley, A.T.
Valenti, M.
Box, G.
de Haven Brandon, A.
Revell, V.L.
Skene, D.J.
Venturi, M.
Rueger, R.
Meresse, V.
Eccles, S.A.
de Bono, J.S.
Kaye, S.B.
Workman, P.
Banerji, U.
Raynaud, F.I.
(2017). Modulation of Plasma Metabolite Biomarkers of the MAPK Pathway with MEK Inhibitor RO4987655: Pharmacodynamic and Predictive Potential in Metastatic Melanoma. Mol cancer ther,
Vol.16
(10),
pp. 2315-2323.
show abstract
full text
MAPK pathway activation is frequently observed in human malignancies, including melanoma, and is associated with sensitivity to MEK inhibition and changes in cellular metabolism. Using quantitative mass spectrometry-based metabolomics, we identified in preclinical models 21 plasma metabolites including amino acids, propionylcarnitine, phosphatidylcholines, and sphingomyelins that were significantly altered in two B-RAF-mutant melanoma xenografts and that were reversed following a single dose of the potent and selective MEK inhibitor RO4987655. Treatment of non-tumor-bearing animals and mice bearing the PTEN-null U87MG human glioblastoma xenograft elicited plasma changes only in amino acids and propionylcarnitine. In patients with advanced melanoma treated with RO4987655, on-treatment changes of amino acids were observed in patients with disease progression and not in responders. In contrast, changes in phosphatidylcholines and sphingomyelins were observed in responders. Furthermore, pretreatment levels of seven lipids identified in the preclinical screen were statistically significantly able to predict objective responses to RO4987655. The RO4987655 treatment-related changes were greater than baseline physiological variability in nontreated individuals. This study provides evidence of a translational exo-metabolomic plasma readout predictive of clinical efficacy together with pharmacodynamic utility following treatment with a signal transduction inhibitor. Mol Cancer Ther; 16(10); 2315-23. ©2017 AACR..
Blagg, J.
Workman, P.
(2017). Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology. Cancer cell,
Vol.32
(1),
pp. 9-25.
show abstract
full text
Small-molecule chemical probes or tools have become progressively more important in recent years as valuable reagents to investigate fundamental biological mechanisms and processes causing disease, including cancer. Chemical probes have also achieved greater prominence alongside complementary biological reagents for target validation in drug discovery. However, there is evidence of widespread continuing misuse and promulgation of poor-quality and insufficiently selective chemical probes, perpetuating a worrisome and misleading pollution of the scientific literature. We discuss current challenges with the selection and use of chemical probes, and suggest how biologists can and should be more discriminating in the probes they employ..
Cheeseman, M.D.
Chessum, N.E.
Rye, C.S.
Pasqua, A.E.
Tucker, M.J.
Wilding, B.
Evans, L.E.
Lepri, S.
Richards, M.
Sharp, S.Y.
Ali, S.
Rowlands, M.
O'Fee, L.
Miah, A.
Hayes, A.
Henley, A.T.
Powers, M.
Te Poele, R.
De Billy, E.
Pellegrino, L.
Raynaud, F.
Burke, R.
van Montfort, R.L.
Eccles, S.A.
Workman, P.
Jones, K.
(2017). Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen. J med chem,
Vol.60
(1),
pp. 180-201.
show abstract
full text
Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography..
Agliano, A.
Balarajah, G.
Ciobota, D.M.
Sidhu, J.
Clarke, P.A.
Jones, C.
Workman, P.
Leach, M.O.
Al-Saffar, N.M.
(2017). Pediatric and adult glioblastoma radiosensitization induced by PI3K/mTOR inhibition causes early metabolic alterations detected by nuclear magnetic resonance spectroscopy. Oncotarget,
Vol.8
(29),
pp. 47969-47983.
show abstract
full text
Poor outcome for patients with glioblastomas is often associated with radioresistance. PI3K/mTOR pathway deregulation has been correlated with radioresistance; therefore, PI3K/mTOR inhibition could render tumors radiosensitive. In this study, we show that NVP-BEZ235, a dual PI3K/mTOR inhibitor, potentiates the effects of irradiation in both adult and pediatric glioblastoma cell lines, resulting in early metabolic changes detected by nuclear magnetic resonance (NMR) spectroscopy. NVP-BEZ235 radiosensitises cells to X ray exposure, inducing cell death through the inhibition of CDC25A and the activation of p21cip1(CDKN1A). Lactate and phosphocholine levels, increased with radiation, are decreased after NVP-BEZ235 and combination treatment, suggesting that inhibiting the PI3K/mTOR pathway reverses radiation induced metabolic changes. Importantly, NVP-BEZ235 potentiates the effects of irradiation in a xenograft model of adult glioblastoma, where we observed a decrease in lactate and phosphocholine levels after seven days of combination treatment. Although tumor size was not affected due to the short length of the treatment, a significant increase in CASP3 mRNA was observed in the combination group. Taken together, our data suggest that NMR metabolites could be used as biomarkers to detect an early response to combination therapy with PI3K/mTOR inhibitors and radiotherapy in adult and pediatric glioblastoma patients..
Dolly, S.O.
Gurden, M.D.
Drosopoulos, K.
Clarke, P.
de Bono, J.
Kaye, S.
Workman, P.
Linardopoulos, S.
(2017). RNAi screen reveals synthetic lethality between cyclin G-associated kinase and FBXW7 by inducing aberrant mitoses. Br j cancer,
Vol.117
(7),
pp. 954-964.
show abstract
full text
BACKGROUND: F-box and WD40 repeat domain-containing 7 (FBXW7) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of multiple oncogenic substrates. The tumour suppressor function is frequently lost in multiple cancers through genetic deletion and mutations in a broad range of tumours. Loss of FBXW7 functionality results in the stabilisation of multiple major oncoproteins, culminating in increased cellular proliferation and pro-survival pathways, cell cycle deregulation, chromosomal instability and altered metabolism. Currently, there is no therapy to specifically target FBXW7-deficient tumours. METHODS: We performed a siRNA kinome screen to identify synthetically lethal hits to FBXW7 deficiency. RESULTS: We identified and validated cyclin G-associated kinase (GAK) as a potential new therapeutic target. Combined loss of FBXW7 and GAK caused cell cycle defects, formation of multipolar mitoses and the induction of apoptosis. The synthetic lethal mechanism appears to be independent of clathrin-mediated receptor endocytosis function of GAK. CONCLUSIONS: These data suggest a putative therapeutic strategy for a large number of different types of human cancers with FBXW7 loss, many of which have a paucity of molecular abnormalities and treatment options..
Pawlyn, C.
Bright, M.D.
Buros, A.F.
Stein, C.K.
Walters, Z.
Aronson, L.I.
Mirabella, F.
Jones, J.R.
Kaiser, M.F.
Walker, B.A.
Jackson, G.H.
Clarke, P.A.
Bergsagel, P.L.
Workman, P.
Chesi, M.
Morgan, G.J.
Davies, F.E.
(2017). Overexpression of EZH2 in multiple myeloma is associated with poor prognosis and dysregulation of cell cycle control. Blood cancer j,
Vol.7
(3),
p. e549.
show abstract
full text
Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies..
Al-Saffar, N.M.
Agliano, A.
Marshall, L.V.
Jackson, L.E.
Balarajah, G.
Sidhu, J.
Clarke, P.A.
Jones, C.
Workman, P.
Pearson, A.D.
Leach, M.O.
(2017). In vitro nuclear magnetic resonance spectroscopy metabolic biomarkers for the combination of temozolomide with PI3K inhibition in paediatric glioblastoma cells. Plos one,
Vol.12
(7),
p. e0180263.
show abstract
full text
Recent experimental data showed that the PI3K pathway contributes to resistance to temozolomide (TMZ) in paediatric glioblastoma and that this effect is reversed by combination treatment of TMZ with a PI3K inhibitor. Our aim is to assess whether this combination results in metabolic changes that are detectable by nuclear magnetic resonance (NMR) spectroscopy, potentially providing metabolic biomarkers for PI3K inhibition and TMZ combination treatment. Using two genetically distinct paediatric glioblastoma cell lines, SF188 and KNS42, in vitro 1H-NMR analysis following treatment with the dual pan-Class I PI3K/mTOR inhibitor PI-103 resulted in a decrease in lactate and phosphocholine (PC) levels (P<0.02) relative to control. In contrast, treatment with TMZ caused an increase in glycerolphosphocholine (GPC) levels (P≤0.05). Combination of PI-103 with TMZ showed metabolic effects of both agents including a decrease in the levels of lactate and PC (P<0.02) while an increase in GPC (P<0.05). We also report a decrease in the protein expression levels of HK2, LDHA and CHKA providing likely mechanisms for the depletion of lactate and PC, respectively. Our results show that our in vitro NMR-detected changes in lactate and choline metabolites may have potential as non-invasive biomarkers for monitoring response to combination of PI3K/mTOR inhibitors with TMZ during clinical trials in children with glioblastoma, subject to further in vivo validation..
Coker, E.A.
Mitsopoulos, C.
Workman, P.
Al-Lazikani, B.
(2017). SiGNet: A signaling network data simulator to enable signaling network inference. Plos one,
Vol.12
(5),
p. e0177701.
show abstract
full text
Network models are widely used to describe complex signaling systems. Cellular wiring varies in different cellular contexts and numerous inference techniques have been developed to infer the structure of a network from experimental data of the network's behavior. To objectively identify which inference strategy is best suited to a specific network, a gold standard network and dataset are required. However, suitable datasets for benchmarking are difficult to find. Numerous tools exist that can simulate data for transcriptional networks, but these are of limited use for the study of signaling networks. Here, we describe SiGNet (Signal Generator for Networks): a Cytoscape app that simulates experimental data for a signaling network of known structure. SiGNet has been developed and tested against published experimental data, incorporating information on network architecture, and the directionality and strength of interactions to create biological data in silico. SiGNet is the first tool to simulate biological signaling data, enabling an accurate and systematic assessment of inference strategies. SiGNet can also be used to produce preliminary models of key biological pathways following perturbation..
Ferraldeschi, R.
Welti, J.
Powers, M.V.
Yuan, W.
Smyth, T.
Seed, G.
Riisnaes, R.
Hedayat, S.
Wang, H.
Crespo, M.
Nava Rodrigues, D.
Figueiredo, I.
Miranda, S.
Carreira, S.
Lyons, J.F.
Sharp, S.
Plymate, S.R.
Attard, G.
Wallis, N.
Workman, P.
de Bono, J.S.
(2016). Second-Generation HSP90 Inhibitor Onalespib Blocks mRNA Splicing of Androgen Receptor Variant 7 in Prostate Cancer Cells. Cancer res,
Vol.76
(9),
pp. 2731-2742.
show abstract
full text
Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731-42. ©2016 AACR..
Rye, C.S.
Chessum, N.E.
Lamont, S.
Pike, K.G.
Faulder, P.
Demeritt, J.
Kemmitt, P.
Tucker, J.
Zani, L.
Cheeseman, M.D.
Isaac, R.
Goodwin, L.
Boros, J.
Raynaud, F.
Hayes, A.
Henley, A.T.
de Billy, E.
Lynch, C.J.
Sharp, S.Y.
Te Poele, R.
Fee, L.O.
Foote, K.M.
Green, S.
Workman, P.
Jones, K.
(2016). Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9. Medchemcomm,
Vol.7
(8),
pp. 1580-1586.
show abstract
full text
Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 μM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM)..
Ang, J.E.
Pandher, R.
Ang, J.C.
Asad, Y.J.
Henley, A.T.
Valenti, M.
Box, G.
de Haven Brandon, A.
Baird, R.D.
Friedman, L.
Derynck, M.
Vanhaesebroeck, B.
Eccles, S.A.
Kaye, S.B.
Workman, P.
de Bono, J.S.
Raynaud, F.I.
(2016). Plasma Metabolomic Changes following PI3K Inhibition as Pharmacodynamic Biomarkers: Preclinical Discovery to Phase I Trial Evaluation. Mol cancer ther,
Vol.15
(6),
pp. 1412-1424.
show abstract
full text
PI3K plays a key role in cellular metabolism and cancer. Using a mass spectrometry-based metabolomics platform, we discovered that plasma concentrations of 26 metabolites, including amino acids, acylcarnitines, and phosphatidylcholines, were decreased in mice bearing PTEN-deficient tumors compared with non-tumor-bearing controls and in addition were increased following dosing with class I PI3K inhibitor pictilisib (GDC-0941). These candidate metabolomics biomarkers were evaluated in a phase I dose-escalation clinical trial of pictilisib. Time- and dose-dependent effects were observed in patients for 22 plasma metabolites. The changes exceeded baseline variability, resolved after drug washout, and were recapitulated on continuous dosing. Our study provides a link between modulation of the PI3K pathway and changes in the plasma metabolome and demonstrates that plasma metabolomics is a feasible and promising strategy for biomarker evaluation. Also, our findings provide additional support for an association between insulin resistance, branched-chain amino acids, and related metabolites following PI3K inhibition. Mol Cancer Ther; 15(6); 1412-24. ©2016 AACR..
Vaughan, L.
Clarke, P.A.
Barker, K.
Chanthery, Y.
Gustafson, C.W.
Tucker, E.
Renshaw, J.
Raynaud, F.
Li, X.
Burke, R.
Jamin, Y.
Robinson, S.P.
Pearson, A.
Maira, M.
Weiss, W.A.
Workman, P.
Chesler, L.
(2016). Inhibition of mTOR-kinase destabilizes MYCN and is a potential therapy for MYCN-dependent tumors. Oncotarget,
Vol.7
(36),
pp. 57525-57544.
show abstract
full text
MYC oncoproteins deliver a potent oncogenic stimulus in several human cancers, making them major targets for drug development, but efforts to deliver clinically practical therapeutics have not yet been realized. In childhood cancer, aberrant expression of MYC and MYCN genes delineates a group of aggressive tumours responsible for a major proportion of pediatric cancer deaths. We designed a chemical-genetic screen that identifies compounds capable of enhancing proteasomal elimination of MYCN oncoprotein. We isolated several classes of compound that selectively kill MYCN expressing cells and we focus on inhibitors of PI3K/mTOR pathway in this study. We show that PI3K/mTOR inhibitors selectively killed MYCN-expressing neuroblastoma tumor cells, and induced significant apoptosis of transgenic MYCN-driven neuroblastoma tumors concomitant with elimination of MYCN protein in vivo. Mechanistically, the ability of these compounds to degrade MYCN requires complete blockade of mTOR but not PI3 kinase activity and we highlight NVP-BEZ235 as a PI3K/mTOR inhibitor with an ideal activity profile. These data establish that MYCN expression is a marker indicative of likely clinical sensitivity to mTOR inhibition, and provide a rationale for the selection of clinical candidate MYCN-destabilizers likely to be useful for the treatment of MYCN-driven cancers..
Jones, A.M.
Westwood, I.M.
Osborne, J.D.
Matthews, T.P.
Cheeseman, M.D.
Rowlands, M.G.
Jeganathan, F.
Burke, R.
Lee, D.
Kadi, N.
Liu, M.
Richards, M.
McAndrew, C.
Yahya, N.
Dobson, S.E.
Jones, K.
Workman, P.
Collins, I.
van Montfort, R.L.
(2016). A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms. Sci rep,
Vol.6,
p. 34701.
show abstract
full text
The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops..
Banerji, U.
Workman, P.
(2016). Critical parameters in targeted drug development: the pharmacological audit trail. Semin oncol,
Vol.43
(4),
pp. 436-445.
show abstract
full text
The Pharmacological Audit Trail (PhAT) comprises a set of critical questions that need to be asked during discovery and development of an anticancer drug. Key aspects include: (1) defining a patient population; (2) establishing pharmacokinetic characteristics; (3) providing evidence of target engagement, pathway modulation, and biological effect with proof of concept pharmacodynamic biomarkers; (4) determining intermediate biomarkers of response; (5) assessing tumor response; and (6) determining how to overcome resistance by combination or sequential therapy and new target/drug discovery. The questions asked in the PhAT should be viewed as a continuum and not used in isolation. Different drug development programmes derive different types of benefit from these questions. The PhAT is critical in making go-no-go decisions in the development of currently studied drugs and will continue to be relevant to discovery and development of future generations of anticancer agents..
Mallinger, A.
Schiemann, K.
Rink, C.
Sejberg, J.
Honey, M.A.
Czodrowski, P.
Stubbs, M.
Poeschke, O.
Busch, M.
Schneider, R.
Schwarz, D.
Musil, D.
Burke, R.
Urbahns, K.
Workman, P.
Wienke, D.
Clarke, P.A.
Raynaud, F.I.
Eccles, S.A.
Esdar, C.
Rohdich, F.
Blagg, J.
(2016). 2,8-Disubstituted-1,6-Naphthyridines and 4,6-Disubstituted-Isoquinolines with Potent, Selective Affinity for CDK8/19. Acs med chem lett,
Vol.7
(6),
pp. 573-578.
show abstract
full text
We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1(SER727) phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing..
Al-Lazikani, B.
Workman, P.
(2016). Minimizing bias in target selection by exploiting multidisciplinary Big Data and the protein interactome. Future med chem,
Vol.8
(14),
pp. 1711-1716.
full text
Clarke, P.A.
Ortiz-Ruiz, M.-.
TePoele, R.
Adeniji-Popoola, O.
Box, G.
Court, W.
Czasch, S.
El Bawab, S.
Esdar, C.
Ewan, K.
Gowan, S.
De Haven Brandon, A.
Hewitt, P.
Hobbs, S.M.
Kaufmann, W.
Mallinger, A.
Raynaud, F.
Roe, T.
Rohdich, F.
Schiemann, K.
Simon, S.
Schneider, R.
Valenti, M.
Weigt, S.
Blagg, J.
Blaukat, A.
Dale, T.C.
Eccles, S.A.
Hecht, S.
Urbahns, K.
Workman, P.
Wienke, D.
(2016). Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases. Elife,
Vol.5.
show abstract
full text
Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors..
Yap, T.A.
Smith, A.D.
Ferraldeschi, R.
Al-Lazikani, B.
Workman, P.
de Bono, J.S.
(2016). Drug discovery in advanced prostate cancer: translating biology into therapy. Nat rev drug discov,
Vol.15
(10),
pp. 699-718.
show abstract
full text
Castration-resistant prostate cancer (CRPC) is associated with a poor prognosis and poses considerable therapeutic challenges. Recent genetic and technological advances have provided insights into prostate cancer biology and have enabled the identification of novel drug targets and potent molecularly targeted therapeutics for this disease. In this article, we review recent advances in prostate cancer target identification for drug discovery and discuss their promise and associated challenges. We review the evolving therapeutic landscape of CRPC and discuss issues associated with precision medicine as well as challenges encountered with immunotherapy for this disease. Finally, we envision the future management of CRPC, highlighting the use of circulating biomarkers and modern clinical trial designs..
Mallinger, A.
Schiemann, K.
Rink, C.
Stieber, F.
Calderini, M.
Crumpler, S.
Stubbs, M.
Adeniji-Popoola, O.
Poeschke, O.
Busch, M.
Czodrowski, P.
Musil, D.
Schwarz, D.
Ortiz-Ruiz, M.-.
Schneider, R.
Thai, C.
Valenti, M.
de Haven Brandon, A.
Burke, R.
Workman, P.
Dale, T.
Wienke, D.
Clarke, P.A.
Esdar, C.
Raynaud, F.I.
Eccles, S.A.
Rohdich, F.
Blagg, J.
(2016). Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19. J med chem,
Vol.59
(3),
pp. 1078-1101.
show abstract
full text
The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer..
Delgado-Goni, T.
Miniotis, M.F.
Wantuch, S.
Parkes, H.G.
Marais, R.
Workman, P.
Leach, M.O.
Beloueche-Babari, M.
(2016). The BRAF Inhibitor Vemurafenib Activates Mitochondrial Metabolism and Inhibits Hyperpolarized Pyruvate-Lactate Exchange in BRAF-Mutant Human Melanoma Cells. Mol cancer ther,
Vol.15
(12),
pp. 2987-2999.
show abstract
full text
Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long-term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity, and potential as noninvasive imaging response biomarkers. 1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine, and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of hexokinase 2, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase, and monocarboxylate transporters (MCT) 1 and 4 in BRAF-mutant but not BRAFWT cells and, interestingly, decreased BRAF-mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate-lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as noninvasive imaging of response. Mol Cancer Ther; 15(12); 2987-99. ©2016 AACR..
Workman, P.
Clarke, P.A.
Al-Lazikani, B.
(2016). Blocking the survival of the nastiest by HSP90 inhibition. Oncotarget,
Vol.7
(4),
pp. 3658-3661.
show abstract
It is now recognised that genetic, epigenetic and phenotypic heterogeneity within individual human cancers is responsible for therapeutic resistance - knowledge that is having a profound impact on current thinking and experimentation. There has been concern that molecularly targeted therapy is doomed to failure, with resistant clones emerging in response to the Darwinian selective pressure of any drug treatment. However, two studies have shown that the evolution of drug resistance can be restrained by co-administration of a pharmacologic inhibitor of the HSP90 molecular chaperone..
Cheeseman, M.D.
Westwood, I.M.
Barbeau, O.
Rowlands, M.
Dobson, S.
Jones, A.M.
Jeganathan, F.
Burke, R.
Kadi, N.
Workman, P.
Collins, I.
van Montfort, R.L.
Jones, K.
(2016). Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70. J med chem,
Vol.59
(10),
pp. 4625-4636.
show abstract
full text
HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner..
Patel, A.
Sharp, S.Y.
Hall, K.
Lewis, W.
Stevens, M.F.
Workman, P.
Moody, C.J.
(2016). Fused imidazoles as potential chemical scaffolds for inhibition of heat shock protein 70 and induction of apoptosis Synthesis and biological evaluation of phenanthro[9,10-d]imidazoles and imidazo[4,5-f][1,10]phenanthrolines. Org biomol chem,
Vol.14
(16),
pp. 3889-3905.
show abstract
The imidazole ring is widespread in biologically active compounds, and hence imidazole-containing scaffolds are useful starting points for drug discovery programmes. We report the synthesis of a series of novel imidazole-containing compounds fused with either phenanthrene or phenanthroline, which show enhanced growth inhibitory potency against human colon, breast and melanoma cancer cell lines, as well as evidence of inhibition of the molecular chaperone heat shock protein 70 (Hsp70) pathway in cells, as shown by depletion of downstream oncogenic client proteins of the Hsp90 chaperone pathway, and induction of apoptosis..
Antolin, A.A.
Workman, P.
Mestres, J.
Al-Lazikani, B.
(2016). Polypharmacology in Precision Oncology: Current Applications and Future Prospects. Curr pharm des,
Vol.22
(46),
pp. 6935-6945.
show abstract
full text
Over the past decade, a more comprehensive, large-scale approach to studying cancer genetics and biology has revealed the challenges of tumor heterogeneity, adaption, evolution and drug resistance, while systems-based pharmacology and chemical biology strategies have uncovered a much more complex interaction between drugs and the human proteome than was previously anticipated. In this mini-review we assess the progress and potential of drug polypharmacology in biomarker-driven precision oncology. Polypharmacology not only provides great opportunities for drug repurposing to exploit off-target effects in a new single-target indication but through simultaneous blockade of multiple targets or pathways offers exciting opportunities to slow, overcome or even prevent inherent or adaptive drug resistance. We highlight the many challenges associated with exploiting known or desired polypharmacology in drug design and development, and assess computational and experimental methods to uncover unknown polypharmacology. A comprehensive understanding of the intricate links between polypharmacology, efficacy and safety is urgently needed if we are to tackle the enduring challenge of cancer drug resistance and to fully exploit polypharmacology for the ultimate benefit of cancer patients..
Sarker, D.
Ang, J.E.
Baird, R.
Kristeleit, R.
Shah, K.
Moreno, V.
Clarke, P.A.
Raynaud, F.I.
Levy, G.
Ware, J.A.
Mazina, K.
Lin, R.
Wu, J.
Fredrickson, J.
Spoerke, J.M.
Lackner, M.R.
Yan, Y.
Friedman, L.S.
Kaye, S.B.
Derynck, M.K.
Workman, P.
de Bono, J.S.
(2015). First-in-human phase I study of pictilisib (GDC-0941), a potent pan-class I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors. Clin cancer res,
Vol.21
(1),
pp. 77-86.
show abstract
PURPOSE: This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal-tolerated dose (MTD), dose-limiting toxicities (DLT), pharmacokinetics, pharmacodynamics, and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent, and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K). PATIENTS AND METHODS: Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily, initially on days 1 to 21 every 28 days and later, using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated (18)F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue. RESULTS: Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea, rash, and fatigue, whereas the DLT was grade 3 maculopapular rash (450 mg, 2 of 3 patients; 330 mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC >20 h·μmol/L. Significant increase in plasma insulin and glucose levels, and >25% decrease in (18)F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively. CONCLUSION: Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily..
Smith, J.R.
de Billy, E.
Hobbs, S.
Powers, M.
Prodromou, C.
Pearl, L.
Clarke, P.A.
Workman, P.
(2015). Restricting direct interaction of CDC37 with HSP90 does not compromise chaperoning of client proteins. Oncogene,
Vol.34
(1),
pp. 15-26.
show abstract
full text
The HSP90 molecular chaperone plays a key role in the maturation, stability and activation of its clients, including many oncogenic proteins. Kinases are a substantial and important subset of clients requiring the key cochaperone CDC37. We sought an improved understanding of protein kinase chaperoning by CDC37 in cancer cells. CDC37 overexpression in human colon cancer cells increased CDK4 protein levels, which was negated upon CDC37 knockdown. Overexpressing CDC37 increased CDK4 protein half-life and enhanced binding of HSP90 to CDK4, consistent with CDC37 promoting kinase loading onto chaperone complexes. Against expectation, expression of C-terminus-truncated CDC37 (ΔC-CDC37) that lacks HSP90 binding capacity did not affect kinase client expression or activity; moreover, as with wild-type CDC37 overexpression, it augmented CDK4-HSP90 complex formation. However, although truncation blocked binding to HSP90 in cells, ΔC-CDC37 also showed diminished client protein binding and was relatively unstable. CDC37 mutants with single and double point mutations at residues M164 and L205 showed greatly reduced binding to HSP90, but retained association with client kinases. Surprisingly, these mutants phenocopied wild-type CDC37 overexpression by increasing CDK4-HSP90 association and CDK4 protein levels in cells. Furthermore, expression of the mutants was sufficient to protect kinase clients CDK4, CDK6, CRAF and ERBB2 from depletion induced by silencing endogenous CDC37, indicating that CDC37's client stabilising function cannot be inactivated by substantially reducing its direct interaction with HSP90. However, CDC37 could not compensate for loss of HSP90 function, showing that CDC37 and HSP90 have their own distinct and non-redundant roles in maintaining kinase clients. Our data substantiate the important function of CDC37 in chaperoning protein kinases. Furthermore, we demonstrate that CDC37 can stabilise kinase clients by a mechanism that is not dependent on a substantial direct interaction between CDC37 and HSP90, but nevertheless requires HSP90 activity. These results have significant implications for therapeutic targeting of CDC37..
Yap, T.A.
Bjerke, L.
Clarke, P.A.
Workman, P.
(2015). Drugging PI3K in cancer: refining targets and therapeutic strategies. Curr opin pharmacol,
Vol.23,
pp. 98-107.
show abstract
full text
The phosphatidylinositol-3 kinase (PI3K) pathway is one of the most frequently activated pathogenic signalling routes in human cancers, making it a rational and important target for innovative anticancer drug development and precision medicine. The three main classes of PI3K inhibitors currently in clinical testing comprise dual pan-Class I PI3K/mTOR inhibitors, pan-Class I PI3K inhibitors lacking significant mTOR activity and isoform-selective PI3K inhibitors. A major step forward in recent years is the progression of over 30 small molecule PI3K inhibitors into clinical trials and the first regulatory approval of the PI3Kδ inhibitor idelalisib for multiple B-cell malignancies. This review article focuses on the progress made in the discovery and development of novel PI3K inhibitors, with an emphasis on antitumour activity and tolerability profiles for agents that have entered clinical trials. We also discuss the key issues of drug resistance, patient selection approaches and rational targeted combinations. Finally, we envision the future development and use of PI3K inhibitors for the treatment of patients with a range of malignancies..
Arrowsmith, C.H.
Audia, J.E.
Austin, C.
Baell, J.
Bennett, J.
Blagg, J.
Bountra, C.
Brennan, P.E.
Brown, P.J.
Bunnage, M.E.
Buser-Doepner, C.
Campbell, R.M.
Carter, A.J.
Cohen, P.
Copeland, R.A.
Cravatt, B.
Dahlin, J.L.
Dhanak, D.
Edwards, A.M.
Frederiksen, M.
Frye, S.V.
Gray, N.
Grimshaw, C.E.
Hepworth, D.
Howe, T.
Huber, K.V.
Jin, J.
Knapp, S.
Kotz, J.D.
Kruger, R.G.
Lowe, D.
Mader, M.M.
Marsden, B.
Mueller-Fahrnow, A.
Müller, S.
O'Hagan, R.C.
Overington, J.P.
Owen, D.R.
Rosenberg, S.H.
Roth, B.
Ross, R.
Schapira, M.
Schreiber, S.L.
Shoichet, B.
Sundström, M.
Superti-Furga, G.
Taunton, J.
Toledo-Sherman, L.
Walpole, C.
Walters, M.A.
Willson, T.M.
Workman, P.
Young, R.N.
Zuercher, W.J.
(2015). The promise and peril of chemical probes. Nat chem biol,
Vol.11
(8),
pp. 536-541.
show abstract
Chemical probes are powerful reagents with increasing impacts on biomedical research. However, probes of poor quality or that are used incorrectly generate misleading results. To help address these shortcomings, we will create a community-driven wiki resource to improve quality and convey current best practice..
Butler, L.M.
Ferraldeschi, R.
Armstrong, H.K.
Centenera, M.M.
Workman, P.
(2015). Maximizing the Therapeutic Potential of HSP90 Inhibitors. Mol cancer res,
Vol.13
(11),
pp. 1445-1451.
show abstract
HSP90 is required for maintaining the stability and activity of a diverse group of client proteins, including protein kinases, transcription factors, and steroid hormone receptors involved in cell signaling, proliferation, survival, oncogenesis, and cancer progression. Inhibition of HSP90 alters the HSP90-client protein complex, leading to reduced activity, misfolding, ubiquitination, and, ultimately, proteasomal degradation of client proteins. HSP90 inhibitors have demonstrated significant antitumor activity in a wide variety of preclinical models, with evidence of selectivity for cancer versus normal cells. In the clinic, however, the efficacy of this class of therapeutic agents has been relatively limited to date, with promising responses mainly observed in breast and lung cancer, but no major activity seen in other tumor types. In addition, adverse events and some significant toxicities have been documented. Key to improving these clinical outcomes is a better understanding of the cellular consequences of inhibiting HSP90 that may underlie treatment response or resistance. This review considers the recent progress that has been made in the study of HSP90 and its inhibitors and highlights new opportunities to maximize their therapeutic potential..
Dale, T.
Clarke, P.A.
Esdar, C.
Waalboer, D.
Adeniji-Popoola, O.
Ortiz-Ruiz, M.-.
Mallinger, A.
Samant, R.S.
Czodrowski, P.
Musil, D.
Schwarz, D.
Schneider, K.
Stubbs, M.
Ewan, K.
Fraser, E.
TePoele, R.
Court, W.
Box, G.
Valenti, M.
de Haven Brandon, A.
Gowan, S.
Rohdich, F.
Raynaud, F.
Schneider, R.
Poeschke, O.
Blaukat, A.
Workman, P.
Schiemann, K.
Eccles, S.A.
Wienke, D.
Blagg, J.
(2015). A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease. Nat chem biol,
Vol.11
(12),
pp. 973-980.
show abstract
full text
There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors..
Mitsopoulos, C.
Schierz, A.C.
Workman, P.
Al-Lazikani, B.
(2015). Distinctive Behaviors of Druggable Proteins in Cellular Networks. Plos comput biol,
Vol.11
(12),
p. e1004597.
show abstract
full text
The interaction environment of a protein in a cellular network is important in defining the role that the protein plays in the system as a whole, and thus its potential suitability as a drug target. Despite the importance of the network environment, it is neglected during target selection for drug discovery. Here, we present the first systematic, comprehensive computational analysis of topological, community and graphical network parameters of the human interactome and identify discriminatory network patterns that strongly distinguish drug targets from the interactome as a whole. Importantly, we identify striking differences in the network behavior of targets of cancer drugs versus targets from other therapeutic areas and explore how they may relate to successful drug combinations to overcome acquired resistance to cancer drugs. We develop, computationally validate and provide the first public domain predictive algorithm for identifying druggable neighborhoods based on network parameters. We also make available full predictions for 13,345 proteins to aid target selection for drug discovery. All target predictions are available through canSAR.icr.ac.uk. Underlying data and tools are available at https://cansar.icr.ac.uk/cansar/publications/druggable_network_neighbourhoods/..
Gurden, M.D.
Westwood, I.M.
Faisal, A.
Naud, S.
Cheung, K.-.
McAndrew, C.
Wood, A.
Schmitt, J.
Boxall, K.
Mak, G.
Workman, P.
Burke, R.
Hoelder, S.
Blagg, J.
Van Montfort, R.L.
Linardopoulos, S.
(2015). Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance. Cancer res,
Vol.75
(16),
pp. 3340-3354.
show abstract
Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells..
Mallinger, A.
Crumpler, S.
Pichowicz, M.
Waalboer, D.
Stubbs, M.
Adeniji-Popoola, O.
Wood, B.
Smith, E.
Thai, C.
Henley, A.T.
Georgi, K.
Court, W.
Hobbs, S.
Box, G.
Ortiz-Ruiz, M.-.
Valenti, M.
De Haven Brandon, A.
TePoele, R.
Leuthner, B.
Workman, P.
Aherne, W.
Poeschke, O.
Dale, T.
Wienke, D.
Esdar, C.
Rohdich, F.
Raynaud, F.
Clarke, P.A.
Eccles, S.A.
Stieber, F.
Schiemann, K.
Blagg, J.
(2015). Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen. J med chem,
Vol.58
(4),
pp. 1717-1735.
show abstract
full text
WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target..
Samant, R.S.
Clarke, P.A.
Workman, P.
(2014). E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells. Proc natl acad sci u s a,
Vol.111
(18),
pp. 6834-6839.
show abstract
The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90-client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition..
Workman, P.
van Montfort, R.
(2014). EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence. Cancer discov,
Vol.4
(6),
pp. 642-645.
show abstract
The crystal structure of a conserved tubulin-binding region of the EML1 protein reveals a highly atypical fold in one of its β-propeller domains. Disruption of the EML1 core region domain in many of the oncogenic EML4-ALK fusion protein variants that drive non-small cell lung cancer explains their dependence on the HSP90 molecular chaperone, provides a basis to allow more precise patient stratification for therapy, and suggests a more general model for other oncogenic fusion proteins..
Blagg, J.
Workman, P.
(2014). Chemical biology approaches to target validation in cancer. Current opinion in pharmacology,
Vol.17,
pp. 87-100.
Workman, P.
(2014). Successes for UK cancer partnership. Nature,
Vol.510
(7504),
pp. 218-218.
Al-Saffar, N.M.
Marshall, L.V.
Jackson, L.E.
Balarajah, G.
Eykyn, T.R.
Agliano, A.
Clarke, P.A.
Jones, C.
Workman, P.
Pearson, A.D.
Leach, M.O.
(2014). Lactate and choline metabolites detected in vitro by nuclear magnetic resonance spectroscopy are potential metabolic biomarkers for PI3K inhibition in pediatric glioblastoma. Plos one,
Vol.9
(8),
p. e103835.
show abstract
The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan-Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma..
Patel, M.N.
Halling-Brown, M.D.
Tym, J.E.
Workman, P.
Al-Lazikani, B.
(2013). Objective assessment of cancer genes for drug discovery. Nat rev drug discov,
Vol.12
(1),
pp. 35-50.
show abstract
Selecting the best targets is a key challenge for drug discovery, and achieving this effectively, efficiently and systematically is particularly important for prioritizing candidates from the sizeable lists of potential therapeutic targets that are now emerging from large-scale multi-omics initiatives, such as those in oncology. Here, we describe an objective, systematic, multifaceted computational assessment of biological and chemical space that can be applied to any human gene set to prioritize targets for therapeutic exploration. We use this approach to evaluate an exemplar set of 479 cancer-associated genes, reveal the tension between biological relevance and chemical tractability, and describe major gaps in available knowledge that could be addressed to aid objective decision-making. We also propose drug repurposing opportunities and identify potentially druggable cancer-associated proteins that have been poorly explored with regard to the discovery of small-molecule modulators, despite their biological relevance..
Knapp, S.
Arruda, P.
Blagg, J.
Burley, S.
Drewry, D.H.
Edwards, A.
Fabbro, D.
Gillespie, P.
Gray, N.S.
Kuster, B.
Lackey, K.E.
Mazzafera, P.
Tomkinson, N.C.
Willson, T.M.
Workman, P.
Zuercher, W.J.
(2013). A public-private partnership to unlock the untargeted kinome. Nat chem biol,
Vol.9
(1),
pp. 3-6.
Al-Lazikani, B.
Workman, P.
(2013). Unpicking the combination lock for mutant BRAF and RAS melanomas. Cancer discov,
Vol.3
(1),
pp. 14-19.
show abstract
Large-scale, unbiased combinatorial drug screening has been used to identify effective genotype-selective therapeutic combinations that show promising activity in preclinical models of mutant BRAF andRAS melanoma that are resistant to the clinical BRAF inhibitor vemurafenib..
Falck Miniotis, M.
Arunan, V.
Eykyn, T.R.
Marais, R.
Workman, P.
Leach, M.O.
Beloueche-Babari, M.
(2013). MEK1/2 inhibition decreases lactate in BRAF-driven human cancer cells. Cancer res,
Vol.73
(13),
pp. 4039-4049.
show abstract
The RAS/BRAF/MEK/ERK signaling pathway is a central driver in cancer with many BRAF and MEK inhibitors being evaluated in clinical trials. Identifying noninvasive biomarkers of early pharmacodynamic responses is important for development of these targeted drugs. As increased aerobic glycolysis is often observed in cancer, we hypothesized that MEK1/2 (MAP2K1/MAP2K2) inhibitors may reduce lactate levels as detected by magnetic resonance spectroscopy (MRS), as a metabolic biomarker for the pharmacodynamic response. MRS was used to monitor intracellular and extracellular levels of lactate in human cancer cells in vitro and in melanoma tumors ex vivo. In addition, we used (1)H MRS and a fluorescent glucose analog to evaluate the effect of MEK inhibition on glucose uptake. MEK1/2 signaling inhibition reduced extracellular lactate levels in BRAF-dependent cells but not BRAF-independent cells. The reduction in extracellular lactate in BRAF-driven melanoma cells was time-dependent and associated with reduced expression of hexokinase-II driven by c-Myc depletion. Taken together, these results reveal how MEK1/2 inhibition affects cancer cell metabolism in the context of BRAF oncogene addiction. Furthermore, they offer a preclinical proof-of-concept for the use of MRS to measure lactate as a noninvasive metabolic biomarker for pharmacodynamic response to MEK1/2 inhibition in BRAF-driven cancers..
Zhang, L.
Fok, J.J.
Mirabella, F.
Aronson, L.I.
Fryer, R.A.
Workman, P.
Morgan, G.J.
Davies, F.E.
(2013). Hsp70 inhibition induces myeloma cell death via the intracellular accumulation of immunoglobulin and the generation of proteotoxic stress. Cancer lett,
Vol.339
(1),
pp. 49-59.
show abstract
Multiple myeloma (MM) cells rely on protein homeostatic mechanisms for survival. These mechanisms could be therapeutically targeted via modulation of the heat shock response. We studied the roles of Hsp72 and Hsc70, and show that the two major cytoplasmic Hsp70s play a key role in regulating protein homeostasis and controlling multiple oncogenic pathways in MM, and their inhibition can lead to myeloma cell death. Our study provides further evidence that targeting Hsp70 represents a novel therapeutic approach which may be effective in the treatment of MM..
Gonzalez de Castro, D.
Clarke, P.A.
Al-Lazikani, B.
Workman, P.
(2013). Personalized cancer medicine: molecular diagnostics, predictive biomarkers, and drug resistance. Clin pharmacol ther,
Vol.93
(3),
pp. 252-259.
show abstract
The progressive elucidation of the molecular pathogenesis of cancer has fueled the rational development of targeted drugs for patient populations stratified by genetic characteristics. Here we discuss general challenges relating to molecular diagnostics and describe predictive biomarkers for personalized cancer medicine. We also highlight resistance mechanisms for epidermal growth factor receptor (EGFR) kinase inhibitors in lung cancer. We envisage a future requiring the use of longitudinal genome sequencing and other omics technologies alongside combinatorial treatment to overcome cellular and molecular heterogeneity and prevent resistance caused by clonal evolution..
Polier, S.
Samant, R.S.
Clarke, P.A.
Workman, P.
Prodromou, C.
Pearl, L.H.
(2013). ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system. Nat chem biol,
Vol.9
(5),
pp. 307-312.
show abstract
full text
Protein kinase clients are recruited to the Hsp90 molecular chaperone system via Cdc37, which simultaneously binds Hsp90 and kinases and regulates the Hsp90 chaperone cycle. Pharmacological inhibition of Hsp90 in vivo results in degradation of kinase clients, with a therapeutic effect in dependent tumors. We show here that Cdc37 directly antagonizes ATP binding to client kinases, suggesting a role for the Hsp90-Cdc37 complex in controlling kinase activity. Unexpectedly, we find that Cdc37 binding to protein kinases is itself antagonized by ATP-competitive kinase inhibitors, including vemurafenib and lapatinib. In cancer cells, these inhibitors deprive oncogenic kinases such as B-Raf and ErbB2 of access to the Hsp90-Cdc37 complex, leading to their degradation. Our results suggest that at least part of the efficacy of ATP-competitive inhibitors of Hsp90-dependent kinases in tumor cells may be due to targeted chaperone deprivation..
Bjerke, L.
Mackay, A.
Nandhabalan, M.
Burford, A.
Jury, A.
Popov, S.
Bax, D.A.
Carvalho, D.
Taylor, K.R.
Vinci, M.
Bajrami, I.
McGonnell, I.M.
Lord, C.J.
Reis, R.M.
Hargrave, D.
Ashworth, A.
Workman, P.
Jones, C.
(2013). Histone H3 3 mutations drive pediatric glioblastoma through upregulation of MYCN. Cancer discov,
Vol.3
(5),
pp. 512-519.
show abstract
UNLABELLED: Children and young adults with glioblastoma (GBM) have a median survival rate of only 12 to 15 months, and these GBMs are clinically and biologically distinct from histologically similar cancers in older adults. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A , occurring either at or close to key residues marked by methylation for regulation of transcription—K27 and G34. Here, we show that the cerebral hemisphere-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem-cell maintenance, cell-fate decisions, and self-renewal.Critically, H3F3A G34 mutations cause profound upregulation of MYCN , a potent oncogene that is causative of GBMs when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population through inhibiting kinases responsible for stabilization of the protein. SIGNIFICANCE: We provide the mechanistic explanation for how the fi rst histone gene mutation inhuman disease biology acts to deliver MYCN, a potent tumorigenic initiator, into a stem-cell compartment of the developing forebrain, selectively giving rise to incurable cerebral hemispheric GBM. Using synthetic lethal approaches to these mutant tumor cells provides a rational way to develop novel and highly selective treatment strategies.
Bjerke, L.
Mackay, A.
Nandhabalan, M.
Burford, A.
Jury, A.
Popov, S.
Bax, D.A.
Carvalho, D.
Taylor, K.R.
Vinci, M.
Bajrami, I.
McGonnell, I.M.
Lord, C.J.
Reis, R.M.
Hargrave, D.
Ashworth, A.
Workman, P.
Jones, C.
(2013). Histone H3 3 Mutations Drive Pediatric Glioblastoma through Upregulation of MYCN. Cancer discovery,
Vol.3
(5),
pp. 512-519.
Workman, P.
Al-Lazikani, B.
Clarke, P.A.
(2013). Genome-based cancer therapeutics: targets, kinase drug resistance and future strategies for precision oncology. Curr opin pharmacol,
Vol.13
(4),
pp. 486-496.
show abstract
Extraordinary progress has been made in our detailed understanding of the genetic and epigenetic mechanisms responsible for oncogenesis and cancer progression. Empowered by next-generation sequencing, many new targets and pathways have been identified to exploit oncogene and non-oncogene addiction and synthetic lethality. Kinase inhibitors feature strongly in the druggable cancer genome and 19 have been approved in oncology. While survival gains are valuable, drug resistance has emerged as the major challenge. The clonal heterogeneity and evolution of cancers is an intrinsic problem, together with feedback loops, kinase switching and activation of alternative targets and pathways. The solution to drug resistance will require the use of rationally targeted combinational regimens. The application of adaptive treatment cycles based on ongoing multi-technology profiling will be the key to long-term therapeutic success..
Saturno, G.
Valenti, M.
De Haven Brandon, A.
Thomas, G.V.
Eccles, S.
Clarke, P.A.
Workman, P.
(2013). Combining trail with PI3 kinase or HSP90 inhibitors enhances apoptosis in colorectal cancer cells via suppression of survival signaling. Oncotarget,
Vol.4
(8),
pp. 1185-1198.
show abstract
TRAIL has been shown to induce apoptosis in cancer cells, but in some cases they fail to respond to this ligand. We explored the ability of representative phosphatidylinositol-3-kinase (PI3 Kinase)/mTOR and HSP90 inhibitors to overcome TRAIL resistance by increasing apoptosis in colorectal cancer models. We determined the sensitivity of 27 human colorectal cancer and 2 non-transformed colon epithelial cell lines to TRAIL treatment. A subset of the cancer cell lines with a range of responses to TRAIL was selected from the panel for treatment with TRAIL combined with the PI3 Kinase/mTOR inhibitor PI-103 or the HSP90 inhibitor 17-AAG (tanespimycin). Two TRAIL-resistant cell lines were selected for in vivo combination studies with TRAIL and 17-AAG. We found that 13 colorectal cancer cell lines and the 2 non-transformed colon epithelial cell lines were resistant to TRAIL. We demonstrated that co-treatment of TRAIL and PI-103 or 17-AAG was synergistic or additive and significantly enhanced apoptosis in colorectal cancer cells. This was associated with decreased expression or activity of survival protein biomarkers such as ERBB2, AKT, IKKα and XIAP. In contrast, the effect of the combination treatments in non-transformed colon cells was minimal. We show here for the first time that co-treatment in vivo with TRAIL and 17-AAG in two TRAIL-resistant human colorectal cancer xenograft models resulted in significantly greater tumor growth inhibition compared to single treatments. We propose that combining TRAIL with PI3 Kinase/mTOR or HSP90 inhibitors has therapeutic potential in the treatment of TRAIL-resistant colorectal cancers..
Powers, M.V.
Valenti, M.
Miranda, S.
Maloney, A.
Eccles, S.A.
Thomas, G.
Clarke, P.A.
Workman, P.
(2013). Mode of cell death induced by the HSP90 inhibitor 17-AAG (tanespimycin) is dependent on the expression of pro-apoptotic BAX. Oncotarget,
Vol.4
(11),
pp. 1963-1975.
show abstract
Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) are of considerable current interest as targeted cancer therapeutic agents because of the ability to destabilize multiple oncogenic client proteins. Despite their resulting pleiotropic effects on multiple oncogenic pathways and hallmark traits of cancer, resistance to HSP90 inhibitors is possible and their ability to induce apoptosis is less than might be expected. Using an isogenic model for BAX knockout in HCT116 human colon carcinoma cells, we demonstrate the induction of BAX-dependent apoptosis at pharmacologically relevant concentrations of the HSP90 inhibitor 17-AAG both in vitro and in tumor xenografts in vivo. Removal of BAX expression by homologous recombination reduces apoptosis in vitro and in vivo but allows a lower level of cell death via a predominantly necrotic mechanism. Despite reducing apoptosis, the loss of BAX does not alter the overall sensitivity to 17-AAG in vitro or in vivo. The results indicate that 17-AAG acts predominantly to cause a cytostatic antiproliferative effect rather than cell death and further suggest that BAX status may not alter the overall clinical response to HSP90 inhibitors. Other agents may be required in combination to enhance tumor-selective killing by these promising drugs. In addition, there are implications for the use of apoptotic endpoints in the assessment of the activity of molecularly targeted agents..
de Billy, E.
Clarke, P.A.
Workman, P.
(2013). HSF1 in Translation. Cancer cell,
Vol.24
(2),
pp. 147-149.
show abstract
The master regulator of the classical cytoprotective "heat shock" response, heat shock factor 1 (HSF1), is increasingly implicated in cancer pathogenesis, but the mechanisms remain poorly understood. A recent study connects increased protein translation to activation of HSF1 in malignant cells and demonstrates the therapeutic benefit of targeting this link..
Workman, P.
Al-Lazikani, B.
(2013). Drugging cancer genomes. Nat rev drug discov,
Vol.12
(12),
pp. 889-890.
Beloueche-Babari, M.
Jamin, Y.
Arunan, V.
Walker-Samuel, S.
Revill, M.
Smith, P.D.
Halliday, J.
Waterton, J.C.
Barjat, H.
Workman, P.
Leach, M.O.
Robinson, S.P.
(2013). Acute tumour response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) evaluated by non-invasive diffusion-weighted MRI. Br j cancer,
Vol.109
(6),
pp. 1562-1569.
show abstract
BACKGROUND: Non-invasive imaging biomarkers underpin the development of molecularly targeted anti-cancer drugs. This study evaluates tumour apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (DW-MRI), as a biomarker of response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in human tumour xenografts. METHODS: Nude mice bearing human BRAF(V600D) WM266.4 melanoma or BRAF(V600E) Colo205 colon carcinoma xenografts were treated for 4 days with vehicle or selumetinib. DW-MRI was performed before and 2 h after the last dose and excised tumours analysed for levels of phospho-ERK1/2, cleaved caspase 3 (CC3) and necrosis. RESULTS: Selumetinib treatment induced tumour stasis and reduced ERK1/2 phosphorylation in both WM266.4 and Colo205 tumour xenografts. Relative to day 0, mean tumour ADC was unchanged in the control groups but was significantly increased by up to 1.6-fold in selumetinib-treated WM266.4 and Colo205 tumours. Histological analysis revealed a significant increase in necrosis in selumetinib-treated WM266.4 and Colo205 xenografts and CC3 staining in selumetinib-treated Colo205 tumours relative to controls. CONCLUSION: Changes in ADC following treatment with the MEK1/2 inhibitor selumetinib in responsive human tumour xenografts were concomitant with induction of tumour cell death. ADC may provide a useful non-invasive pharmacodynamic biomarker for early clinical assessment of response to selumetinib and other MEK-ERK1/2 signalling-targeted therapies..
Couty, S.
Westwood, I.M.
Kalusa, A.
Cano, C.
Travers, J.
Boxall, K.
Chow, C.L.
Burns, S.
Schmitt, J.
Pickard, L.
Barillari, C.
McAndrew, P.C.
Clarke, P.A.
Linardopoulos, S.
Griffin, R.J.
Aherne, G.W.
Raynaud, F.I.
Workman, P.
Jones, K.
Van Montfort, R.L.
(2013). The discovery of potent ribosomal S6 kinase inhibitors by high-throughput screening and structure-guided drug design. Oncotarget,
Vol.4,
pp. 1-15.
show abstract
full text
The ribosomal P70 S6 kinases play a crucial role in PI3K/mTOR regulated signalling pathways and are therefore potential targets for the treatment of a variety of diseases including diabetes and cancer. In this study we describe the identification of three series of chemically distinct S6K1 inhibitors. In addition, we report a novel PKA-S6K1 chimeric protein with five mutations in or near its ATP-binding site, which was used to determine the binding mode of two of the three inhibitor series, and provided a robust system to aid the optimisation of the oxadiazole-substituted benzimidazole inhibitor series. We show that the resulting oxadiazole-substituted aza-benzimidazole is a potent and ligand efficient S6 kinase inhibitor, which blocks the phosphorylation of RPS6 at Ser235/236 in TSC negative HCV29 human bladder cancer cells by inhibiting S6 kinase activity and thus provides a useful tool compound to investigate the function of S6 kinases..
Bavetsias, V.
Faisal, A.
Crumpler, S.
Brown, N.
Kosmopoulou, M.
Joshi, A.
Atrash, B.
Pérez-Fuertes, Y.
Schmitt, J.A.
Boxall, K.J.
Burke, R.
Sun, C.
Avery, S.
Bush, K.
Henley, A.
Raynaud, F.I.
Workman, P.
Bayliss, R.
Linardopoulos, S.
Blagg, J.
(2013). Aurora isoform selectivity: design and synthesis of imidazo[4,5-b]pyridine derivatives as highly selective inhibitors of Aurora-A kinase in cells. J med chem,
Vol.56
(22),
pp. 9122-9135.
show abstract
full text
Aurora-A differs from Aurora-B/C at three positions in the ATP-binding pocket (L215, T217, and R220). Exploiting these differences, crystal structures of ligand-Aurora protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibitors. Guided by a computational modeling approach, appropriate C7-imidazo[4,5-b]pyridine derivatization led to the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B. In HCT116 human colon carcinoma cells, 28c and 40f inhibited the Aurora-A L215R and R220K mutants with IC50 values similar to those seen for the Aurora-A wild type. However, the Aurora-A T217E mutant was significantly less sensitive to inhibition by 28c and 40f compared to the Aurora-A wild type, suggesting that the T217 residue plays a critical role in governing the observed isoform selectivity for Aurora-A inhibition. These compounds are useful small-molecule chemical tools to further explore the function of Aurora-A in cells..
Samant, R.S.
Clarke, P.A.
Workman, P.
(2012). The expanding proteome of the molecular chaperone HSP90. Cell cycle,
Vol.11
(7),
pp. 1301-1308.
show abstract
The molecular chaperone HSP90 maintains the activity and stability of a diverse set of "client" proteins that play key roles in normal and disease biology. Around 20 HSP90 inhibitors that deplete the oncogenic clientele have entered clinical trials for cancer. However, the full extent of the HSP90-dependent proteome, which encompasses not only clients but also proteins modulated by downstream transcriptional responses, is still incompletely characterized and poorly understood. Earlier large-scale efforts to define the HSP90 proteome have been valuable but are incomplete because of limited technical sensitivity. Here we discuss previous large-scale surveys of proteome perturbations induced by HSP90 inhibitors in light of a significant new study using state-of-the-art SILAC technology combined with more sensitive high-resolution mass spectrometry (MS) that extends the catalog of proteomic changes in inhibitor-treated cancer cells. Among wide-ranging changes, major functional responses include downregulation of protein kinase activity and the DNA damage response alongside upregulation of the protein degradation machinery. Despite this improved proteomic coverage, there was surprisingly little overlap with previous studies. This may be due in part to technical issues but is likely also due to the variability of the HSP90 proteome with the inhibitor conditions used, the cancer cell type and the genetic status of client proteins. We suggest future proteomic studies to address these factors, to help distinguish client protein components from indirect transcriptional components and to address other key questions in fundamental and translational HSP90 research. Such studies should also reveal new biomarkers for patient selection and novel targets for therapeutic intervention..
Neckers, L.
Workman, P.
(2012). Hsp90 molecular chaperone inhibitors: are we there yet?. Clin cancer res,
Vol.18
(1),
pp. 64-76.
show abstract
Heat shock protein (Hsp) 90 is an ATP-dependent molecular chaperone that is exploited by malignant cells to support activated oncoproteins, including many cancer-associated kinases and transcription factors, and it is essential for oncogenic transformation. Originally viewed with skepticism, Hsp90 inhibitors are now being actively pursued by the pharmaceutical industry, with 17 agents having entered clinical trials. Investigators established Hsp90's druggability using the natural products geldanamycin and radicicol, which mimic the unusual ATP structure adopted in the chaperone's N-terminal nucleotide-binding pocket and cause potent and selective blockade of ATP binding/hydrolysis, inhibit chaperone function, deplete oncogenic clients, and show antitumor activity. Preclinical data obtained with these natural products have heightened interest in Hsp90 as a drug target, and 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) has shown clinical activity (as defined by Response Evaluation Criteria in Solid Tumors) in HER2+ breast cancer. Many optimized synthetic, small-molecule Hsp90 inhibitors from diverse chemotypes are now in clinical trials. Here, we review the discovery and development of Hsp90 inhibitors and assess their potential. There has been significant learning from studies of the basic biology of Hsp90, as well as translational drug development involving this chaperone, enhanced by the use of Hsp90 inhibitors as chemical probes. Success will likely lie in treating cancers that are addicted to particular driver oncogene products (e.g., HER2, ALK, EGFR, and BRAF) that are sensitive Hsp90 clients, as well as malignancies (especially multiple myeloma) in which buffering of proteotoxic stress is critical for survival. We discuss approaches for enhancing the effectiveness of Hsp90 inhibitors and highlight new chaperone and stress-response pathway targets, including HSF1 and Hsp70..
Travers, J.
Sharp, S.
Workman, P.
(2012). HSP90 inhibition: two-pronged exploitation of cancer dependencies. Drug discov today,
Vol.17
(5-6),
pp. 242-252.
show abstract
The early clinical hypothesis for inhibiting HSP90 in cancers was based on the dependence of certain key client proteins in malignant cells--including a host of well-characterized oncoproteins--on the activity of HSP90 for their function and stability. The additional concept has been established that cancer cells have heightened dependence on the efficient maintenance of intracellular proteomic homeostasis, central components of which are HSP90 and other heat shock proteins. We evaluate the evidence that inhibiting HSP90 in cancer exploits both of these biological vulnerabilities very effectively, we review the current status of the discovery and development of HSP90 inhibitors and we identify routes to improve their clinical efficacy, based on emerging knowledge..
Hoelder, S.
Clarke, P.A.
Workman, P.
(2012). Discovery of small molecule cancer drugs: successes, challenges and opportunities. Mol oncol,
Vol.6
(2),
pp. 155-176.
show abstract
The discovery and development of small molecule cancer drugs has been revolutionised over the last decade. Most notably, we have moved from a one-size-fits-all approach that emphasized cytotoxic chemotherapy to a personalised medicine strategy that focuses on the discovery and development of molecularly targeted drugs that exploit the particular genetic addictions, dependencies and vulnerabilities of cancer cells. These exploitable characteristics are increasingly being revealed by our expanding understanding of the abnormal biology and genetics of cancer cells, accelerated by cancer genome sequencing and other high-throughput genome-wide campaigns, including functional screens using RNA interference. In this review we provide an overview of contemporary approaches to the discovery of small molecule cancer drugs, highlighting successes, current challenges and future opportunities. We focus in particular on four key steps: Target validation and selection; chemical hit and lead generation; lead optimization to identify a clinical drug candidate; and finally hypothesis-driven, biomarker-led clinical trials. Although all of these steps are critical, we view target validation and selection and the conduct of biology-directed clinical trials as especially important areas upon which to focus to speed progress from gene to drug and to reduce the unacceptably high attrition rate during clinical development. Other challenges include expanding the envelope of druggability for less tractable targets, understanding and overcoming drug resistance, and designing intelligent and effective drug combinations. We discuss not only scientific and technical challenges, but also the assessment and mitigation of risks as well as organizational, cultural and funding problems for cancer drug discovery and development, together with solutions to overcome the 'Valley of Death' between basic research and approved medicines. We envisage a future in which addressing these challenges will enhance our rapid progress towards truly personalised medicine for cancer patients..
Pintzas, A.
Zhivotovsky, B.
Workman, P.
Clarke, P.A.
Linardopoulos, S.
Martinou, J.-.
Lacal, J.C.
Robine, S.
Nasioulas, G.
Andera, L.
(2012). Sensitization of (colon) cancer cells to death receptor related therapies: a report from the FP6-ONCODEATH research consortium. Cancer biol ther,
Vol.13
(7),
pp. 458-466.
show abstract
The objective of the ONCODEATH consortium [EU Research Consortium "ONCODEATH" (2006-2010)] was to achieve sensitization of solid tumor cells to death receptor related therapies using rational mechanism-based drug combinations of targeted therapies. In this collaborative effort, during a period of 42 mo, cell and animal model systems of defined oncogenes were generated. Exploitation of generated knowledge and tools enabled the consortium to achieve the following research objectives: (1) elucidation of tumor components which confer sensitivity or resistance to TRAIL-induced cell death; (2) providing detailed knowledge on how small molecule Hsp90, Aurora, Choline kinase, BRAF inhibitors, DNA damaging agents, HDAC and DNMT inhibitors affect the intrinsic apoptotic amplification and execution machineries; (3) optimization of combined action of TRAIL with these therapeutics for optimum effects with minimum concentrations and toxicity in vivo. These findings provide mechanistic basis for a pharmacogenomic approach, which could be exploited further therapeutically, in order to reach novel personalized therapies for cancer patients..
Moore, A.S.
Faisal, A.
Gonzalez de Castro, D.
Bavetsias, V.
Sun, C.
Atrash, B.
Valenti, M.
de Haven Brandon, A.
Avery, S.
Mair, D.
Mirabella, F.
Swansbury, J.
Pearson, A.D.
Workman, P.
Blagg, J.
Raynaud, F.I.
Eccles, S.A.
Linardopoulos, S.
(2012). Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns. Leukemia,
Vol.26
(7),
pp. 1462-1470.
show abstract
full text
Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML..
Carden, C.P.
Stewart, A.
Thavasu, P.
Kipps, E.
Pope, L.
Crespo, M.
Miranda, S.
Attard, G.
Garrett, M.D.
Clarke, P.A.
Workman, P.
de Bono, J.S.
Gore, M.
Kaye, S.B.
Banerji, U.
(2012). The association of PI3 kinase signaling and chemoresistance in advanced ovarian cancer. Mol cancer ther,
Vol.11
(7),
pp. 1609-1617.
show abstract
Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN..
de Billy, E.
Travers, J.
Workman, P.
(2012). Shock about heat shock in cancer. Oncotarget,
Vol.3
(8),
pp. 741-743.
Francis, N.-.
Rowlands, M.
Workman, P.
Jones, K.
Aherne, W.
(2012). Small-molecule inhibitors of the protein methyltransferase SET7/9 identified in a high-throughput screen. J biomol screen,
Vol.17
(8),
pp. 1102-1109.
show abstract
Aberrant expression of chromatin-modifying enzymes (CMEs) is associated with a range of human diseases, including cancer. CMEs are now an important target area in drug discovery. Although the role that histone and protein (lysine) methyltransferases (PMTs) play in the regulation of transcription and cell growth is increasingly recognized, few small-molecule inhibitors of this class of enzyme have been reported. Here we describe an assay suitable for primary compound screening for the identification of PMT inhibitors. The assay followed the methylation of histones in the presence of the PMT SET7/9 and the radioactive cofactor S-adenosyl-methionine using scintillating microplates (FlashPlate) and was used to screen approximately 65 000 compounds (% coefficient of variation = 10%; Z' = 0.6). The hits identified from a library of more than 63 000 diverse small molecules included a series of rhodanine compounds with micromolar activity. A screen of the National Cancer Institute Diversity Set (2000 compounds) identified an orsein derivative that inhibited SET7/9 (~20 µM) and showed modest growth inhibition associated with the expected cellular phenotype of reduced histone methylation in a human tumor cell line. The assay represents a useful tool for the identification of inhibitors of PMT activity..
Al-Lazikani, B.
Banerji, U.
Workman, P.
(2012). Combinatorial drug therapy for cancer in the post-genomic era. Nat biotechnol,
Vol.30
(7),
pp. 679-692.
show abstract
Over the past decade, whole genome sequencing and other 'omics' technologies have defined pathogenic driver mutations to which tumor cells are addicted. Such addictions, synthetic lethalities and other tumor vulnerabilities have yielded novel targets for a new generation of cancer drugs to treat discrete, genetically defined patient subgroups. This personalized cancer medicine strategy could eventually replace the conventional one-size-fits-all cytotoxic chemotherapy approach. However, the extraordinary intratumor genetic heterogeneity in cancers revealed by deep sequencing explains why de novo and acquired resistance arise with molecularly targeted drugs and cytotoxic chemotherapy, limiting their utility. One solution to the enduring challenge of polygenic cancer drug resistance is rational combinatorial targeted therapy..
Martins, A.S.
Davies, F.E.
Workman, P.
(2012). Inhibiting the molecular evolution of cancer through HSP90. Oncotarget,
Vol.3
(10),
pp. 1054-1056.
Stockwell, S.R.
Platt, G.
Barrie, S.E.
Zoumpoulidou, G.
Poele, R.H.
Aherne, G.W.
Wilson, S.C.
Sheldrake, P.
McDonald, E.
Venet, M.
Soudy, C.
Elustondo, F.
Rigoreau, L.
Blagg, J.
Workman, P.
Garrett, M.D.
Mittnacht, S.
(2012). Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling. Plos one,
Vol.7
(1).
Beloueche-Babari, M.
Arunan, V.
Troy, H.
te Poele, R.H.
te Fong, A.-.
Jackson, L.E.
Payne, G.S.
Griffiths, J.R.
Judson, I.R.
Workman, P.
Leach, M.O.
Chung, Y.-.
(2012). Histone deacetylase inhibition increases levels of choline kinase α and phosphocholine facilitating noninvasive imaging in human cancers. Cancer res,
Vol.72
(4),
pp. 990-1000.
show abstract
Histone deacetylase (HDAC) inhibitors are currently approved for cutaneous T-cell lymphoma and are in mid-late stage trials for other cancers. The HDAC inhibitors LAQ824 and SAHA increase phosphocholine (PC) levels in human colon cancer cells and tumor xenografts as observed by magnetic resonance spectroscopy (MRS). In this study, we show that belinostat, an HDAC inhibitor with an alternative chemical scaffold, also caused a rise in cellular PC content that was detectable by (1)H and (31)P MRS in prostate and colon carcinoma cells. In addition, (1)H MRS showed an increase in branched chain amino acid and alanine concentrations. (13)C-choline labeling indicated that the rise in PC resulted from increased de novo synthesis and correlated with an induction of choline kinase α expression. Furthermore, metabolic labeling experiments with (13)C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratios in vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate noninvasive imaging biomarker for monitoring the action of HDAC inhibitors..
Yap, T.A.
Walton, M.I.
Grimshaw, K.M.
Te Poele, R.H.
Eve, P.D.
Valenti, M.R.
de Haven Brandon, A.K.
Martins, V.
Zetterlund, A.
Heaton, S.P.
Heinzmann, K.
Jones, P.S.
Feltell, R.E.
Reule, M.
Woodhead, S.J.
Davies, T.G.
Lyons, J.F.
Raynaud, F.I.
Eccles, S.A.
Workman, P.
Thompson, N.T.
Garrett, M.D.
(2012). AT13148 is a novel, oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity. Clin cancer res,
Vol.18
(14),
pp. 3912-3923.
show abstract
PURPOSE: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. EXPERIMENTAL DESIGN: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. RESULTS: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. CONCLUSIONS: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial..
Workman, P.
Clarke, P.A.
Al-Lazikani, B.
(2012). Personalized medicine: patient-predictive panel power. Cancer cell,
Vol.21
(4),
pp. 455-458.
show abstract
Two recent papers published in Nature demonstrate the power of systematic high-throughput pharmacologic profiling of very large, diverse, molecularly-characterized human cancer cell line panels to reveal linkages between genetic profile and targeted-drug sensitivity. Known oncogene addictions are confirmed while surprising complexities and biomarker relationships with clinical potential are revealed..
Samant, R.S.
Workman, P.
(2012). MOLECULAR BIOLOGY Choose your protein partners. Nature,
Vol.490
(7420),
pp. 351-352.
Clarke, P.A.
Workman, P.
(2012). Phosphatidylinositide-3-kinase inhibitors: addressing questions of isoform selectivity and pharmacodynamic/predictive biomarkers in early clinical trials. J clin oncol,
Vol.30
(3),
pp. 331-333.
Rodrigues, L.M.
Chung, Y.-.
Al Saffar, N.M.
Sharp, S.Y.
Jackson, L.E.
Banerji, U.
Stubbs, M.
Leach, M.O.
Griffiths, J.R.
Workman, P.
(2012). Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy. Bmc res notes,
Vol.5,
p. 250.
show abstract
BACKGROUND: The importance of ERBB2/NEU/HER2 in the response of breast tumours to the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) has been demonstrated in the clinic. ERBB2 is an oncoprotein client that is highly dependent on HSP90. This and other oncogenic client proteins (e.g. B-RAF, C-RAF, ALK and CDK4) are depleted by 17-AAG in both animal tumours and patients. Here we investigate by Magnetic Resonance Spectroscopy (MRS) the metabolic response of 17-AAG in spontaneous, NEU/HER2 driven mammary tumours in transgenic MMTV-NEU-NT mice and in cells isolated and cultured from these tumours. METHODS: Mammary tumours were monitored by 31P MRS in vivo and in tumour extracts, comparing control and 17-AAG treated mice. A cell line derived from NEU/HER2 mammary tumours was also cultured and the effect of 17-AAG was measured by 31P MRS in cell extracts. Molecular biomarkers were assessed by immunoblotting in extracts from cells and tumours. For comparison of tumour volume, metabolite concentrations and Western blot band intensities, two-tailed unpaired t-tests were used. RESULTS: The NEU/HER2 mammary tumours were very sensitive to 17-AAG and responded in a dose-dependent manner to 3 daily doses of 20, 40 and 80mg/kg of 17-AAG, all of which caused significant regression. At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose. Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment. Western blots confirmed the expected action of 17-AAG in inducing HSP72 and significantly depleting HSP90 client proteins, including NEU/HER2 both in tumours and in isolated cells. CONCLUSIONS: The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment..
Bavetsias, V.
Crumpler, S.
Sun, C.
Avery, S.
Atrash, B.
Faisal, A.
Moore, A.S.
Kosmopoulou, M.
Brown, N.
Sheldrake, P.W.
Bush, K.
Henley, A.
Box, G.
Valenti, M.
de Haven Brandon, A.
Raynaud, F.I.
Workman, P.
Eccles, S.A.
Bayliss, R.
Linardopoulos, S.
Blagg, J.
(2012). Optimization of imidazo[4,5-b]pyridine-based kinase inhibitors: identification of a dual FLT3/Aurora kinase inhibitor as an orally bioavailable preclinical development candidate for the treatment of acute myeloid leukemia. J med chem,
Vol.55
(20),
pp. 8721-8734.
show abstract
Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K(d) = 7.5 nM, Aurora-B K(d) = 48 nM), FLT3 kinase (K(d) = 6.2 nM), and FLT3 mutants including FLT3-ITD (K(d) = 38 nM) and FLT3(D835Y) (K(d) = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children..
Yap, T.A.
Workman, P.
(2012). Exploiting the cancer genome: strategies for the discovery and clinical development of targeted molecular therapeutics. Annu rev pharmacol toxicol,
Vol.52,
pp. 549-573.
show abstract
Our biological understanding of the molecular basis of cancer has benefited from advances in basic research, accelerated recently by cancer genome sequencing and other high-throughput, genome-wide profiling technologies. Given the diverse heterogeneity among tumors, the traditional cytotoxic chemotherapy and one-size-fits-all approaches to cancer discovery and development are not appropriate for molecularly targeted agents. Selection of new drug targets is based on achieving cancer selectivity through exploiting specific dependencies and vulnerabilities predicted from tumor genetics. Discovery of highly target-selective agents is enhanced by integrating multiple modern technologies, particularly structure-based design. Efficient clinical evaluation requires smart, hypothesis-testing studies using validated pharmacodynamic and predictive biomarkers. We discuss and exemplify biomarker-driven clinical development and the concept of the Pharmacologic Audit Trail. We detail the exciting approaches offered by drugging the cancer genome, focusing on blocking oncogene addiction, drugging the oncogenic lipid kinome, addressing nononcogene addiction, exploiting synthetic lethality, and overcoming apoptotic resistance, leading to personalized molecular medicine..
Zaidi, S.
McLaughlin, M.
Bhide, S.A.
Eccles, S.A.
Workman, P.
Nutting, C.M.
Huddart, R.A.
Harrington, K.J.
(2012). The HSP90 inhibitor NVP-AUY922 radiosensitizes by abrogation of homologous recombination resulting in mitotic entry with unresolved DNA damage. Plos one,
Vol.7
(4),
p. e35436.
show abstract
BACKGROUND: Heat shock protein 90 (HSP90) is a molecular chaperone responsible for the conformational maintenance of a number of client proteins that play key roles in cell cycle arrest, DNA damage repair and apoptosis following radiation. HSP90 inhibitors exhibit antitumor activity by modulating the stabilisation and activation of HSP90 client proteins. We sought to evaluate NVP-AUY922, the most potent HSP90 inhibitor yet reported, in preclinical radiosensitization studies. PRINCIPAL FINDINGS: NVP-AUY922 potently radiosensitized cells in vitro at low nanomolar concentrations with a concurrent depletion of radioresistance-linked client proteins. Radiosensitization by NVP-AUY922 was verified for the first time in vivo in a human head and neck squamous cell carcinoma xenograft model in athymic mice, as measured by delayed tumor growth and increased surrogate end-point survival (p = <0.0001). NVP-AUY922 was shown to ubiquitously inhibit resolution of dsDNA damage repair correlating to delayed Rad51 foci formation in all cell lines tested. Additionally, NVP-AUY922 induced a stalled mitotic phenotype, in a cell line-dependent manner, in HeLa and HN5 cell lines irrespective of radiation exposure. Cell cycle analysis indicated that NVP-AUY922 induced aberrant mitotic entry in all cell lines tested in the presence of radiation-induced DNA damage due to ubiquitous CHK1 depletion, but resultant downstream cell cycle effects were cell line dependent. CONCLUSIONS: These results identify NVP-AUY922 as the most potent HSP90-mediated radiosensitizer yet reported in vitro, and for the first time validate it in a clinically relevant in vivo model. Mechanistic analysis at clinically achievable concentrations demonstrated that radiosensitization is mediated by the combinatorial inhibition of cell growth and survival pathways, ubiquitous delay in Rad51-mediated homologous recombination and CHK1-mediated G(2)/M arrest, but that the contribution of cell cycle perturbation to radiosensitization may be cell line specific..
Workman, P.
Clarke, P.
(2012). PI3 Kinase in Cancer: From Biology to Clinic. Am soc clin oncol educ book,
,
pp. e93-e98.
show abstract
The discovery and clinical development of small-molecule inhibitors of the phosphatidylinositide 3-kinase (PI3 kinase) family of lipid kinases have marked a remarkable 20-year journey that follows the progressive developments in cancer biology over the last few decades: from hypothesis-driven, basic cancer research that began with viral oncogenesis and developed in the 1960s and 70s, through the discovery of individual mutated oncogenes and tumor suppressor genes in 1970 and 80s and the linkage of these cancer genes to signal transduction pathways in the 1990s, to all large-scale genome-wide sequencing, functional screening, and network biology efforts today. Thus, PI3 kinase research began with the discovery in 1985 of a new type of enzyme activity associated with viral oncogenesis. It benefited greatly from the discovery of wortmannin and LY294002 as PI3 kinase inhibitors and chemical tools in late 1980s to mid-90s. Alongside these tools, genetic validation of PI3 kinase as a target initially involved activation by upstream oncogenic receptor tyrosine kinases and RAS mutation, together with overexpression and amplification of the p110α catalytic isoform of PI3 kinase and frequent loss of the tumor suppressor and negative regulator of PI3 kinase activity, PTEN. As PI3 kinase drug development began, further stimulus came from the discovery through genome sequencing of mutations in PIK3CA, which encodes p110α and is the most frequently mutated kinase in the human genome. From these beginnings, there are now many PI3 kinase inhibitors in clinical trials and more in preclinical development. We review progress, current challenges, and future opportunities in this article..
Sharp, S.Y.
Roe, S.M.
Kazlauskas, E.
CikotienÄ—, I.
Workman, P.
Matulis, D.
Prodromou, C.
(2012). Co-crystalization and in vitro biological characterization of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazole Hsp90 inhibitors. Plos one,
Vol.7
(9),
p. e44642.
show abstract
A potential therapeutic strategy for targeting cancer that has gained much interest is the inhibition of the ATP binding and ATPase activity of the molecular chaperone Hsp90. We have determined the structure of the human Hsp90α N-terminal domain in complex with a series of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazoles. The structures provide the molecular details for the activity of these inhibitors. One of these inhibitors, ICPD 34, causes a structural change that affects a mobile loop, which adopts a conformation similar to that seen in complexes with ADP, rather than the conformation generally seen with the pyrazole/isoxazole-resorcinol class of inhibitors. Competitive binding to the Hsp90 N-terminal domain was observed in a biochemical assay, and these compounds showed antiproliferative activity and induced apoptosis in the HCT116 human colon cancer cell line. These inhibitors also caused induction of the heat shock response with the upregulation of Hsp72 and Hsp27 protein expression and the depletion of Hsp90 clients, CRAF, ERBB2 and CDK4, thus confirming that antiproliferative activity was through the inhibition of Hsp90. The presence of increased levels of the cleavage product of PARP indicated apoptosis in response to Hsp90 inhibitors. This work provides a framework for the further optimization of thiadiazole inhibitors of Hsp90. Importantly, we demonstrate that the thiadiazole inhibitors display a more limited core set of interactions relative to the clinical trial candidate NVP-AUY922, and consequently may be less susceptible to resistance derived through mutations in Hsp90..
Rao, S.
Welsh, L.
Cunningham, D.
Te-Poele, R.H.
Benson, M.
Norman, A.
Saffery, C.
Giddings, I.
Workman, P.
Clarke, P.A.
(2011). Correlation of Overall Survival With Gene Expression Profiles in a Prospective Study of Resectable Esophageal Cancer. Clinical colorectal cancer,
Vol.10
(1),
pp. 48-9.
Suwaki, N.
Vanhecke, E.
Atkins, K.M.
Graf, M.
Swabey, K.
Huang, P.
Schraml, P.
Moch, H.
Cassidy, A.M.
Brewer, D.
Al-Lazikani, B.
Workman, P.
De-Bono, J.
Kaye, S.B.
Larkin, J.
Gore, M.E.
Sawyers, C.L.
Nelson, P.
Beer, T.M.
Geng, H.
Gao, L.
Qian, D.Z.
Alumkal, J.J.
Thomas, G.
Thomas, G.V.
(2011). A HIF-regulated VHL-PTP1B-Src signaling axis identifies a therapeutic target in renal cell carcinoma. Sci transl med,
Vol.3
(85),
p. 85ra47.
show abstract
full text
Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials..
Beloueche-Babari, M.
Workman, P.
Leach, M.O.
(2011). Exploiting tumor metabolism for non-invasive imaging of the therapeutic activity of molecularly targeted anticancer agents. Cell cycle,
Vol.10
(17),
pp. 2883-2893.
show abstract
Rational drug discovery and development requires biomarkers to inform on target modulation and treatment efficacy. Many aspects of metabolism are altered in cancer, compared to normal tissues, and are often regulated by oncogene activation. Non-invasive imaging of spatio-temporal effects of molecularly targeted anticancer agents on tumor metabolism has considerable potential in the development and use of personalized molecular medicine approaches to cancer treatment. Here we describe how non-invasive monitoring of metabolism, using primarily magnetic resonance spectroscopy (MRS), can be used to follow treatment with novel molecularly targeted anticancer agents. We discuss how the regulation of metabolic pathways by oncogenic signaling can affect MRS-detectable metabolic signals together with the physiological factors that can influence the measured changes. Finally, the translation of these metabolic measurements from pre-clinical models to patients will be discussed..
Travers, J.
Blagg, J.
Workman, P.
(2011). EPIGENETICS Targeting leukemia on the DOT. Nature chemical biology,
Vol.7
(10),
pp. 663-4.
Yap, T.A.
Walton, M.I.
Hunter, L.-.
Valenti, M.
de Haven Brandon, A.
Eve, P.D.
Ruddle, R.
Heaton, S.P.
Henley, A.
Pickard, L.
Vijayaraghavan, G.
Caldwell, J.J.
Thompson, N.T.
Aherne, W.
Raynaud, F.I.
Eccles, S.A.
Workman, P.
Collins, I.
Garrett, M.D.
(2011). Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930. Mol cancer ther,
Vol.10
(2),
pp. 360-371.
show abstract
AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials..
Raynaud, F.I.
Workman, P.
(2011). Discovering and developing PI3 kinase inhibitors for cancer: rapid progress through academic-biotech-pharma interactions. Mol cancer ther,
Vol.10
(11),
pp. 2017-2018.
Workman, P.
Clarke, P.A.
(2011). Resisting Targeted Therapy: Fifty Ways to Leave Your EGFR. Cancer cell,
Vol.19
(4),
pp. 437-440.
show abstract
Despite the promise of the new generation of molecularly targeted drugs, intrinsic and acquired resistance is proving to be as problematic as with cytotoxic drugs. Two recent papers have identified novel ways by which non-small cell lung cancers can exhibit resistance to EGFR inhibitors and suggest new therapeutic workarounds..
Day, J.E.
Sharp, S.Y.
Rowlands, M.G.
Aherne, W.
Hayes, A.
Raynaud, F.I.
Lewis, W.
Roe, S.M.
Prodromou, C.
Pearl, L.H.
Workman, P.
Moody, C.J.
(2011). Targeting the Hsp90 Molecular Chaperone with Novel Macrolactams Synthesis, Structural, Binding, and Cellular Studies. Acs chemical biology,
Vol.6
(12),
pp. 1339-1347.
Large, J.M.
Torr, J.E.
Raynaud, F.I.
Clarke, P.A.
Hayes, A.
Stefano, F.
Urban, F.
Shuttleworth, S.J.
Saghir, N.
Sheldrake, P.
Workman, P.
McDonald, E.
(2011). Preparation and evaluation of trisubstituted pyrimidines as phosphatidylinositol 3-kinase inhibitors 3-Hydroxyphenol analogues and bioisosteric replacements. Bioorg med chem,
Vol.19
(2),
pp. 836-851.
show abstract
Two classes of trisubstituted pyrimidines related to PI-103 1 have been prepared and their inhibitory activities against phosphatidylinositol 3-kinase (PI3K) p110α were determined. From those with direct 6-aryl substitution compound 11a was the most potent inhibitor with an IC₅₀ value of 62 nM, and showed similar activity against other class 1a PI3K isoforms tested, p110β and p110γ. When a linking chain was introduced, as in the second exemplified class, compound 15f inhibited p110α with IC₅₀ 142 nM, and showed greater selectivity towards p110α. Compounds of both classes showed promising inhibition of cellular proliferation in IGROV-1 ovarian cancer cells. Among compounds designed to replace the 3-phenolic motif with structural isosteres, analogues incorporating a 4-indazolyl group possessed enzyme and cellular activities comparable to the parent phenols..
Pacey, S.
Wilson, R.H.
Walton, M.
Eatock, M.M.
Hardcastle, A.
Zetterlund, A.
Arkenau, H.T.
Moreno-Farre, J.
Banerji, U.
Roels, B.
Peachey, H.
Aherne, W.
de Bono, J.S.
Raynaud, F.
Workman, P.
Judson, I.
(2011). A phase I study of the heat shock protein 90 inhibitor alvespimycin (17-DMAG) given intravenously to patients with advanced solid tumors. Clin cancer res,
Vol.17
(6),
pp. 1561-1570.
show abstract
A phase I study to define toxicity and recommend a phase II dose of the HSP90 inhibitor alvespimycin (17-DMAG; 17-dimethylaminoethylamino-17-demethoxygeldanamycin). Secondary endpoints included evaluation of pharmacokinetic profile, tumor response, and definition of a biologically effective dose (BED)..
Wilson, S.C.
Atrash, B.
Barlow, C.
Eccles, S.
Fischer, P.M.
Hayes, A.
Kelland, L.
Jackson, W.
Jarman, M.
Mirza, A.
Moreno, J.
Nutley, B.P.
Raynaud, F.I.
Sheldrake, P.
Walton, M.
Westwood, R.
Whittaker, S.
Workman, P.
McDonald, E.
(2011). Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors. Bioorg med chem,
Vol.19
(22),
pp. 6949-6965.
show abstract
The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSβR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 μM, and CDK9-cyclinT with IC(50)=0.11 μM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 μM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts..
Shuttleworth, S.J.
Silva, F.A.
Cecil, A.R.
Tomassi, C.D.
Hill, T.J.
Raynaud, F.I.
Clarke, P.A.
Workman, P.
(2011). Progress in the Preclinical Discovery and Clinical Development of Class I and Dual Class I/IV Phosphoinositide 3-Kinase (PI3K) Inhibitors. Curr med chem,
.
show abstract
The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer..
Darby, J.F.
Workman, P.
(2011). CHEMICAL BIOLOGY Many faces of a cancer-supporting protein. Nature,
Vol.478
(7369),
pp. 334-2.
Workman, P.
Davies, F.E.
(2011). A stressful life (or death): Combinatorial proteotoxic approaches to cancer-selective therapeutic vulnerability. Oncotarget,
.
Faisal, A.
Vaughan, L.
Bavetsias, V.
Sun, C.
Atrash, B.
Avery, S.
Workman, P.
Blagg, J.
Raynaud, R.I.
Eccles, S.A.
Chesler, L.
Linardopoulos, S.
(2011). The Aurora kinase inhibitor CCT137690 downregulates MYCN and sensitizes MYCN-amplified neuroblastoma in vivo. Mol canc ther,
.
Shuttleworth, S.J.
Silva, F.A.
Cecil, A.R.
Tomassi, C.D.
Hill, T.J.
Raynaud, F.I.
Clarke, P.A.
Workman, P.
(2011). Progress in the preclinical discovery and clinical development of class I and dual class I/IV phosphoinositide 3-kinase (PI3K) inhibitors. Curr med chem,
Vol.18
(18),
pp. 2686-2714.
show abstract
full text
The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer..
Al-Saffar, N.M.
Jackson, L.E.
Raynaud, F.I.
Clarke, P.A.
RamÃrez de Molina, A.
Lacal, J.C.
Workman, P.
Leach, M.O.
(2010). The phosphoinositide 3-kinase inhibitor PI-103 downregulates choline kinase alpha leading to phosphocholine and total choline decrease detected by magnetic resonance spectroscopy. Cancer res,
Vol.70
(13),
pp. 5507-5517.
show abstract
The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors..
Workman, P.
van Montfort, R.L.
(2010). PI(3) KINASES Revealing the delta lady. Nature chemical biology,
Vol.6
(2),
pp. 82-2.
Carden, C.P.
Sarker, D.
Postel-Vinay, S.
Yap, T.A.
Attard, G.
Banerji, U.
Garrett, M.D.
Thomas, G.V.
Workman, P.
Kaye, S.B.
de Bono, J.S.
(2010). Can molecular biomarker-based patient selection in Phase I trials accelerate anticancer drug development?. Drug discov today,
Vol.15
(3-4),
pp. 88-97.
show abstract
Anticancer drug development remains slow, costly and inefficient. One way of addressing this might be the use of predictive biomarkers to select patients for Phase I/II trials. Such biomarkers, which predict response to molecular-targeted agents, have the potential to enrich these trials with patients more likely to benefit. Doing so could maximize the efficiency of anticancer drug development by facilitating earlier clinical qualification of predictive biomarkers and generating valuable information on cancer biology. In this review, we suggest a new model of early clinical trial design, which incorporates patient selection through predictive molecular biomarkers for selected targeted agents..
Rowlands, M.
McAndrew, C.
Prodromou, C.
Pearl, L.
Kalusa, A.
Jones, K.
Workman, P.
Aherne, W.
(2010). Detection of the ATPase activity of the molecular chaperones Hsp90 and Hsp72 using the TranscreenerTM ADP assay kit. J biomol screen,
Vol.15
(3),
pp. 279-286.
show abstract
The molecular chaperone heat shock protein 90 (Hsp90) is required for the correct folding and stability of a number of client proteins that are important for the growth and maintenance of cancer cells. Heat shock protein 72 (Hsp72), a co-chaperone of Hsp90, is also emerging as an attractive cancer drug target. Both proteins bind and hydrolyze adenosine triphosphate (ATP), and ATPase activity is essential for their function. Inhibition of Hsp90 ATPase activity leads to the degradation of client proteins, resulting in cell growth inhibition and apoptosis. Several small-molecule inhibitors of the ATPase activity of Hsp90 have been described and are currently being evaluated clinically for the treatment of cancer. A number of methods for the measurement of ATPase activity have been previously used, but not all of these are ideally suited to screening cascades in drug discovery projects. The authors have evaluated the use of commercial reagents (Transcreener ADP) for the measurement of ATPase activity of both yeast and human Hsp90 (ATP K(m) approximately 500 microM) and human Hsp72 (ATP K(m) ~1 microM). The low ATPase activity of human Hsp90 and its stimulation by the co-chaperone Aha1 was measured with ease using reduced incubation times, generating robust data (Z' = 0.75). The potency of several small-molecule inhibitors of both Hsp90 and Hsp72 was determined using the Transcreener reagents and compared well to that determined using other assay formats..
Gaspar, N.
Sharp, S.Y.
Eccles, S.A.
Gowan, S.
Popov, S.
Jones, C.
Pearson, A.
Vassal, G.
Workman, P.
(2010). Mechanistic evaluation of the novel HSP90 inhibitor NVP-AUY922 in adult and pediatric glioblastoma. Mol cancer ther,
Vol.9
(5),
pp. 1219-1233.
show abstract
The dismal prognosis of glioblastoma (GB) indicates the urgent need for new therapies for these tumors. Heat shock protein 90 (HSP90) inhibitors induce the proteasome-mediated degradation of many oncogenic client proteins involved in all of the hallmark characteristics of cancer. Here, we explored the mechanistic potential of the potent synthetic diarylisoxazole amide resorcinol HSP90 inhibitor, NVP-AUY922, in adult and pediatric GB. In vitro antiproliferative potency (nanomolar range) was seen in both adult and pediatric human GB cell lines with different molecular pathologies. A cytostatic effect was observed in all GB lines; more apoptosis was observed at lower concentrations in the SF188 pediatric GB line and at 144 hours in the slower growing KNS42 pediatric GB line, as compared with the adult GB lines U87MG and SF268. In vitro combination studies with inhibitors of phosphoinositide 3-kinase/mammalian target of rapamycin (PI-103) or mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (PD-0325901) supported the hypothesis that sustained inhibition of ERK up to 72 hours and at least temporary inhibition of AKT were necessary to induce apoptosis in GB lines. In athymic mice bearing established s.c U87MG GB xenografts, NVP-AUY922 (50 mg/kg i.p x 3 days) caused the inhibition of ERK1/2 and AKT phosphorylation and induced apoptosis, whereas 17-AAG used at maximum tolerated dose was less effective. NVP-AUY922 antitumor activity with objective tumor regression resulted from antiproliferative, proapoptotic, and antiangiogenic effects, the latter shown by decreased microvessel density and HIF1alpha levels. Our results have established a mechanistic proof of concept for the potential of novel synthetic HSP90 inhibitors in adult and pediatric GB, alone or in combination with phosphoinositide 3-kinase/mammalian target of rapamycin and mitogen-activated protein/ERK kinase inhibitors..
Pacey, S.
Gore, M.
Chao, D.
Banerji, U.
Larkin, J.
Sarker, S.
Owen, K.
Asad, Y.
Raynaud, F.
Walton, M.
Judson, I.
Workman, P.
Eisen, T.
(2010). A Phase II trial of 17-allylamino, 17-demethoxygeldanamycin (17-AAG, tanespimycin) in patients with metastatic melanoma. Invest new drugs,
.
show abstract
Purpose A Phase II study to screen for anti-melanoma activity of the heat shock protein 90 (HSP90) inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin) was performed. The primary endpoint was the rate of disease stabilisation in patients with progressive, metastatic melanoma treated with 17-AAG. Secondary endpoints were to determine: the toxicity of 17-AAG, the duration of response(s), median survival and further study the pharmacokinetics and pharmacodynamics of 17-AAG. Patients and Methods Patients with metastatic melanoma (progressive disease documented /=25% at 6 months was considered compatible with 17-AAG having activity. Results Fourteen patients (8 male: 6 female) were entered, eleven received 17-AAG (performance status 0 or 1). Median age was 60 (range 29-81) years. The majority (93%) received prior chemotherapy and had stage M1c disease (71%). Toxicity was rarely >/= Grade 2 in severity and commonly included fatigue, headache and gastrointestinal disturbances. One of eleven patients treated with 17-AAG had stable disease for 6 months and median survival for all patients was 173 days. The study was closed prematurely prior to completion of the first stage of recruitment and limited planned pharmacokinetic and pharmacodynamic analyses. Conclusion Some evidence of 17-AAG activity was observed although early study termination meant study endpoints were not reached. Stable disease rates can be incorporated into trials screening for anti-melanoma activity and further study of HSP90 inhibitors in melanoma should be considered..
Grimshaw, K.M.
Hunter, L.-.
Yap, T.A.
Heaton, S.P.
Walton, M.I.
Woodhead, S.J.
Fazal, L.
Reule, M.
Davies, T.G.
Seavers, L.C.
Lock, V.
Lyons, J.F.
Thompson, N.T.
Workman, P.
Garrett, M.D.
(2010). AT7867 Is a Potent and Oral Inhibitor of AKT and p70 S6 Kinase That Induces Pharmacodynamic Changes and Inhibits Human Tumor Xenograft Growth. Molecular cancer therapeutics,
Vol.9
(5),
pp. 1100-11.
Yap, T.A.
Sandhu, S.K.
Workman, P.
de Bono, J.S.
(2010). Envisioning the future of early anticancer drug development. Nat rev cancer,
Vol.10
(7),
pp. 514-523.
show abstract
The development of novel molecularly targeted cancer therapeutics remains slow and expensive with many late-stage failures. There is an urgent need to accelerate this process by improving early clinical anticancer drug evaluation through modern and rational trial designs that incorporate predictive, pharmacokinetic, pharmacodynamic, pharmacogenomic and intermediate end-point biomarkers. In this article, we discuss current approaches and propose strategies that will potentially maximize benefit to patients and expedite the regulatory approvals of new anticancer drugs..
Beloueche-Babari, M.
Arunan, V.
Jackson, L.E.
Perusinghe, N.
Sharp, S.Y.
Workman, P.
Leach, M.O.
(2010). Modulation of melanoma cell phospholipid metabolism in response to heat shock protein 90 inhibition. Oncotarget,
Vol.1
(3),
pp. 185-197.
show abstract
full text
Molecular chaperone heat shock protein 90 (Hsp90) inhibitors are promising targeted cancer therapeutic drugs, with the advantage that they deplete multiple oncogenic client proteins and modulate all the classical hallmarks of cancer. They are now in clinical trial and show potential for activity in melanoma and other malignancies. Here we explore the metabolic response to Hsp90 inhibition in human melanoma cells using magnetic resonance spectroscopy. We show that, concomitant with growth inhibition and re-differentiation, Hsp90 inhibition in human melanoma cells is associated with increased glycerophosphocholine content. This was seen with both the clinical geldanamycin-based Hsp90 drug 17-AAG and the structurally dissimilar Hsp90 inhibitor CCT018159. The effect was noted in both BRAF mutant SKMEL28 and BRAF wildtype CHL-1 melanoma cells. Elevated content of the -CH2+CH3 fatty acyl chains and cytoplasmic mobile lipid droplets was also observed in 17-AAG-treated SKMEL28 cells. Importantly, the phospholipase A2 inhibitor bromoenol lactone prevented the rise in glycerophosphocholine seen with 17-AAG, suggesting a role for phospholipase A2 activation in the Hsp90 inhibitor-induced metabolic response. Our findings provide a basis for using metabolic changes as non-invasive indicators of Hsp90 inhibition and potentially as biomarkers of anticancer activity with Hsp90 drugs in malignant melanoma and possibly in other cancers..
Workman, P.
Travers, J.
(2010). CANCER Drug-tolerant insurgents. Nature,
Vol.464
(7290),
pp. 844-845.
Hayward, D.G.
Newbatt, Y.
Pickard, L.
Byrne, E.
Mao, G.
Burns, S.
Sahota, N.K.
Workman, P.
Collins, I.
Aherne, W.
Fry, A.M.
(2010). Identification by High-Throughput Screening of Viridin Analogs as Biochemical and Cell-Based Inhibitors of the Cell Cycle-Regulated Nek2 Kinase. Journal of biomolecular screening,
Vol.15
(8),
pp. 918-10.
Davenport, E.L.
Zeisig, A.
Aronson, L.I.
Moore, H.E.
Hockley, S.
Gonzalez, D.
Smith, E.M.
Powers, M.V.
Sharp, S.Y.
Workman, P.
Morgan, G.J.
Davies, F.E.
(2010). Targeting heat shock protein 72 enhances Hsp90 inhibitor-induced apoptosis in myeloma. Leukemia,
Vol.24
(10),
pp. 1804-1807.
Workman, P.
Clarke, P.A.
Raynaud, F.I.
van Montfort, R.L.
(2010). Drugging the PI3 kinome: from chemical tools to drugs in the clinic. Cancer res,
Vol.70
(6),
pp. 2146-2157.
show abstract
The phosphatidylinositide 3-kinase (PI3K) pathway is very commonly activated in a wide range of human cancers and is a major driving force in oncogenesis. One of the class I lipid kinase members of the PI3K family, p110alpha, is probably the most commonly mutated kinase in the human genome. Alongside genetic, molecular biological, and biochemical studies, chemical inhibitors have been extremely helpful tools in understanding the role of PI3K enzymes in signal transduction and downstream physiological and pathological processes, and also in validating PI3Ks as therapeutic targets. Although they have been valuable in the past, the early and still frequently employed inhibitors, wortmannin and LY294002, have significant limitations as chemical tools. Here, we discuss the case history of the discovery and properties of an increasingly used chemical probe, the pan-class I PI3K and mammalian target of rapamycin (mTOR) inhibitor PI-103 (a pyridofuropyrimidine), and its very recent evolution into the thienopyrimidine drug GDC-0941, which exhibits excellent oral anticancer activity in preclinical models and is now undergoing phase I clinical trials in cancer patients. We also illustrate the impact of structural biology on the design of PI3K inhibitors and on the interpretation of their effects. The challenges and outlook for drugging the PI3 kinome are discussed in the more general context of the role of structural biology and chemical biology in innovative drug discovery..
Powers, M.V.
Jones, K.
Barillari, C.
Westwood, I.
van Montfort, R.L.
Workman, P.
(2010). Targeting HSP70: the second potentially druggable heat shock protein and molecular chaperone?. Cell cycle,
Vol.9
(8),
pp. 1542-1550.
show abstract
The HSF1-mediated stress response pathway is steadily gaining momentum as a critical source of targets for cancer therapy. Key mediators of this pathway include molecular chaperones such as heat shock protein (HSP) 90. There has been considerable progress in targeting HSP90 and the preclinical efficacy and signs of early clinical activity of HSP90 inhibitors have provided proof-of-concept for targeting this group of proteins. The HSP70 family of molecular chaperones are also key mediators of the HSF-1-stress response pathway and have multiple additional roles in protein folding, trafficking and degradation, as well as regulating apoptosis. Genetic and biochemical studies have supported the discovery of HSP70 inhibitors which have the potential for use as single agents or in combination to enhance the effects of classical chemotherapeutic or molecularly targeted agents including HSP90 inhibitors. Here we provide a perspective on the progress made so far in designing agents which target the HSP70 family..
Ewan, K.
Pajak, B.
Stubbs, M.
Todd, H.
Barbeau, O.
Quevedo, C.
Botfield, H.
Young, R.
Ruddle, R.
Samuel, L.
Battersby, A.
Raynaud, F.
Allen, N.
Wilson, S.
Latinkic, B.
Workman, P.
McDonald, E.
Blagg, J.
Aherne, W.
Dale, T.
(2010). A Useful Approach to Identify Novel Small-Molecule Inhibitors of Wnt-Dependent Transcription. Cancer research,
Vol.70
(14),
pp. 5963-11.
Gaspar, N.
Marshall, L.
Perryman, L.
Bax, D.A.
Little, S.E.
Viana-Pereira, M.
Sharp, S.Y.
Vassal, G.
Pearson, A.D.
Reis, R.M.
Hargrave, D.
Workman, P.
Jones, C.
(2010). MGMT-Independent Temozolomide Resistance in Pediatric Glioblastoma Cells Associated with a PI3-Kinase-Mediated HOX/Stem Cell Gene Signature. Cancer research,
Vol.70
(22),
pp. 9243-10.
Workman, P.
van Montfort, R.L.
(2010). Unveiling the secrets of the ancestral PI3 kinase Vps34. Cancer cell,
Vol.17
(5),
pp. 421-423.
show abstract
Vps34 is the primordial member of the PI3 kinase family involved in vesicular trafficking, nutrient signaling, and autophagy. A report in Science unveils the Vps34 structure, providing new insights into the catalytic mechanism, explaining why Vsp34 is so difficult to inhibit, and facilitating design of chemical tools and potential drugs..
Bavetsias, V.
Large, J.M.
Sun, C.
Bouloc, N.
Kosmopoulou, M.
Matteucci, M.
Wilsher, N.E.
Martins, V.
Reynisson, J.
Atrash, B.
Faisal, A.
Urban, F.
Valenti, M.
de Haven Brandon, A.
Box, G.
Raynaud, F.I.
Workman, P.
Eccles, S.A.
Bayliss, R.
Blagg, J.
Linardopoulos, S.
McDonald, E.
(2010). Imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases: lead optimization studies toward the identification of an orally bioavailable preclinical development candidate. J med chem,
Vol.53
(14),
pp. 5213-5228.
show abstract
Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC(50) = 0.015 +/- 0.003 muM, Aurora-B IC(50) = 0.025 muM, Aurora-C IC(50) = 0.019 muM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss..
Workman, P.
Aboagye, E.O.
Balkwill, F.
Balmain, A.
Bruder, G.
Chaplin, D.J.
Double, J.A.
Everitt, J.
Farningham, D.A.
Glennie, M.J.
Kelland, L.R.
Robinson, V.
Stratford, I.J.
Tozer, G.M.
Watson, S.
Wedge, S.R.
Eccles, S.A.
Committee of the National Cancer Research Institute,
(2010). Guidelines for the welfare and use of animals in cancer research. Br j cancer,
Vol.102
(11),
pp. 1555-1577.
show abstract
Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice..
Workman, P.
Collins, I.
(2010). Probing the probes: fitness factors for small molecule tools. Chem biol,
Vol.17
(6),
pp. 561-577.
show abstract
Chemical probes for interrogating biological processes are of considerable current interest. Cell permeable small molecule tools have a major role in facilitating the functional annotation of the human genome, understanding both physiological and pathological processes, and validating new molecular targets. To be valuable, chemical tools must satisfy necessary criteria and recent publications have suggested objective guidelines for what makes a useful chemical probe. Although recognizing that such guidelines may be valuable, we caution against overly restrictive rules that may stifle innovation in favor of a "fit-for-purpose" approach. Reviewing the literature and providing examples from the cancer field, we recommend a series of "fitness factors" to be considered when assessing chemical probes. We hope this will encourage innovative chemical biology research while minimizing the generation of poor quality and misleading biological data, thus increasing understanding of the particular biological area, to the benefit of basic research and drug discovery..
Day, J.E.
Sharp, S.Y.
Rowlands, M.G.
Aherne, W.
Workman, P.
Moody, C.J.
(2010). Targeting the Hsp90 Chaperone: Synthesis of Novel Resorcylic Acid Macrolactone Inhibitors of Hsp90. Chemistry-a european journal,
Vol.16
(9),
pp. 2758-6.
Day, J.E.
Sharp, S.Y.
Rowlands, M.G.
Aherne, W.
Lewis, W.
Roe, S.M.
Prodromou, C.
Pearl, L.H.
Workman, P.
Moody, C.J.
(2010). Inhibition of Hsp90 with Resorcylic Acid Macrolactones: Synthesis and Binding Studies. Chemistry-a european journal,
Vol.16
(34),
pp. 10366-7.
Guillard, S.
Clarke, P.A.
Te Poele, R.
Mohri, Z.
Bjerke, L.
Valenti, M.
Raynaud, F.
Eccles, S.A.
Workman, P.
(2009). Molecular pharmacology of phosphatidylinositol 3-kinase inhibition in human glioma. Cell cycle,
Vol.8
(3),
pp. 443-453.
show abstract
Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of the isoform-selective class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited ribosomal protein S6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr(308)-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G(1)/S phase progression and increased expression of the negative cell cycle regulator p27(kip1). A reversible PI-103-mediated G(1) cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with a PI-103-like profile as therapeutic agents for the treatment of glioma..
Smith, J.R.
Workman, P.
(2009). Targeting CDC37: an alternative, kinase-directed strategy for disruption of oncogenic chaperoning. Cell cycle,
Vol.8
(3),
pp. 362-372.
show abstract
Interest in HSP90 inhibitors has grown rapidly in the last decade. The heightened dependence of malignant cells on molecular chaperones to maintain multiple oncogenic signalling pathways gives HSP90 broad anticancer appeal. New HSP90-directed agents are continually emerging, several of which are under clinical evaluation. In parallel, dissection of the functional mechanism of the chaperone system has emphasised the importance of cochaperones that regulate HSP90. As we begin to fully elucidate the roles of these HSP90 accessory proteins, it is becoming increasingly clear that they too have potential as additional routes to disrupt chaperone activity. CDC37, a predominantly kinase client-associated cochaperone that promotes malignant transformation, has particular promise. Recently, we demonstrated that, similar to HSP90 inhibitors, siRNA-mediated CDC37 silencing caused the proteasomal degradation of kinase client proteins and inhibited the proliferation of cancer cells. Importantly, depleting CDC37 does not induce the unwanted, antiapoptotic heat shock response that is characteristic of pharmacologic HSP90 inhibition. Furthermore, CDC37 silencing sensitises cancer cells to HSP90 inhibitors by potentiating kinase client depletion and the induction of apoptosis, suggesting that simultaneously modulating HSP90 and CDC37 could be beneficial. Here we discuss the therapeutic possibilities of targeting CDC37 for cancer treatment in light of recent significant findings..
Gaspar, N.
Sharp, S.Y.
Pacey, S.
Jones, C.
Walton, M.
Vassal, G.
Eccles, S.
Pearson, A.
Workman, P.
(2009). Acquired resistance to 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) in glioblastoma cells. Cancer res,
Vol.69
(5),
pp. 1966-1975.
show abstract
Heat shock protein 90 (HSP90) inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), which is currently in phase II/phase III clinical trials, are promising new anticancer agents. Here, we explored acquired resistance to HSP90 inhibitors in glioblastoma (GB), a primary brain tumor with poor prognosis. GB cells were exposed continuously to increased 17-AAG concentrations. Four 17-AAG-resistant GB cell lines were generated. High-resistance levels with resistance indices (RI = resistant line IC(50)/parental line IC(50)) of 20 to 137 were obtained rapidly (2-8 weeks). After cessation of 17-AAG exposure, RI decreased and then stabilized. Cross-resistance was found with other ansamycin benzoquinones but not with the structurally unrelated HSP90 inhibitors, radicicol, the purine BIIB021, and the resorcinylic pyrazole/isoxazole amide compounds VER-49009, VER-50589, and NVP-AUY922. An inverse correlation between NAD(P)H/quinone oxidoreductase 1 (NQO1) expression/activity and 17-AAG IC(50) was observed in the resistant lines. The NQO1 inhibitor ES936 abrogated the differential effects of 17-AAG sensitivity between the parental and resistant lines. NQO1 mRNA levels and NQO1 DNA polymorphism analysis indicated different underlying mechanisms: reduced expression and selection of the inactive NQO1*2 polymorphism. Decreased NQO1 expression was also observed in a melanoma line with acquired resistance to 17-AAG. No resistance was generated with VER-50589 and NVP-AUY922. In conclusion, low NQO1 activity is a likely mechanism of acquired resistance to 17-AAG in GB, melanoma, and, possibly, other tumor types. Such resistance can be overcome with novel HSP90 inhibitors..
Lee, C.P.
Payne, G.S.
Oregioni, A.
Ruddle, R.
Tan, S.
Raynaud, F.I.
Eaton, D.
Campbell, M.J.
Cross, K.
Halbert, G.
Tracy, M.
McNamara, J.
Seddon, B.
Leach, M.O.
Workman, P.
Judson, I.
(2009). A phase I study of the nitroimidazole hypoxia marker SR4554 using 19F magnetic resonance spectroscopy. Br j cancer,
Vol.101
(11),
pp. 1860-1868.
show abstract
SR4554 is a fluorine-containing 2-nitroimidazole, designed as a hypoxia marker detectable with 19F magnetic resonance spectroscopy (MRS). In an initial phase I study of SR4554, nausea/vomiting was found to be dose-limiting, and 1400 mg m(-2) was established as MTD. Preliminary MRS studies demonstrated some evidence of 19F retention in tumour. In this study we investigated higher doses of SR4554 and intratumoral localisation of the 19F MRS signal..
de Billy, E.
Powers, M.V.
Smith, J.R.
Workman, P.
(2009). Drugging the heat shock factor 1 pathway Exploitation of the critical cancer cell dependence on the guardian of the proteome. Cell cycle,
Vol.8
(23),
pp. 3806-3808.
Massey, A.
Brough, P.
Jensen, M.
Ruetz, S.
Schoepfer, J.
Workman, P.
Garcia-Echeverria, C.
Drysdale, M.
(2009). NVP-BEP795 and NVP-BEP800: Novel, fragment derived orally bioavailable small molecule Hsp90 inhibitors with potent antitumor activity. Cancer research,
Vol.69.
Carden, C.P.
Banerji, U.
Kaye, S.B.
Workman, P.
de Bono, J.S.
(2009). From darkness to light with biomarkers in early clinical trials of cancer drugs. Clin pharmacol ther,
Vol.85
(2),
pp. 131-133.
show abstract
Compared with conventional chemotherapy, rationally designed molecularly targeted agents may be more likely to have antitumor activity in selected tumor subgroups driven by the oncogenic signals targeted by these compounds and a different side-effect profile. The use of biomarkers in the early clinical trials of these new anticancer agents has the potential to increase study participants' benefit from early clinical trials, accelerate the drug development process, maximize the ability to generate important biological information about human cancer, and decrease the risk of late and costly drug attrition..
Rogers, S.J.
Box, C.
Chambers, P.
Barbachano, Y.
Nutting, C.M.
RhÅ·s-Evans, P.
Workman, P.
Harrington, K.J.
Eccles, S.A.
(2009). Determinants of response to epidermal growth factor receptor tyrosine kinase inhibition in squamous cell carcinoma of the head and neck. J pathol,
Vol.218
(1),
pp. 122-130.
show abstract
Dramatic responses to epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitors may be seen in non-small cell lung cancers (NSCLCs) with a sensitizing mutation of the EGFR TK domain. It is not known how to predict response in patients with squamous cell carcinoma of the head and neck (SCCHN), where EGFR TK mutations are less frequent and where response rates in unselected patients are disappointing. We have characterized the intrinsic sensitivity of a panel of 18 SCCHN cell lines to gefitinib, an EGFR TK inhibitor, and have investigated correlations between putative markers of response and intrinsic sensitivity. Induction of G1 arrest was only seen in cell lines with GI(50) < 1 microM. Expression of EGFR, by three techniques, correlated with sensitivity to gefitinib. ERB-B2 expression appeared to influence sensitivity to gefitinib but ERB-B3 expression did not. While EGFR tyrosine kinase mutations were not detected, EGFR gene amplification was confirmed by fluorescence in situ hybridization in the most sensitive cell line. The number of cytosine adenine dinucleotide repeats in intron 1 of the EGFR gene did not correlate with sensitivity. E-cadherin expression was detected in cell lines with a range of sensitivities, whereas amphiregulin was secreted predominantly by sensitive cell lines. MET expression was an independent predictor of sensitivity to gefitinib, although neither expression nor phosphorylation of insulin-like growth factor 1 receptor correlated with intrinsic resistance. Breast receptor kinase (BRK) was more highly expressed in the sensitive cell lines, but siRNA knockdown of neither BRK nor MET affected sensitivity. Our data suggest that overexpression of EGFR and multiple related cell surface receptors may be associated with sensitivity to gefitinib and that differences between our data and the literature highlight that biomarkers of response are tumour type- and cell line-dependent..
van Montfort, R.L.
Workman, P.
(2009). Structure-based design of molecular cancer therapeutics. Trends biotechnol,
Vol.27
(5),
pp. 315-328.
show abstract
Structure-based approaches now impact across the whole continuum of drug discovery, from new target selection through the identification of hits to the optimization of lead compounds. Optimal application of structure-based design involves close integration with other discovery technologies, including fragment-based and virtual screening. Here, we illustrate the use of structural information and of structure-based drug design approaches in the discovery of small-molecule inhibitors for cancer drug targets and provide an outlook on the exploitation of structural information in future cancer drug discovery. Examples include high profile protein kinase targets and structurally related PI3 kinases, histone deacetylases, poly(ADP-ribose)polymerase and the molecular chaperone HSP90. Structure-based design approaches have also been successfully applied to the protein-protein interaction targets p53-MDM2 and the Bcl-2 family..
Baird, R.D.
Kitzen, J.
Clarke, P.A.
Planting, A.
Reade, S.
Reid, A.
Welsh, L.
López Lázaro, L.
de las Heras, B.
Judson, I.R.
Kaye, S.B.
Eskens, F.
Workman, P.
deBono, J.S.
Verweij, J.
(2009). Phase I safety, pharmacokinetic, and pharmacogenomic trial of ES-285, a novel marine cytotoxic agent, administered to adult patients with advanced solid tumors. Mol cancer ther,
Vol.8
(6),
pp. 1430-1437.
show abstract
A dose-escalation, phase I study evaluated the safety, pharmacokinetics, pharmacogenomics, and efficacy of ES-285, a novel agent isolated from a marine mollusc, in adult cancer patients. Patients received a 24-hour i.v. infusion of ES-285 once every 3 weeks until disease progression or unacceptable toxicity. The starting dose was 4 mg/m(2). Dose escalation in cohorts of at least three patients proceeded according to the worst toxicity observed in the previous cohort. Twenty-eight patients were treated with 72 courses of ES-285 across eight dose levels. No dose-limiting toxicities were seen between 4 and 128 mg/m(2). Two of four patients treated at 256 mg/m(2) had dose-limiting reversible grade 3 transaminitis; one patient at 256 mg/m(2) also had transient grade 3 central neurotoxicity. One of three patients subsequently treated at 200 mg/m(2) died following drug-related central neurotoxicity. Other toxicities included phlebitis, nausea, fatigue, and fever. Pharmacokinetic studies indicated dose proportionality with high volume of distribution (median V(ss) at 256 mg/m(2) was 2,389 liters; range, 1,615-4,051 liters) and long elimination half life (median t(1/2) at 256 mg/m(2) was 28 h; range, 21-32 h). The three patients with dose-limiting toxicity had the highest drug exposure. Pharmacogenomic studies of paired surrogate tissue samples identified changes in gene expression following treatment that correlated with increasing dose. Disease stabilization for 6 to 18 weeks was recorded in nine patients. Using this schedule, 128 mg/m(2) was considered safe and feasible. At this dose, pharmacologically relevant concentrations of the drug were safely achieved with pharmacogenomic studies indicating changes in the expression of genes of potential mechanistic relevance..
Raynaud, F.I.
Eccles, S.A.
Patel, S.
Alix, S.
Box, G.
Chuckowree, I.
Folkes, A.
Gowan, S.
De Haven Brandon, A.
Di Stefano, F.
Hayes, A.
Henley, A.T.
Lensun, L.
Pergl-Wilson, G.
Robson, A.
Saghir, N.
Zhyvoloup, A.
McDonald, E.
Sheldrake, P.
Shuttleworth, S.
Valenti, M.
Wan, N.C.
Clarke, P.A.
Workman, P.
(2009). Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941. Mol cancer ther,
Vol.8
(7),
pp. 1725-1738.
show abstract
The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110alpha with IC(50) < or = 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials..
Brough, P.A.
Barril, X.
Borgognoni, J.
Chene, P.
Davies, N.G.
Davis, B.
Drysdale, M.J.
Dymock, B.
Eccles, S.A.
Garcia-Echeverria, C.
Fromont, C.
Hayes, A.
Hubbard, R.E.
Jordan, A.M.
Jensen, M.R.
Massey, A.
Merrett, A.
Padfield, A.
Parsons, R.
Radimerski, T.
Raynaud, F.I.
Robertson, A.
Roughley, S.D.
Schoepfer, J.
Simmonite, H.
Sharp, S.Y.
Surgenor, A.
Valenti, M.
Walls, S.
Webb, P.
Wood, M.
Workman, P.
Wright, L.
(2009). Combining hit identification strategies: fragment-based and in silico approaches to orally active 2-aminothieno[2,3-d]pyrimidine inhibitors of the Hsp90 molecular chaperone. J med chem,
Vol.52
(15),
pp. 4794-4809.
show abstract
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model..
Bax, D.A.
Gaspar, N.
Little, S.E.
Marshall, L.
Perryman, L.
Regairaz, M.
Viana-Pereira, M.
Vuononvirta, R.
Sharp, S.Y.
Reis-Filho, J.S.
Stávale, J.N.
Al-Sarraj, S.
Reis, R.M.
Vassal, G.
Pearson, A.D.
Hargrave, D.
Ellison, D.W.
Workman, P.
Jones, C.
(2009). EGFRvIII deletion mutations in pediatric high-grade glioma and response to targeted therapy in pediatric glioma cell lines. Clin cancer res,
Vol.15
(18),
pp. 5753-5761.
show abstract
The epidermal growth factor receptor (EGFR) is amplified and overexpressed in adult glioblastoma, with response to targeted inhibition dependent on the underlying biology of the disease. EGFR has thus far been considered to play a less important role in pediatric glioma, although extensive data are lacking. We have sought to clarify the role of EGFR in pediatric high-grade glioma (HGG)..
Smith, J.R.
Clarke, P.A.
de Billy, E.
Workman, P.
(2009). Silencing the cochaperone CDC37 destabilizes kinase clients and sensitizes cancer cells to HSP90 inhibitors. Oncogene,
Vol.28
(2),
pp. 157-169.
show abstract
full text
The cochaperone CDC37 promotes the association of HSP90 with the protein kinase subset of client proteins to maintain their stability and signalling functions. HSP90 inhibitors induce depletion of clients, which include several oncogenic kinases. We hypothesized that the targeting of CDC37 using siRNAs would compromise the maturation of these clients and increase the sensitivity of cancer cells to HSP90 inhibitors. Here, we show that silencing of CDC37 in human colon cancer cells diminished the association of kinase clients with HSP90 and reduced levels of the clients ERBB2, CRAF, CDK4 and CDK6, as well as phosphorylated AKT. CDC37 silencing promoted the proteasome-mediated degradation of kinase clients, suggesting a degradation pathway independent from HSP90 binding. Decreased cell signalling through kinase clients was also demonstrated by reduced phosphorylation of downstream substrates and colon cancer cell proliferation was subsequently reduced by the inhibition of the G1/S-phase transition. Furthermore, combining CDC37 silencing with the HSP90 inhibitor 17-AAG induced more extensive and sustained depletion of kinase clients and potentiated cell cycle arrest and apoptosis. These results support an essential role for CDC37 in concert with HSP90 in maintaining oncogenic protein kinase clients and endorse the therapeutic potential of targeting CDC37 in cancer..
Gorsuch, S.
Bavetsias, V.
Rowlands, M.G.
Aherne, G.W.
Workman, P.
Jarman, M.
McDonald, E.
(2009). Synthesis of isothiazol-3-one derivatives as inhibitors of histone acetyltransferases (HATs). Bioorg med chem,
Vol.17
(2),
pp. 467-474.
show abstract
High-throughput screening led to the identification of isothiazolones 1 and 2 as inhibitors of histone acetyltransferase (HAT) with IC50s of 3 microM and 5 microM, respectively. Analogues of these hit compounds with variations of the N-phenyl group, and with variety of substituents at C-4, C-5 of the thiazolone ring, were prepared and assayed for inhibition of the HAT enzyme PCAF. Potency is modestly favoured when the N-aryl group is electron deficient (4-pyridyl derivative 10 has IC(50)=1.5 microM); alkyl substitution at C-4 has little effect, whilst similar substitution at C-5 causes a significant drop in potency. The ring-fused compound 38 has activity (IC(50)=6.1 microM) to encourage further exploration of this bicyclic structure. The foregoing SAR is consistent with an inhibitory mechanism involving cleavage of the S-N bond of the isothiazolone ring by a catalytically important thiol residue..
Reynisson, J.
Court, W.
O'Neill, C.
Day, J.
Patterson, L.
McDonald, E.
Workman, P.
Katan, M.
Eccles, S.A.
(2009). The identification of novel PLC-gamma inhibitors using virtual high throughput screening. Bioorg med chem,
Vol.17
(8),
pp. 3169-3176.
show abstract
Phospholipase C-gamma (PLC-gamma) has been identified as a possible biological target for anticancer drug therapy but suitable inhibitors are lacking. Therefore, in order to identify active compounds (hits) virtual high throughput screening was performed. The crystal structure of the PLC-delta isoform was used as a model docking scaffold since no crystallographic data are available on its gamma counterpart. A pilot screen was performed using approximately 9.2x10(4) compounds, where the robustness of the methodology was tested. This was followed by the main screening effort where approximately 4.4x10(5) compounds were used. In both cases, plausible compounds were identified (virtual hits) and a selection of these was experimentally tested. The most potent compounds were in the single digit micro-molar range as determined from the biochemical (Flashplate) assay. This translated into approximately 15 microM in a functional assay in cells. About 30% of the virtual hits showed activity against PLC-gamma (IC(50)<50 microM)..
Powers, M.V.
Clarke, P.A.
Workman, P.
(2009). Death by chaperone HSP90, HSP70 or both?. Cell cycle,
Vol.8
(4),
pp. 518-526.
show abstract
HSP70 family members are highly conserved proteins that function as molecular chaperones. Their principle role is to aid protein folding and promote the correct cellular localizations of their respective substrates. The function of HSP70 isoforms can be exhibited independently or with the HSP90 chaperone system in which HSP70 is important for substrate recruitment. In addition to their chaperone role, HSP70 isoforms promote cell survival by inhibiting apoptosis at multiple points within both the intrinsic and extrinsic cell death pathways. Consistent with this cytoprotective function, increased expression of HSP70 isoforms is commonly associated with the malignant phenotype. We recently reported that dual silencing of the major constitutive (HSC70) and inducible (HSP72) isoforms of HSP70 in cancer cells could phenocopy the effects of a pharmacologic HSP90 inhibitor to induce proteasome-dependent degradation of HSP90 client proteins CRAF, CDK4 and ERBB2. This was accompanied by a G(1) cell cycle arrest and extensive apoptosis which was not seen in non-tumorigenic human cell lines. Here we discuss the possible implications of our research for the development of HSP70 family modulators which offer not only the possibility of inhibiting HSP70 activity but also the simultaneous inhibition of HSP90, resulting in extensive tumor-specific apoptosis..
Davies, N.G.
Barril, X.
Drysdale, M.J.
Brough, P.A.
Eccles, S.A.
Dymock, B.
Garcia-Echeverria, C.
Massey, A.
Radimerski, T.
Raynaud, F.I.
Jensen, M.R.
Schoepfer, J.
Sharp, S.Y.
Workman, P.
(2009). NVP-BEP800/VER-82576: A potent orally active Hsp90 chaperone inhibitor evolved by SBDD from Vernalis fragment ("SeeDs") hits and in silico screening hits. Abstracts of papers of the american chemical society,
Vol.238,
p. 1.
Prodromou, C.
Nuttall, J.M.
Millson, S.H.
Roe, S.M.
Sim, T.S.
Tan, D.
Workman, P.
Pearl, L.H.
Piper, P.W.
(2009). Structural basis of the radicicol resistance displayed by a fungal hsp90. Acs chem biol,
Vol.4
(4),
pp. 289-297.
show abstract
Heat shock protein 90 (Hsp90) is a promising cancer drug target, as multiple oncogenic proteins are destabilized simultaneously when it loses its activity in tumor cells. Highly selective Hsp90 inhibitors, including the natural antibiotics geldanamycin (GdA) and radicicol (RAD), inactivate this essential molecular chaperone by occupying its nucleotide binding site. Often cancer drug therapy is compromised by the development of resistance, but a resistance to these Hsp90 inhibitors should not arise readily by mutation of those amino acids within Hsp90 that facilitate inhibitor binding, as these are required for the essential ATP binding/ATPase steps of the chaperone cycle and are tightly conserved. Despite this, the Hsp90 of a RAD-producing fungus is shown to possess an unusually low binding affinity for RAD but not GdA. Within its nucleotide binding site a normally conserved leucine is replaced by isoleucine, though the chaperone ATPase activity is not severely affected. Inserted into the Hsp90 of yeast, this conservative leucine to isoleucine substitution recreated this lowered affinity for RAD in vitro. It also generated a substantially enhanced resistance to RAD in vivo. Co-crystal structures reveal that the change to isoleucine is associated with a localized increase in the hydration of an Hsp90-bound RAD but not GdA. To the best of our knowledge, this is the first demonstration that it is possible for Hsp90 inhibitor resistance to arise by subtle alteration to the structure of Hsp90 itself..
Bax, D.A.
Little, S.E.
Gaspar, N.
Perryman, L.
Marshall, L.
Viana-Pereira, M.
Jones, T.A.
Williams, R.D.
Grigoriadis, A.
Vassal, G.
Workman, P.
Sheer, D.
Reis, R.M.
Pearson, A.D.
Hargrave, D.
Jones, C.
(2009). Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development. Plos one,
Vol.4
(4),
p. e5209.
show abstract
Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines..
Tan, D.S.
Thomas, G.V.
Garrett, M.D.
Banerji, U.
de Bono, J.S.
Kaye, S.B.
Workman, P.
(2009). Biomarker-driven early clinical trials in oncology: a paradigm shift in drug development. Cancer j,
Vol.15
(5),
pp. 406-420.
show abstract
Early clinical trials represent a crucial bridge between preclinical drug discovery and the especially resource-intense randomized phase III trial-the definitive regulatory hurdle for drug approval. High attrition rates and rising costs, when coupled with the extraordinary opportunities opened up by cancer genomics and the promise of personalized medicine call for new approaches in the conduct and design of phase I/II trials. The key challenge lies in increasing the odds for successful and efficient transition of a compound through the drug development pipeline. The incorporation of scientifically and analytically validated biomarkers into rationally designed hypothesis-testing clinical trials offers a promising way forward to achieving this objective. In this article, we provide an overview of biomarkers in early clinical trials, including examples where they have been particularly successful, and the caveats and pitfalls associated with indiscriminate application. We describe the use of pharmacodynamic end points to demonstrate the proof of modulation of target, pathway, and biologic effect, as well as predictive biomarkers for patient selection and trial enrichment. Establishing the pharmacologic audit trail provides a means to assess and manage risk in a drug development program and thus increases the rationality of the decision-making process. Accurate preclinical models are important for pharmacokinetic-pharmacodynamic-efficacy modeling and biomarker validation. The degree of scientific and analytical validation should ensure that biomarkers are fit-for purpose, according to the stage of development and the impact on the trial; specifically they are either exploratory or used to make decisions within the trial. To be maximally useful at an early stage, these must be in place before the commencement of phase I trials. Validation and qualification of biomarkers then continues through clinical development. We highlight the impact of modern technology platforms, such as genomics, proteomics, circulating tumor cells, and minimally invasive functional and molecular imaging, with respect to their potential role in improving the success rate and speed of drug development and in interrogating the consequences of therapeutic intervention and providing a unique insight into human disease biology. With these technologies already having an impact in the clinic today, we predict that further future advances will come from the application of network analysis to clinical trials, leading to individualized systems-based medicine for cancer..
Workman, P.
(2009). Drugging the cancer genome. Drug discovery today,
(July 2009).
Leyton, J.
Smith, G.
Lees, M.
Perumal, M.
Nguyen, Q.-.
Aigbirhio, F.I.
Golovko, O.
He, Q.
Workman, P.
Aboagye, E.O.
(2008). Noninvasive imaging of cell proliferation following mitogenic extracellular kinase inhibition by PD0325901. Molecular cancer therapeutics,
Vol.7
(9),
pp. 3112-10.
Chung, Y.-.
Troy, H.
Kristeleit, R.
Aherne, W.
Jackson, E.
Atadja, P.
Griffiths, J.R.
Judson, I.R.
Workman, P.
Leach, M.O.
Beloueche-Babari, M.
(2008). Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of a novel histone deacetylase inhibitor, LAQ824, in human colon carcinoma cells and xenografts. Neoplasia,
Vol.10
(4),
pp. 303-11.
full text
Jefferies, H.B.
Cooke, F.T.
Jat, P.
Boucheron, C.
Koizumi, T.
Hayakawa, M.
Kaizawa, H.
Ohishi, T.
Workman, P.
Waterfield, M.D.
Parker, P.J.
(2008). A selective PIKfyve inhibitor blocks PtdIns(3,5) P2 production and disrupts endomembrane transport and retroviral budding. Embo reports,
Vol.9
(2),
pp. 164-7.
full text
Ashworth, A.
Balkwill, F.
Bast, R.C.
Berek, J.S.
Kaye, A.
Boyd, J.A.
Mills, G.
Weinstein, J.N.
Woolley, K.
Workman, P.
(2008). Opportunities and challenges in ovarian cancer research, a perspective from the 11th Ovarian cancer action/HHMT Forum, Lake Como, March 2007. Gynecol oncol,
Vol.108
(3),
pp. 652-657.
show abstract
Advances in surgery and chemotherapy have improved the 5-year survival for patients with epithelial ovarian cancer, but have not impacted on the ultimate rate of cure in a disease that is diagnosed in late stage and that recurs in the majority of patients. "Omic" technologies promise to define genetically driven aberrant signaling pathways in malignant cells, provided that bioinformatic expertise can be focused on a cancer that is neither common nor rare. Molecular therapeutics must be linked to molecular diagnostics to permit individualized therapy. Not only epithelial cancer cells but also stroma, vasculature and the immune response must be targeted. Closer collaboration between academic institutions, biotech and pharma will be required to facilitate this process and to interest the private sector in an orphan disease. New preclinical models may permit more efficient development of drugs and siRNA that can target dormant drug resistant stem cells. Strategies must be developed to deal with the heterogeneity of different grades and histotypes. Identification of women at increased risk will facilitate prevention and early detection in subsets of patients. BRCA1/2 might be sequenced in all ovarian cancer patients to identify new kindreds. Epidemiologic algorithms are being developed and validated. Awareness must be raised that oral contraceptives can reduce risk of developing ovarian cancer by 50%. Early detection is likely to require panels of complementary biomarkers, analyzed by sophisticated statistical techniques, to improve sensitivity while maintaining extremely high specificity. As ovarian cancer becomes a chronic disease, greater emphasis will be placed on the challenges facing survivors..
Banerji, U.
Affolter, A.
Judson, I.
Marais, R.
Workman, P.
(2008). BRAF and NRAS mutations in melanoma: potential relationships to clinical response to HSP90 inhibitors. Mol cancer ther,
Vol.7
(4),
pp. 737-739.
show abstract
Oncogenic BRAF and NRAS mutations are frequent in malignant melanoma. BRAF that is activated by the common V600E and other mutations, as well as by upstream NRAS mutations, has been shown to require the molecular chaperone heat shock protein 90 (HSP90) for stabilization and is depleted by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)]. Here, we explore the possible relationship between tumor BRAF and NRAS mutations and clinical response to 17-AAG in six patients with metastatic malignant melanoma who received pharmacologically active doses of 17-AAG as part of a phase I clinical trial. One patient with disease stabilization for 49 months had a (G13D)NRAS mutation and (WT)BRAF. A second patient who had stable disease for 15 months had a (V600E)BRAF mutation and (WT)NRAS. These preliminary results suggest that BRAF and NRAS mutation status should be determined in prospective phase II studies of HSP90 inhibitors in melanoma..
Yap, T.A.
Garrett, M.D.
Walton, M.I.
Raynaud, F.
de Bono, J.S.
Workman, P.
(2008). Targeting the PI3K-AKT-mTOR pathway: progress, pitfalls, and promises. Curr opin pharmacol,
Vol.8
(4),
pp. 393-412.
show abstract
The strategy of 'drugging the cancer kinome' has led to the successful development and regulatory approval of several novel molecular targeted agents. The spotlight is now shifting to the phosphatidylinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway as a key potential target. This review details the role of the pathway in oncogenesis and the rationale for inhibiting its vital components. The focus will be on the progress made in the development of novel therapies for cancer treatment, with emphasis placed on agents that have entered clinical development. Strategies involving horizontal and vertical blockade of the pathway, as well as the use of biomarkers to select appropriate patients and to provide proof of target modulation will also be highlighted. Finally, we discuss the issues and limitations involved with targeting the PI3K-AKT-mTOR pathway, and predict what the future may hold for these novel anticancer therapeutics..
Workman, P.
de Bono, J.
(2008). Targeted therapeutics for cancer treatment: major progress towards personalised molecular medicine. Curr opin pharmacol,
Vol.8
(4),
pp. 359-362.
Powers, M.V.
Clarke, P.A.
Workman, P.
(2008). Dual targeting of HSC70 and HSP72 inhibits HSP90 function and induces tumor-specific apoptosis. Cancer cell,
Vol.14
(3),
pp. 250-262.
show abstract
Heat-shock protein 70 (HSP70) isoforms contribute to tumorigenesis through their well-documented antiapoptotic activity and via their role as cochaperones for the HSP90 molecular chaperone. HSP70 expression is induced following treatment with HSP90 inhibitors, which may attenuate the cell death effects of this class of inhibitor. Here we show that silencing either heat-shock cognate 70 (HSC70) or HSP72 expression in human cancer cell lines has no effect on HSP90 activity or cell proliferation. However, simultaneously reducing the expression of both of these isoforms induces proteasome-dependent degradation of HSP90 client proteins, G1 cell-cycle arrest, and extensive tumor-specific apoptosis. Importantly, simultaneous silencing of HSP70 isoforms in nontumorigenic cell lines does not result in comparable growth arrest or induction of apoptosis, indicating a potential therapeutic window..
Banerji, U.
Sain, N.
Sharp, S.Y.
Valenti, M.
Asad, Y.
Ruddle, R.
Raynaud, F.
Walton, M.
Eccles, S.A.
Judson, I.
Jackman, A.L.
Workman, P.
(2008). An in vitro and in vivo study of the combination of the heat shock protein inhibitor 17-allylamino-17-demethoxygeldanamycin and carboplatin in human ovarian cancer models. Cancer chemother pharmacol,
Vol.62
(5),
pp. 769-778.
show abstract
To study the interactions of the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) and carboplatin in vitro and in vivo..
Caldwell, J.J.
Davies, T.G.
Donald, A.
McHardy, T.
Rowlands, M.G.
Aherne, G.W.
Hunter, L.K.
Taylor, K.
Ruddle, R.
Raynaud, F.I.
Verdonk, M.
Workman, P.
Garrett, M.D.
Collins, I.
(2008). Identification of 4-(4-aminopiperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidines as selective inhibitors of protein kinase B through fragment elaboration. J med chem,
Vol.51
(7),
pp. 2147-2157.
show abstract
Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera..
Rayter, S.
Elliott, R.
Travers, J.
Rowlands, M.G.
Richardson, T.B.
Boxall, K.
Jones, K.
Linardopoulos, S.
Workman, P.
Aherne, W.
Lord, C.J.
Ashworth, A.
(2008). A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D. Oncogene,
Vol.27
(8),
pp. 1036-1044.
show abstract
The PPM1D gene is aberrantly amplified in a range of common cancers and encodes a protein phosphatase that is a potential therapeutic target. However, the issue of whether inhibition of PPM1D in human tumour cells that overexpress this protein compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of PPM1D can indeed reduce the viability of human tumour cells and that this effect is selective; tumour cell lines that overexpress PPM1D are sensitive to PPM1D inhibition whereas cell lines with normal levels are not. Loss of viability associated with PPM1D RNAi in human tumour cells occurs via the activation of the kinase P38. To identify chemical inhibitors of PPM1D, a high-throughput screening of a library of small molecules was performed. This strategy successfully identified a compound that selectively reduces viability of human tumour cell lines that overexpress PPM1D. As expected of a specific inhibitor, the toxicity to PPM1D overexpressing cell lines after inhibitor treatment is P38 dependent. These results further validate PPM1D as a therapeutic target and identify a proof-of-principle small molecule inhibitor..
Holmes, J.L.
Sharp, S.Y.
Hobbs, S.
Workman, P.
(2008). Silencing of HSP90 cochaperone AHA1 expression decreases client protein activation and increases cellular sensitivity to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin. Cancer res,
Vol.68
(4),
pp. 1188-1197.
show abstract
AHA1 (activator of HSP90 ATPase) is a cochaperone of the ATP-dependent molecular chaperone, HSP90, which is involved in the maturation, stabilization/degradation, and function of oncogenic proteins. HSP90 operates in a multimeric complex driven by the binding and hydrolysis of ATP. Treatment of cells with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) results in the degradation of client proteins via the ubiquitin-proteasome pathway. As AHA1 increases the ATPase activity of HSP90, we hypothesized that modulation of AHA1 expression could influence the activity of client proteins and/or the cellular response to 17-AAG. We show that the basal expression of AHA1 is different across a panel of human cancer cell lines, and that treatment with 17-AAG resulted in sustained AHA1 up-regulation. Increasing the expression of AHA1 did not affect the sensitivity to 17-AAG, but did increase C-RAF activity and the levels of phosphorylated MEK1/2 and ERK1/2 without affecting total levels of these proteins or of client proteins C-RAF, ERBB2, or CDK4. Conversely, small interfering RNA-selective knockdown of >80% of AHA1 expression decreased C-RAF activity and reduced the levels of MEK1/2 and ERK1/2 phosphorylation. Moreover, the AHA1 knockdown resulted in a significant (P < 0.05) increase in sensitivity to 17-AAG, due in part to a 2- to 3-fold increase in apoptosis. These results show that the reduction of AHA1 levels could decrease the phosphorylation of key signal transduction proteins, and for the first time, separate the activation and stabilization functions of HSP90. Furthermore, AHA1 knockdown could sensitize cancer cells to 17-AAG. We conclude that modulation of AHA1 might be a potential therapeutic strategy to increase sensitivity to HSP90 inhibitors..
Pearl, L.H.
Prodromou, C.
Workman, P.
(2008). The Hsp90 molecular chaperone: an open and shut case for treatment. Biochem j,
Vol.410
(3),
pp. 439-453.
show abstract
The molecular chaperone Hsp90 (90 kDa heat-shock protein) is a remarkably versatile protein involved in the stress response and in normal homoeostatic control mechanisms. It interacts with 'client proteins', including protein kinases, transcription factors and others, and either facilitates their stabilization and activation or directs them for proteasomal degradation. By this means, Hsp90 displays a multifaceted ability to influence signal transduction, chromatin remodelling and epigenetic regulation, development and morphological evolution. Hsp90 operates as a dimer in a conformational cycle driven by ATP binding and hydrolysis at the N-terminus. The cycle is also regulated by a group of co-chaperones and accessory proteins. Here we review the biology of the Hsp90 molecular chaperone, emphasizing recent progress in our understanding of structure-function relationships and the identification of new client proteins. In addition we describe the exciting progress that has been made in the development of Hsp90 inhibitors, which are now showing promise in the clinic for cancer treatment. We also identify the gaps in our current understanding and highlight important topics for future research..
Eccles, S.A.
Massey, A.
Raynaud, F.I.
Sharp, S.Y.
Box, G.
Valenti, M.
Patterson, L.
de Haven Brandon, A.
Gowan, S.
Boxall, F.
Aherne, W.
Rowlands, M.
Hayes, A.
Martins, V.
Urban, F.
Boxall, K.
Prodromou, C.
Pearl, L.
James, K.
Matthews, T.P.
Cheung, K.-.
Kalusa, A.
Jones, K.
McDonald, E.
Barril, X.
Brough, P.A.
Cansfield, J.E.
Dymock, B.
Drysdale, M.J.
Finch, H.
Howes, R.
Hubbard, R.E.
Surgenor, A.
Webb, P.
Wood, M.
Wright, L.
Workman, P.
(2008). NVP-AUY922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis. Cancer res,
Vol.68
(8),
pp. 2850-2860.
show abstract
We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K(d) = 1.7 nmol/L) and proliferation of human tumor cells with GI(50) values of approximately 2 to 40 nmol/L, inducing G(1)-G(2) arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI(50). This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1alpha, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials..
de Bono, J.S.
Kristeleit, R.
Tolcher, A.
Fong, P.
Pacey, S.
Karavasilis, V.
Mita, M.
Shaw, H.
Workman, P.
Kaye, S.
Rowinsky, E.K.
Aherne, W.
Atadja, P.
Scott, J.W.
Patnaik, A.
(2008). Phase I pharmacokinetic and pharmacodynamic study of LAQ824, a hydroxamate histone deacetylase inhibitor with a heat shock protein-90 inhibitory profile, in patients with advanced solid tumors. Clin cancer res,
Vol.14
(20),
pp. 6663-6673.
show abstract
To determine the safety, maximum tolerated dose, and pharmacokinetic-pharmacodynamic profile of a histone deacetylase inhibitor, LAQ824, in patients with advanced malignancy. Patients and Methods: LAQ824 was administered i.v. as a 3-h infusion on days 1, 2, and 3 every 21 days. Western blot assays of peripheral blood mononuclear cell lysates and tumor biopsies pretherapy and posttherapy evaluated target inhibition and effects on heat shock protein-90 (HSP90) client proteins and HSP72..
Banerji, U.
de Bono, J.
Judson, I.
Kaye, S.
Workman, P.
(2008). Biomarkers in early clinical trials: the committed and the skeptics. Clin cancer res,
Vol.14
(8),
p. 2512.
Brough, P.A.
Aherne, W.
Barril, X.
Borgognoni, J.
Boxall, K.
Cansfield, J.E.
Cheung, K.-.
Collins, I.
Davies, N.G.
Drysdale, M.J.
Dymock, B.
Eccles, S.A.
Finch, H.
Fink, A.
Hayes, A.
Howes, R.
Hubbard, R.E.
James, K.
Jordan, A.M.
Lockie, A.
Martins, V.
Massey, A.
Matthews, T.P.
McDonald, E.
Northfield, C.J.
Pearl, L.H.
Prodromou, C.
Ray, S.
Raynaud, F.I.
Roughley, S.D.
Sharp, S.Y.
Surgenor, A.
Walmsley, D.L.
Webb, P.
Wood, M.
Workman, P.
Wright, L.
(2008). 4,5-diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for the treatment of cancer. J med chem,
Vol.51
(2),
pp. 196-218.
show abstract
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%..
Vaughan, C.K.
Mollapour, M.
Smith, J.R.
Truman, A.
Hu, B.
Good, V.M.
Panaretou, B.
Neckers, L.
Clarke, P.A.
Workman, P.
Piper, P.W.
Prodromou, C.
Pearl, L.H.
(2008). Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37. Mol cell,
Vol.31
(6),
pp. 886-895.
show abstract
Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes..
Williamson, D.
Selfe, J.
Gordon, T.
Lu, Y.J.
Pritchard-Jones, K.
Murai, K.
Jones, P.
Workman, P.
Shipley, J.
(2007). Role for amplification and expression of glypican-5 in rhabdomyosarcoma. Cancer res,
Vol.67
(1),
pp. 57-65.
show abstract
Overexpression of genes, through genomic amplification and other mechanisms, can critically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that are primarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was defined as containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-17-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in all cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-17-92, in primary samples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the growth factors fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and altered the cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4 in Xenopus blastocysts was also enhanced. Our data are consistent with a role of GPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads to mesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeutic intervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene..
Sharp, S.Y.
Boxall, K.
Rowlands, M.
Prodromou, C.
Roe, S.M.
Maloney, A.
Powers, M.
Clarke, P.A.
Box, G.
Sanderson, S.
Patterson, L.
Matthews, T.P.
Cheung, K.M.
Ball, K.
Hayes, A.
Raynaud, F.
Marais, R.
Pearl, L.
Eccles, S.
Aherne, W.
McDonald, E.
Workman, P.
(2007). In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors. Cancer res,
Vol.67
(5),
pp. 2206-2216.
show abstract
The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development..
Maloney, A.
Clarke, P.A.
Naaby-Hansen, S.
Stein, R.
Koopman, J.O.
Akpan, A.
Yang, A.
Zvelebil, M.
Cramer, R.
Stimson, L.
Aherne, W.
Banerji, U.
Judson, I.
Sharp, S.
Powers, M.
deBilly, E.
Salmons, J.
Walton, M.
Burlingame, A.
Waterfield, M.
Workman, P.
(2007). Gene and protein expression profiling of human ovarian cancer cells treated with the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin. Cancer res,
Vol.67
(7),
pp. 3239-3253.
show abstract
The promising antitumor activity of 17-allylamino-17-demethoxygeldanamycin (17AAG) results from inhibition of the molecular chaperone heat shock protein 90 (HSP90) and subsequent degradation of multiple oncogenic client proteins. Gene expression microarray and proteomic analysis were used to profile molecular changes in the A2780 human ovarian cancer cell line treated with 17AAG. Comparison of results with an inactive analogue and an alternative HSP90 inhibitor radicicol indicated that increased expression of HSP72, HSC70, HSP27, HSP47, and HSP90beta at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were increased in tumor biopsies following treatment of patients with 17AAG. A group of MYC-regulated mRNAs was decreased by 17AAG. Of particular interest and novelty were changes in expression of chromatin-associated proteins. Expression of the heterochromatin protein 1 was increased, and expression of the histone acetyltransferase 1 and the histone arginine methyltransferase PRMT5 was decreased by 17AAG. PRMT5 was shown to be a novel HSP90-binding partner and potential client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction on cell proliferation with the histone deacetylase inhibitor trichostatin A. This mRNA and protein expression analysis has provided new insights into the complex molecular pharmacology of 17AAG and suggested new genes and proteins that may be involved in response to the drug or be potential biomarkers of drug action..
Davenport, E.L.
Moore, H.E.
Dunlop, A.S.
Sharp, S.Y.
Workman, P.
Morgan, G.J.
Davies, F.E.
(2007). Heat shock protein inhibition is associated with activation of the unfolded protein response pathway in myeloma plasma cells. Blood,
Vol.110
(7),
pp. 2641-2649.
show abstract
Plasma cells producing high levels of paraprotein are dependent on the unfolded protein response (UPR) and chaperone proteins to ensure correct protein folding and cell survival. We hypothesized that disrupting client-chaperone interactions using heat shock protein 90 (Hsp90) inhibitors would result in an inability to handle immunoglobulin production with the induction of the UPR and myeloma cell death. To study this, myeloma cells were treated with Hsp90 inhibitors as well as known endoplasmic reticulum stress inducers and proteasome inhibitors. Treatment with thapsigargin and tunicamycin led to the activation of all 3 branches of the UPR, with early splicing of XBP1 indicative of IRE1 activation, upregulation of CHOP consistent with ER resident kinase (PERK) activation, and activating transcription factor 6 (ATF6) splicing. 17-AAG and radicicol also induced splicing of XBP1, with the induction of CHOP and activation of ATF6, whereas bortezomib resulted in the induction of CHOP and activation of ATF6 with minimal effects on XBP1. After treatment with all drugs, expression levels of the molecular chaperones BiP and GRP94 were increased. All drugs inhibited proliferation and induced cell death with activation of JNK and caspase cleavage. In conclusion, Hsp90 inhibitors induce myeloma cell death at least in part via endoplasmic reticulum stress and the UPR death pathway..
Bavetsias, V.
Sun, C.
Bouloc, N.
Reynisson, J.
Workman, P.
Linardopoulos, S.
McDonald, E.
(2007). Hit generation and exploration: imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases. Bioorg med chem lett,
Vol.17
(23),
pp. 6567-6571.
show abstract
A hit generation and exploration approach led to the discovery of 31 (2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide), a potent, novel inhibitor of Aurora-A, Aurora-B and Aurora-C kinases with IC(50) values of 0.042, 0.198 and 0.227microM, respectively. Compound 31 inhibits cell proliferation and has good microsomal stability..
Hayakawa, M.
Kaizawa, H.
Kawaguchi, K.
Ishikawa, N.
Koizumi, T.
Ohishi, T.
Yamano, M.
Okada, M.
Ohta, M.
Tsukamoto, S.
Raynaud, F.I.
Waterfield, M.D.
Parker, P.
Workman, P.
(2007). Synthesis and biological evaluation of imidazo[1,2-a]pyridine derivatives as novel PI3 kinase p110alpha inhibitors. Bioorg med chem,
Vol.15
(1),
pp. 403-412.
show abstract
3-{1-[(4-Fluorophenyl)sulfonyl]-1H-pyrazol-3-yl}-2-methylimidazo[1,2-a]pyridine, 2a, was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 0.67microM, through screening in a scintillation proximity assay. Optimization of the substituents of 2a increased the p110alpha inhibitory activity by more than 300-fold (2g: IC(50)=0.0018microM). Further structural modification of 2g afforded thiazole derivative 12, which has potent p110alpha inhibitory activity (IC(50) of 0.0028microM) and is highly selective for p110alpha over other PI3K isoforms. Compound 12 also inhibited serum-induced cell proliferation of A375 and HeLa cells in vitro with IC(50) values of 0.14microM and 0.21microM, respectively, and suppressed tumor growth by 37% in a mouse HeLa xenograft model when dosed intraperitoneally at 25mg/kg. These results suggest that selective p110alpha inhibitors may have potential as cancer therapeutic agents..
Hayakawa, M.
Kaizawa, H.
Moritomo, H.
Koizumi, T.
Ohishi, T.
Yamano, M.
Okada, M.
Ohta, M.
Tsukamoto, S.
Raynaud, F.I.
Workman, P.
Waterfield, M.D.
Parker, P.
(2007). Synthesis and biological evaluation of pyrido[3',2':4,5]furo[3,2-d]pyrimidine derivatives as novel PI3 kinase p110alpha inhibitors. Bioorg med chem lett,
Vol.17
(9),
pp. 2438-2442.
show abstract
4-Morpholin-4-ylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine 2a was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 1.4 microM. By structural modification of 2a, the 2-aryl-4-morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidine derivative 10e was discovered as a p110alpha inhibitor with approximately 400-fold greater potency than 2a. Evaluation of isoform selectivity showed that 10e is a potent inhibitor of p110beta. Furthermore, 10e showed anti-proliferative activity in various cell lines, including multi-drug resistant MCF7/ADR-res cells, and was effective against HeLa human cervical tumor xenografts in nude mice..
Gaspar, N.
Sharp, S.
Vassal, G.
Pearson, A.
Workman, P.
(2007). Preclinical evaluation of HSP90 inhibitors in adult and paediatric glioblastomas. Pediatric blood & cancer,
Vol.49
(4),
pp. 451-1.
Hayakawa, M.
Kawaguchi, K.
Kaizawa, H.
Koizumi, T.
Ohishi, T.
Yamano, M.
Okada, M.
Ohta, M.
Tsukamoto, S.
Raynaud, F.I.
Parker, P.
Workman, P.
Waterfield, M.D.
(2007). Synthesis and biological evaluation of sulfonylhydrazone-substituted imidazo[1,2-a]pyridines as novel PI3 kinase p110alpha inhibitors. Bioorg med chem,
Vol.15
(17),
pp. 5837-5844.
show abstract
We have previously reported the imidazo[1,2-a]pyridine derivative 4 as a novel p110alpha inhibitor; however, although 4 is a potent inhibitor of p110alpha enzymatic activity and tumor cell proliferation in vitro, it is unstable in solution and ineffective in vivo. To increase stability the pyrazole of 4 was replaced with a hydrazone and a moderately potent p110alpha inhibitor 7a was obtained. Subsequent optimization of 7a afforded exceptionally potent p110alpha inhibitors, including 8c and 8h, with IC(50) values of 0.30 nM and 0.26 nM, respectively; to the best of our knowledge, these compounds are the most potent PI3K p110alpha inhibitors reported to date. Compound 8c was also stable in solution and exhibited significant anti-tumor effectiveness in vivo..
Clarke, P.A.
Guillard, S.
Di Stefano, F.
Poele, R.T.
Valenti, M.
Brandon, A.D.
Eccles, S.
Raynaud, F.
Workman, P.
(2007). Characterization of a potent inhibitor of class 1 phosphatidylinositide-3 '-kinases (PI3K) in human glioma. Molecular cancer therapeutics,
Vol.6
(12),
pp. 3414S-1.
Lyons, J.F.
Grimshaw, K.M.
Woodhead, S.J.
Feltell, R.E.
Reule, M.
Smyth, T.
Seavers, L.C.
Harada, I.
Higgins, J.
Smith, D.M.
Fazal, L.
Workman, P.
Collins, I.
Garrett, M.D.
Thompson, N.T.
(2007). AT13148, an orally bioavailable AKT kinase inhibitor with potent anti-tumor activity in both in vitro and in vivo models exhibiting AKT pathway deregulation. Molecular cancer therapeutics,
Vol.6
(12),
pp. 3512S-1.
Brough, P.
Massey, A.
Drysdale, M.
Barril, X.
Dymock, B.
Eccles, S.A.
Finch, H.
Hubbard, R.E.
McDonald, E.
Raynaud, F.
Sharp, S.Y.
Surgenor, A.
Wood, M.
Workman, P.
Wright, L.
(2007). NVP-AUY922/VER-52296: A novel 3,4 diarylisoxazole Hsp90 chaperone inhibitor discovered via structure-based drug design. Molecular cancer therapeutics,
Vol.6
(12),
pp. 3521S-1.
Eccles, S.A.
Sharp, S.Y.
Raynaud, F.I.
Valenti, M.
Patterson, L.
Gowan, S.
Boxall, K.
Aherne, W.
Rowlands, M.
Hayes, A.
Martins, V.
Urban, F.
Prodromou, C.
Pearl, L.
James, K.
Matthews, T.P.
Cheung, K.-.
Kalusa, A.
Jones, K.
McDonald, E.
Brough, P.A.
Massey, A.
Dymock, B.
Drysdale, M.
Workman, P.
(2007). NVP-AUY922, a novel diarylisoxazole resorcinol HSP90 inhibitor, potently inhibits growth and metastasis of human tumor xenografts. Molecular cancer therapeutics,
Vol.6
(12),
pp. 3522S-2.
Raynaud, F.I.
Hayes, A.
Martins, V.
Smith, N.F.
Sharp, S.Y.
Valenti, M.
Henley, A.
Matthews, T.P.
Cheung, K.-.
Kalusa, A.
Jones, K.
James, K.
McDonald, E.
Brough, P.A.
Massey, A.
Dymock, B.
Drysdale, M.
Eccles, S.A.
Workman, P.
(2007). Cassette dosing in tumor bearing animals for the discovery of NVP-AUY922, a novel HSP90 inhibitor. Molecular cancer therapeutics,
Vol.6
(12),
pp. 3524S-1.
Baird, R.
Planting, A.
Reid, A.
Kitzen, J.
Reade, S.
Clarke, P.
Welsh, L.
Lazaro, L.L.
Heras, B.D.
Judson, I.
Pico, C.
Workman, P.
Eskens, F.
Kaye, S.
de Bono, J.
Verweij, J.
(2007). Phase I safety, pharmacokinetic, and pharmacogenomic trial of ES-285, a novel marine cytotoxic agent administered as an infusion over 24 h every 21 days in patients with solid tumors. Molecular cancer therapeutics,
Vol.6
(12),
pp. 3381S-1.
Smith, N.F.
Raynaud, F.I.
Workman, P.
(2007). The application of cassette dosing for pharmacokinetic screening in small-molecule cancer drug discovery. Mol cancer ther,
Vol.6
(2),
pp. 428-440.
show abstract
Pharmacokinetic evaluation is an essential component of drug discovery and should be conducted early in the process so that those compounds with the best chance of success are prioritized and progressed. However, pharmacokinetic analysis has become a serious bottleneck during the 'hit-to-lead' and lead optimization phases due to the availability of new targets and the large numbers of compounds resulting from advances in synthesis and screening technologies. Cassette dosing, which involves the simultaneous administration of several compounds to a single animal followed by rapid sample analysis by liquid chromatography/tandem mass spectrometry, was developed to increase the throughput of in vivo pharmacokinetic screening. Although cassette dosing is advantageous in terms of resources and throughput, there are possible complications associated with this approach, such as the potential for compound interactions. Following an overview of the cassette dosing literature, this article focuses on the application of the technique in anticancer drug discovery. Specific examples are discussed, including the evaluation of cassette dosing to assess pharmacokinetic properties in the development of cyclin-dependent kinase and heat shock protein 90 inhibitors. Subject to critical analysis and validation in each case, the use of cassette dosing is recommended in appropriate chemical series to enhance the efficiency of drug discovery and reduce animal usage..
Hardcastle, A.
Tomlin, P.
Norris, C.
Richards, J.
Cordwell, M.
Boxall, K.
Rowlands, M.
Jones, K.
Collins, I.
McDonald, E.
Workman, P.
Aherne, W.
(2007). A duplexed phenotypic screen for the simultaneous detection of inhibitors of the molecular chaperone heat shock protein 90 and modulators of cellular acetylation. Mol cancer ther,
Vol.6
(3),
pp. 1112-1122.
show abstract
Histone deacetylases (HDACs), histone acetyltransferases (HATs), and the molecular chaperone heat shock protein 90 (HSP90) are attractive anticancer drug targets. High-throughput screening plays a pivotal role in modern molecular mechanism-based drug discovery. Cell-based screens are particularly useful in that they identify compounds that are permeable and active against the selected target or pathway in a cellular context. We have previously developed time-resolved fluorescence cell immunosorbent assays (TRF-Cellisas) for compound screening and pharmacodynamic studies. These assays use a primary antibody to the single protein of interest and a matched secondary immunoglobulin labeled with an europium chelate (Eu). The availability of species-specific secondary antibodies labeled with different lanthanide chelates provides the potential for multiplexing this type of assay. The approach has been applied to the development of a 384-well duplexed cell-based screen to simultaneously detect compounds that induce the co-chaperone HSP70 as a molecular marker of potential inhibitors of HSP90 together with those that modulate cellular acetylation (i.e., potential inhibitors of histone deacetylase or histone acetyltransferase activity). The duplexed assay proved reliable in high-throughput format and approximately 64,000 compounds were screened. Following evaluation in secondary assays, 3 of 13 hits from the HSP70 arm were confirmed. Two of these directly inhibited the intrinsic ATPase activity of HSP90 whereas the third seems to have a different mechanism of action. In the acetylation arm, two compounds increased cellular acetylation, one of which inhibited histone deacetylase activity. A third compound decreased cellular histone acetylation, potentially through a novel mechanism of action..
Sharp, S.Y.
Prodromou, C.
Boxall, K.
Powers, M.V.
Holmes, J.L.
Box, G.
Matthews, T.P.
Cheung, K.M.
Kalusa, A.
James, K.
Hayes, A.
Hardcastle, A.
Dymock, B.
Brough, P.A.
Barril, X.
Cansfield, J.E.
Wright, L.
Surgenor, A.
Foloppe, N.
Hubbard, R.E.
Aherne, W.
Pearl, L.
Jones, K.
McDonald, E.
Raynaud, F.
Eccles, S.
Drysdale, M.
Workman, P.
(2007). Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues. Mol cancer ther,
Vol.6
(4),
pp. 1198-1211.
show abstract
Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors..
Gowan, S.M.
Hardcastle, A.
Hallsworth, A.E.
Valenti, M.R.
Hunter, L.J.
de Haven Brandon, A.K.
Garrett, M.D.
Raynaud, F.
Workman, P.
Aherne, W.
Eccles, S.A.
(2007). Application of meso scale technology for the measurement of phosphoproteins in human tumor xenografts. Assay drug dev technol,
Vol.5
(3),
pp. 391-401.
show abstract
In this age of molecularly targeted drug discovery, robust techniques are required to measure pharmacodynamic (PD) responses in tumors so that drug exposures can be associated with their effects on molecular biomarkers and efficacy. Our aim was to develop a rapid screen to monitor PD responses within xenografted human tumors as an important step towards a clinically applicable technology. Currently there are various methods available to measure PD end points, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction, gene expression profiling, and western blotting. These may require relatively large samples of tumor, surrogate tissue, or peripheral blood lymphocytes with subsequent analyses taking several days. The phosphoinositide 3-kinase (PI3-kinase) pathway is frequently deregulated in cancer and is also important in diabetes and autoimmune conditions. In this paper, optimization of the Meso Scale Discovery (MSD) (Gaithersburg, MD) platform to quantify changes in phospho-AKT and phospho-glycogen synthase kinase-3beta in response to a PI3-kinase inhibitor, LY294002, is described, initially in vitro and then within xenografted solid tumors. This method is highly practical with high throughput since large number of samples can be run simultaneously in 96-well format. The assays are robust (coefficient of variation for phospho-AKT 13.4%) and offer significant advantages (in terms of speed and quantitation) over western blots. This optimized procedure can be used for both in vitro and in vivo analysis, unlike an established fixed-cell ELISA with a time-resolved fluorescent end point..
Workman, P.
(2007). Drugging the cancer genome: New challenges of infrequent and combinatorial targets. Curr opin invest dr,
Vol.8
(6),
pp. 445-446.
Workman, P.
Powers, M.V.
(2007). Chaperoning cell death: a critical dual role for Hsp90 in small-cell lung cancer. Nat chem biol,
Vol.3
(8),
pp. 455-457.
show abstract
Chemical probes reveal Hsp90 to be a key molecule for the control of apoptosis in small-cell lung cancer - with important implications for Hsp90 biology and cancer treatment..
Workman, P.
Burrows, F.
Neckers, L.
Rosen, N.
(2007). Drugging the cancer chaperone HSP90: combinatorial therapeutic exploitation of oncogene addiction and tumor stress. Ann n y acad sci,
Vol.1113,
pp. 202-216.
show abstract
The molecular chaperone HSP90 has emerged as an exciting target for cancer treatment. We review the potential advantages of HSP90 inhibitors, particularly the simultaneous combinatorial depletion of multiple oncogenic "client" proteins, leading to blockade of many cancer-causing pathways and the antagonism of all of the hallmark pathological traits of malignancy. Cancer selectivity is achieved by exploiting cancer "dependencies," including oncogene addiction and the stressed state of malignant cells. The multiple downstream effects of HSP90 inhibitors should make the development of resistance more difficult than with agents having more restricted effects. We review the various classes of HSP90 inhibitor that have been developed, including the natural products geldanamycin and radicicol and also the purine scaffold and pyrazole/isoxazole class of synthetic small molecule inhibitors. A first-in-class HSP90 drug, the geldanamycin analog 17-AAG, has provided proof of concept for HSP90 inhibition in patients at well tolerated doses and therapeutic activity has been seen. Other inhibitors show promise in preclinical and clinical development. Opportunities and challenges for HSP90 inhibitors are discussed, including use in combination with other agents. Most of the current HSP90 inhibitors act by blocking the essential nucleotide binding and ATPase activity required for chaperone function. Potential new approaches are discussed, for example, interference with cochaperone binding and function in the superchaperone complex. Biomarkers for use with HSP90 inhibitors are described. We stress how basic and translational research has been mutually beneficial and indicate future directions to enhance our understanding of molecular chaperones and their exploitation in cancer and other diseases..
Sarker, D.
Pacey, S.
Workman, P.
(2007). Use of pharmacokinetic/pharmacodynamic biomarkers to support rational cancer drug development. Biomark med,
Vol.1
(3),
pp. 399-417.
show abstract
The process of drug development in oncology has struggled to alter at a pace in keeping with the rapid discovery and testing of agents that act on a wide variety of molecular targets. The rational development of such agents requires an understanding of drug effect(s) on their purported target. It is likely that testing these drugs in a framework designed to examine cytotoxic agents will fail to establish their full potential. We discuss how data gained from biomarker investigation might impact on drug development and provide examples that highlight the development, validation and use of pharmacokinetic, and especially pharmacodynamic biomarkers as drug development moves from the laboratory into clinical testing. The challenges of performing assays to satisfy regulatory requirements have been the subject of much debate. We recommend the implementation of appropriate, fit-for-purpose biomarkers in clinical trials of all new cancer drugs..
Hockley, S.L.
Arlt, V.M.
Brewer, D.
Te Poele, R.
Workman, P.
Giddings, I.
Phillips, D.H.
(2007). AHR- and DNA-damage-mediated gene expression responses induced by benzo(a)pyrene in human cell lines. Chem res toxicol,
Vol.20
(12),
pp. 1797-1810.
show abstract
Carcinogens induce complex transcriptional responses in cells that may hold key mechanistic information. Benzo(a)pyrene (BaP) modulation of transcription may occur through the activation of the aryl hydrocarbon receptor (AHR) or through responses to DNA damage. To characterize further the expression profiles induced by BaP in HepG2 and MCF-7 cells obtained in our previous study, they were compared to those induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which activates AHR but does not bind to DNA, and anti-benzo(a)pyrene- trans-7,8-dihydrodiol-9,10-epoxide (BPDE), which binds directly to DNA but does not activate AHR. A total of 22 genes had altered expression in MCF-7 cells after both BaP and TCDD exposure, and a total of 29 genes had altered expression in HepG2 cells. In both cell lines, xenobiotic metabolism was upregulated through induction of NQO1, MGST1, and CYP1B1. A total of 78 expression changes were induced by both BaP and BPDE in MCF-7 cells, and a total of 29 expression changes were induced by both BaP and BPDE in HepG2 cells. These genes were predominantly involved in cell cycle regulation, apoptosis, and DNA repair. BaP and BPDE caused the repression of histone genes in both cell lines, suggesting that regulation of these genes is an important component of the DNA damage response. Interestingly, overlap of the BPDE and TCDD gene expression profiles was also observed. Furthermore, some genes were modulated by BaP but not by TCDD or BPDE, including induction of CRY1 and MAK, which may represent novel signaling pathways that are independent of both AHR activation and DNA damage. Promoter analysis identified candidate genes for direct transcriptional regulation by either AHR or p53. These analyses have further dissected and characterized the complex cellular response to BaP..
Chan, F.
Sun, C.
Perumal, M.
Nguyen, Q.D.
Bavetsias, V.
McDonald, E.
Martins, V.
Wilsher, N.E.
Raynaud, F.I.
Valenti, M.
Eccles, S.
Te Poele, R.
Workman, P.
Aboagye, E.O.
Linardopoulos, S.
(2007). Mechanism of action of the Aurora kinase inhibitor CCT129202 and in vivo quantification of biological activity. Mol cancer ther,
Vol.6
(12 Pt 1),
pp. 3147-3157.
show abstract
The Aurora family of serine/threonine kinases is important for the regulation of centrosome maturation, chromosome segregation, and cytokinesis during mitosis. Overexpression of Aurora kinases in mammalian cells leads to genetic instability and transformation. Increased levels of Aurora kinases have also been linked to a broad range of human tumors. Here, we describe the properties of CCT129202, a representative of a structurally novel series of imidazopyridine small-molecule inhibitors of Aurora kinase activity. This compound showed high selectivity for the Aurora kinases over a panel of other kinases tested and inhibits proliferation in multiple cultured human tumor cell lines. CCT129202 causes the accumulation of human tumor cells with >or=4N DNA content, leading to apoptosis. CCT120202-treated human tumor cells showed a delay in mitosis, abrogation of nocodazole-induced mitotic arrest, and spindle defects. Growth of HCT116 xenografts in nude mice was inhibited after i.p. administration of CCT129202. We show that p21, the cyclin-dependent kinase inhibitor, is induced by CCT129202. Up-regulation of p21 by CCT129202 in HCT116 cells led to Rb hypophosphorylation and E2F inhibition, contributing to a decrease in thymidine kinase 1 transcription. This has facilitated the use of 3'-deoxy-3'[(18)F]fluorothymidine-positron emission tomography to measure noninvasively the biological activity of the Aurora kinase inhibitor CCT129202 in vivo..
Workman, P.
de Billy, E.
(2007). Putting the heat on cancer. Nat med,
Vol.13
(12),
pp. 1415-1417.
Benson, C.
White, J.
De Bono, J.
O'Donnell, A.
Raynaud, F.
Cruickshank, C.
McGrath, H.
Walton, M.
Workman, P.
Kaye, S.
Cassidy, J.
Gianella-Borradori, A.
Judson, I.
Twelves, C.
(2007). A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days. Br j cancer,
Vol.96
(1),
pp. 29-37.
show abstract
full text
Seliciclib (CYC202; R-roscovitine) is the first selective, orally bioavailable inhibitor of cyclin-dependent kinases 1, 2, 7 and 9 to enter clinical trial. Preclinical studies showed antitumour activity in a broad range of human tumour xenografts. A phase I trial was performed with a 7-day b.i.d. p.o. schedule. Twenty-one patients (median age 62 years, range: 39-73 years) were treated with doses of 100, 200 and 800 b.i.d. Dose-limiting toxicities were seen at 800 mg b.i.d.; grade 3 fatigue, grade 3 skin rash, grade 3 hyponatraemia and grade 4 hypokalaemia. Other toxicities included reversible raised creatinine (grade 2), reversible grade 3 abnormal liver function and grade 2 emesis. An 800 mg portion was investigated further in 12 patients, three of whom had MAG3 renograms. One patient with a rapid increase in creatinine on day 3 had a reversible fall in renal perfusion, with full recovery by day 14, and no changes suggestive of renal tubular damage. Further dose escalation was precluded by hypokalaemia. Seliciclib reached peak plasma concentrations between 1 and 4 h and elimination half-life was 2-5 h. Inhibition of retinoblastoma protein phosphorylation was not demonstrated in peripheral blood mononuclear cells. No objective tumour responses were noted, but disease stabilisation was recorded in eight patients; this lasted for a total of six courses (18 weeks) in a patient with ovarian cancer..
Raynaud, F.I.
Eccles, S.
Clarke, P.A.
Hayes, A.
Nutley, B.
Alix, S.
Henley, A.
Di-Stefano, F.
Ahmad, Z.
Guillard, S.
Bjerke, L.M.
Kelland, L.
Valenti, M.
Patterson, L.
Gowan, S.
de Haven Brandon, A.
Hayakawa, M.
Kaizawa, H.
Koizumi, T.
Ohishi, T.
Patel, S.
Saghir, N.
Parker, P.
Waterfield, M.
Workman, P.
(2007). Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases. Cancer res,
Vol.67
(12),
pp. 5840-5850.
show abstract
Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic..
Whittaker, S.R.
Te Poele, R.H.
Chan, F.
Linardopoulos, S.
Walton, M.I.
Garrett, M.D.
Workman, P.
(2007). The cyclin-dependent kinase inhibitor seliciclib (R-roscovitine; CYC202) decreases the expression of mitotic control genes and prevents entry into mitosis. Cell cycle,
Vol.6
(24),
pp. 3114-3131.
show abstract
The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G(2)/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1 alpha. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials..
Davies, T.G.
Verdonk, M.L.
Graham, B.
Saalau-Bethell, S.
Hamlett, C.C.
McHardy, T.
Collins, I.
Garrett, M.D.
Workman, P.
Woodhead, S.J.
Jhoti, H.
Barford, D.
(2007). A structural comparison of inhibitor binding to PKB, PKA and PKA-PKB chimera. J mol biol,
Vol.367
(3),
pp. 882-894.
show abstract
Although the crystal structure of the anti-cancer target protein kinase B (PKBbeta/Akt-2) has been useful in guiding inhibitor design, the closely related kinase PKA has generally been used as a structural mimic due to its facile crystallization with a range of ligands. The use of PKB-inhibitor crystallography would bring important benefits, including a more rigorous understanding of factors dictating PKA/PKB selectivity, and the opportunity to validate the utility of PKA-based surrogates. We present a "back-soaking" method for obtaining PKBbeta-ligand crystal structures, and provide a structural comparison of inhibitor binding to PKB, PKA, and PKA-PKB chimera. One inhibitor presented here exhibits no PKB/PKA selectivity, and the compound adopts a similar binding mode in all three systems. By contrast, the PKB-selective inhibitor A-443654 adopts a conformation in PKB and PKA-PKB that differs from that with PKA. We provide a structural explanation for this difference, and highlight the ability of PKA-PKB to mimic the true PKB binding mode in this case..
Powers, M.V.
Workman, P.
(2007). Inhibitors of the heat shock response: biology and pharmacology. Febs lett,
Vol.581
(19),
pp. 3758-3769.
show abstract
A number of human diseases can be linked to aberrations in protein folding which cause an imbalance in protein homeostasis. Molecular chaperones, including heat shock proteins, act to assist protein folding, stability and activity in the cell. Attention has begun to focus on modulating the expression and/or activity of this group of proteins for the treatment of a wide variety of human diseases. This review will describe the progress made to date in developing pharmacological modulators of the heat shock response, including both agents which affect the entire heat shock response and those that specifically target the HSP70 and HSP90 chaperone families..
Sarker, D.
Workman, P.
(2007). Pharmacodynamic biomarkers for molecular cancer therapeutics. Adv cancer res,
Vol.96,
pp. 213-268.
show abstract
Rational and efficient development of new molecular cancer therapeutics requires discovery, validation, and implementation of informative biomarkers. Measurement of molecular target status, pharmacokinetic (PK) parameters of drug exposure, and pharmacodynamic (PD) endpoints of drug effects on target, pathway, and downstream biological processes are extremely important. These can be linked to therapeutic effects in what we term a "pharmacological audit trail." Using biomarkers in preclinical drug discovery and development facilitates optimization of PK, PD, and therapeutic properties so that the best agent is selected for clinical evaluation. Applying biomarkers in early clinical trials helps identify the most appropriate patients; provides proof of concept for target modulation; helps test the underlying hypothesis; informs the rational selection of dose and schedule; aids decision making, including key go/no go questions; and may explain or predict clinical outcomes. Despite many successes such as trastuzumab and imatinib, exemplifying the value of targeting specific cancer defects, only 5% of oncology drugs that enter the clinic make it to marketing approval. Use of biomarkers should reduce this high level of attrition and bring forward key decisions (e.g., "fail fast"), thereby reducing the spiraling costs of drug development and increasing the likelihood of getting innovative and active drugs to cancer patients. In this chapter, we focus primarily on PD endpoints that demonstrate target modulation, including both invasive molecular assays and functional imaging technology. We also discuss related clinical trial design issues. Implementation of biomarkers in trials remains disappointingly low and we emphasize the need for greater cooperation between various stakeholders to improve this..
McErlean, C.S.
Proisy, N.
Davis, C.J.
Boland, N.A.
Sharp, S.Y.
Boxall, K.
Slawin, A.M.
Workman, P.
Moody, C.J.
(2007). Synthetic ansamycins prepared by a ring-expanding Claisen rearrangement Synthesis and biological evaluation of ring and conformational analogues of the Hsp90 molecular chaperone inhibitor geldanamycin. Org biomol chem,
Vol.5
(3),
pp. 531-546.
show abstract
A series of ansa-quinones has been prepared by chemical synthesis, and evaluated by biological techniques. Thus, 19-membered ansa-lactams, simplified analogues of the naturally occurring Hsp90 molecular chaperone inhibitor geldanamycin, were obtained by concise routes, the key steps being the combination of a ring-closing metathesis to give a 17-membered ring followed by Claisen rearrangement to effect ring expansion. The methodology was also used to prepare an "unnatural" 18-membered ring analogue. In ATPase enzyme assays, the synthetic ansa-quinones were weak inhibitors of Hsp90..
Garrett, M.D.
Raynaud, F.
Workman, P.
Aherne, W.
Eccles, S.A.
(2007). Application of meso scale technology for the measurement of phosphoproteins in human tumour xenografts. Assay drug development technology,
Vol.5,
pp. 391-401.
Smith, J.R.
Workman, P.
(2007). Targeting the cancer chaperone HSP90. Drug discovery oday: therapeutic strategies,
Vol.4,
pp. 219-227.
Al-Saffar, N.M.
Troy, H.
RamÃrez de Molina, A.
Jackson, L.E.
Madhu, B.
Griffiths, J.R.
Leach, M.O.
Workman, P.
Lacal, J.C.
Judson, I.R.
Chung, Y.L.
(2006). Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of the choline kinase inhibitor MN58b in human carcinoma models. Cancer res,
Vol.66
(1),
pp. 427-434.
show abstract
MN58b is a novel anticancer drug that inhibits choline kinase, resulting in inhibition of phosphocholine synthesis. The aim of this work was to develop a noninvasive and robust pharmacodynamic biomarker for target inhibition and, potentially, tumor response following MN58b treatment. Human HT29 (colon) and MDA-MB-231 (breast) carcinoma cells were examined by proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment with MN58b both in culture and in xenografts. An in vitro time course study of MN58b treatment was also carried out in MDA-MB-231 cells. In addition, enzymatic assays of choline kinase activity in cells were done. A decrease in phosphocholine and total choline levels (P < 0.05) was observed in vitro in both cell lines after MN58b treatment, whereas the inactive analogue ACG20b had no effect. In MDA-MB-231 cells, phosphocholine fell significantly as early as 4 hours following MN58b treatment, whereas a drop in cell number was observed at 48 hours. Significant correlation was also found between phosphocholine levels (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008) following MN58b treatment. Phosphomonoesters also decreased significantly (P < 0.05) in both HT29 and MDA-MB-231 xenografts with no significant changes in controls. 31P-MRS and 1H-MRS of tumor extracts showed a significant decrease in phosphocholine (P < or = 0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors..
Beloueche-Babari, M.
Jackson, L.E.
Al-Saffar, N.M.
Eccles, S.A.
Raynaud, F.I.
Workman, P.
Leach, M.O.
Ronen, S.M.
(2006). Identification of magnetic resonance detectable metabolic changes associated with inhibition of phosphoinositide 3-kinase signaling in human breast cancer cells. Mol cancer ther,
Vol.5
(1),
pp. 187-196.
show abstract
Phosphoinositide 3-kinase (PI3K) is an attractive target for novel mechanism-based anticancer treatment. We used magnetic resonance (MR) spectroscopy (MRS) to detect biomarkers of PI3K signaling inhibition in human breast cancer cells. MDA-MB-231, MCF-7, and Hs578T cells were treated with the prototype PI3K inhibitor LY294002, and the (31)P MR spectra of cell extracts were monitored. In every case, LY294002 treatment was associated with a significant decrease in phosphocholine levels by up to 2-fold (P < 0.05). In addition, a significant increase in glycerophosphocholine levels by up to 5-fold was also observed (P
Barril, X.
Beswick, M.C.
Collier, A.
Drysdale, M.J.
Dymock, B.W.
Fink, A.
Grant, K.
Howes, R.
Jordan, A.M.
Massey, A.
Surgenor, A.
Wayne, J.
Workman, P.
Wright, L.
(2006). 4-amino derivatives of the Hsp90 inhibitor CCT018159. Bioorganic & medicinal chemistry letters,
Vol.16
(9),
pp. 2543-6.
Leyton, J.
Alao, J.P.
Da Costa, M.
Stavropoulou, A.V.
Latigo, J.R.
Perumal, M.
Pillai, R.
He, Q.
Atadja, P.
Lam, E.W.
Workman, P.
Vigushin, D.M.
Aboagye, E.O.
(2006). In vivo biological activity of the histone deacetylase inhibitor LA0824 is detectable with 3 '-deoxy-3 '-[F-18]fluorothymidine positron emission tomography. Cancer research,
Vol.66
(15),
pp. 7621-9.
Workman, P.
(2006). Strategies to transfer success in the development of molecularly targeted therapy to the challenge of paediatric cancers. Ejc supplements,
Vol.4
(12),
pp. 77-1.
Bhattacharya, B.
Krishnan, B.
Kaye, S.
Ormerod, M.
Workman, P.
Jackman, A.
(2006). The phosphatidylinositide 3-kinase (PI3-kinase) inhibitor PI103 sensitises some ovarian carcinoma (OC) cell lines to paclitaxel or carboplatin. Ejc supplements,
Vol.4
(12),
pp. 80-2.
Stimson, L.
Hardcastle, A.
Fong, P.
Gowan, S.
Drummond, A.
Davidson, A.
Moffat, D.
Workman, P.
Aherne, W.
(2006). Downstream mechanistic markers of HDAC inhibition for drug discovery and clinical evaluation. Ejc supplements,
Vol.4
(12),
pp. 116-1.
Francis, N.-.
Rowlands, M.
Travers, J.
Reynissen, J.
Hardcastle, A.
Stimson, L.
Gamblin, S.
Jones, K.
Workman, P.
Aherne, W.
(2006). A high throughput screen and secondary assays for the identification and evaluation of histone methyltransferase inhibitors. Ejc supplements,
Vol.4
(12),
pp. 130-1.
Beloueche-Babari, M.
Jackson, L.E.
Workman, P.
Leach, M.O.
(2006). Inhibition of MEK1/2 signalling with CI-1040 in human melanoma cells leads to alterations in phosphocholine metabolism. Ejc supplements,
Vol.4
(12),
pp. 170-2.
Raynaud, F.
Eccles, S.
Clarke, P.
Hayes, A.
Di Stefano, F.
Ahmad, Z.
Guillard, S.
Patel, S.
Workman, P.
(2006). In vitro and in vivo pharmacological properties of the potent phosphatidylinositol 3-kinase (PI3K) family inhibitor PI103. Ejc supplements,
Vol.4
(12),
pp. 172-1.
Holmes, J.
Sharp, S.
Workman, P.
(2006). Modulation of the HSP90 co-chaperone AHA1 affects client protein activity and increases cellular sensitivity to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Ejc supplements,
Vol.4
(12),
pp. 183-1.
Honma, M.
Stubbs, M.
Collins, I.
Workman, P.
Aherne, W.
Watt, F.M.
(2006). Identification of novel keratinocyte differentiation modulating compounds by high-throughput screening. Journal of biomolecular screening,
Vol.11
(8),
pp. 977-8.
Collins, I.
Workman, P.
(2006). Design and development of signal transduction inhibitors for cancer treatment: Experience and challenges with kinase targets. Current signal transduction therapy,
Vol.1
(1),
pp. 13-11.
Sanderson, S.
Valenti, M.
Gowan, S.
Patterson, L.
Ahmad, Z.
Workman, P.
Eccles, S.A.
(2006). Benzoquinone ansamycin heat shock protein 90 inhibitors modulate multiple functions required for tumor angiogenesis. Mol cancer ther,
Vol.5
(3),
pp. 522-532.
show abstract
Heat shock protein 90 (Hsp90) is a molecular chaperone involved in maintaining the correct conformation and stability of its client proteins. This study investigated the effects of Hsp90 inhibitors on client protein expression and key cellular functions required for tumor angiogenesis. The benzoquinone ansamycin Hsp90 inhibitors geldanamycin and/or its derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin inhibited production of vascular endothelial growth factor (VEGF)-A by tumor cells and blocked proliferative responses of human endothelial cells at nanomolar concentrations. 17-AAG also significantly reduced endothelial cell migration, tubular differentiation, invasion through Matrigel, and secretion of urokinase-type plasminogen activator at concentrations at or below those that inhibited proliferation. 17-AAG significantly reduced expression of VEGF receptor (VEGFR)-2 and established Hsp90 client proteins in human endothelial cells in vitro as well as in mouse vena cava, mesenteric vessels, and blood vessels within human tumor xenografts in vivo; this was associated with decreased tumor microvessel density. Finally, we showed for the first time that Hsp90 inhibitors also reduce expression of VEGFR-1 on human vascular endothelial cells, VEGFR-3 on lymphatic endothelial cells in vitro, and all three VEGFRs on mouse vasculature in vivo. Thus, we identify Hsp90 inhibitors as important regulators of many aspects of tumor angiogenesis (and potentially lymphangiogenesis) and suggest that they may provide therapeutic benefit not only via direct effects on tumor cells but also indirectly by inhibiting the production of angiogenic cytokines and responses of activated endothelial cells that contribute to tumor progression and metastasis..
Workman, P.
Aboagye, E.O.
Chung, Y.-.
Griffiths, J.R.
Hart, R.
Leach, M.O.
Maxwell, R.J.
McSheehy, P.M.
Price, P.M.
Zweit, J.
Cancer Research UK Pharmacodynamic/Pharmacokinetic Technologies Advisory Committee,
(2006). Minimally invasive pharmacokinetic and pharmacodynamic technologies in hypothesis-testing clinical trials of innovative therapies. J natl cancer inst,
Vol.98
(9),
pp. 580-598.
show abstract
Clinical trials of new cancer drugs should ideally include measurements of parameters such as molecular target expression, pharmacokinetic (PK) behavior, and pharmacodynamic (PD) endpoints that can be linked to measures of clinical effect. Appropriate PK/PD biomarkers facilitate proof-of-concept demonstrations for target modulation; enhance the rational selection of an optimal drug dose and schedule; aid decision-making, such as whether to continue or close a drug development project; and may explain or predict clinical outcomes. In addition, measurement of PK/PD biomarkers can minimize uncertainty associated with predicting drug safety and efficacy, reduce the high levels of drug attrition during development, accelerate drug approval, and decrease the overall costs of drug development. However, there are many challenges in the development and implementation of biomarkers that probably explain their disappointingly low implementation in phase I trials. The Pharmacodynamic/Pharmacokinetic Technologies Advisory committee of Cancer Research UK has found that submissions for phase I trials of new cancer drugs in the United Kingdom often lack detailed information about PK and/or PD endpoints, which leads to suboptimal information being obtained in those trials or to delays in starting the trials while PK/PD methods are developed and validated. Minimally invasive PK/PD technologies have logistic and ethical advantages over more invasive technologies. Here we review these technologies, emphasizing magnetic resonance spectroscopy and positron emission tomography, which provide detailed functional and metabolic information. Assays that measure effects of drugs on important biologic pathways and processes are likely to be more cost-effective than those that measure specific molecular targets. Development, validation, and implementation of minimally invasive PK/PD methods are encouraged..
Newbatt, Y.
Burns, S.
Hayward, R.
Whittaker, S.
Kirk, R.
Marshall, C.
Springer, C.
McDonald, E.
Marais, R.
Workman, P.
Aherne, W.
(2006). Identification of inhibitors of the kinase activity of oncogenic (V600E)BRAF in an enzyme cascade high-throughput screen. J biomol screen,
Vol.11
(2),
pp. 145-154.
show abstract
The Cancer Genome Project has identified several oncogenic mutations in BRAT that represent important opportunities for cancer drug discovery. The (V600E)BRAF mutation accounts for approximately 90% of the mutations identified. A strong case has emerged from molecular, cellular, and structural studies for the identification and development of inhibitors of this mutated BRAF protein. The authors have developed and run a high-throughput screen to find inhibitors of (V600E)BRAF using an enzyme cascade assay in which oncogenic BRAF activates MEK1, which in turn activates ERK2, which then phosphorylates the transcription factor ELK1. A phosphospecific antibody, Europium-labeled secondary antibody, and a time-resolved fluorescent readout were used to measure phosphorylation of ELK1. Overall assay variation was 12.4%. The assay was used to screen 64,000 compounds with an overall Z' factor of 0.58 +/- 0.12. A series of 3,5,di-substituted pyridines were identified as inhibitors of the cascade assay. These compounds did not inhibit a shortened activated MEK1 to ELK1 cascade but were active (0.5-27.9 mu M) in a (V600E)BRAF assay and represent a potential starting point for future drug discovery and development..
Sain, N.
Krishnan, B.
Ormerod, M.G.
De Rienzo, A.
Liu, W.M.
Kaye, S.B.
Workman, P.
Jackman, A.L.
(2006). Potentiation of paclitaxel activity by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin in human ovarian carcinoma cell lines with high levels of activated AKT. Mol cancer ther,
Vol.5
(5),
pp. 1197-1208.
show abstract
Activation of the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway is a mechanism of cytotoxic drug resistance in ovarian cancer, and inhibitors of this pathway can sensitize to cytotoxic drugs. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) depletes some proteins involved in PI3K/AKT signaling, e.g., ERBB2, epidermal growth factor receptor (EGFR), and phosphorylated AKT (p-AKT). 17-AAG and paclitaxel were combined (at a fixed 1:1 ratio of their IC(50)) in four ovarian cancer cell lines that differ in expression of p-AKT, EGFR, and ERBB2. The EGFR-overexpressing A431 and KB epidermoid cell lines were also included. Combination indices (CI) were calculated using the median-effect equation and interpreted in the context of 17-AAG-mediated inhibition of PI3K signaling. Synergy was observed in IGROV-1- and ERBB2-overexpressing SKOV-3 ovarian cancer cells that express a high level of constitutively activated p-AKT [CI at fraction unaffected (fu)(0.5) = 0.50 and 0.53, respectively]. Slight synergy was observed in A431 cells (moderate p-AKT/overexpressed EGFR; CI at fu(0.5) = 0.76) and antagonism in CH1 (moderate p-AKT), HX62 cells (low p-AKT), and KB cells (low p-AKT/overexpressed EGFR; CI at fu(50) = 3.0, 3.5, and 2.0, respectively). The observed effects correlated with changes in the rate of apoptosis induction. 17-AAG induced a decrease in HSP90 client proteins (e.g., C-RAF, ERBB2, and p-AKT) or in downstream markers of their activity (e.g., phosphorylated extracellular signal-regulated kinase or p-AKT) in SKOV-3, IGROV-1, and CH1 cells at IC(50) concentrations. A non-growth-inhibitory concentration (6 nmol/L) reduced the phosphorylation of AKT (but not extracellular signal-regulated kinase) and sensitized SKOV-3 cells to paclitaxel. In conclusion, 17-AAG may sensitize a subset of ovarian cancer to paclitaxel, particularly those tumors in which resistance is driven by ERBB2 and/or p-AKT..
Smith, N.F.
Hayes, A.
James, K.
Nutley, B.P.
McDonald, E.
Henley, A.
Dymock, B.
Drysdale, M.J.
Raynaud, F.I.
Workman, P.
(2006). Preclinical pharmacokinetics and metabolism of a novel diaryl pyrazole resorcinol series of heat shock protein 90 inhibitors. Mol cancer ther,
Vol.5
(6),
pp. 1628-1637.
show abstract
CCT018159 was recently identified as a novel inhibitor of heat shock protein (Hsp) 90, a promising target for cancer therapy. Pharmacokinetic and metabolic properties are likely to be important for efficacy and need to be optimized during drug development. Here, we define the preclinical metabolism and pharmacokinetics of CCT018159 and some early derivatives. In addition, we assess in vitro metabolic stability screening and in vivo cassette dosing (simultaneous administration of several compounds to a single animal) as approaches to investigate these compounds. The plasma clearance following individual i.v. administration to mice was rapid (0.128-0.816 L/h), exceeding hepatic blood flow. For CCT066950 and CCT066952, this could be attributed in part to extensive (>80%) blood cell binding. Oral bioavailability ranged from 1.8% to 29.6%. Tissue distribution of CCT066952 was rapid and moderate, and renal excretion of the compounds was minimal (<1% of dose excreted). Compounds underwent rapid glucuronidation both in vivo and following incubation with mouse liver microsomes. However, whereas CCT066965 was metabolized to the greatest extent in vitro, this compound displayed the slowest plasma clearance. The rank order of the compounds from the highest to lowest area under the curve was the same following discrete and cassette dosing. Furthermore, pharmacokinetic variables were similar whether the compounds were dosed alone or in combination. We conclude that the pharmacokinetics of CCT018159 are complex. Cassette dosing is currently the best option available to assess the pharmacokinetics of this promising series of compounds in relatively high throughput and is now being applied to identify compounds with optimal pharmacokinetic properties during structural analogue synthesis..
Workman, P.
(2006). Using biomarkers in drug development. Clin adv hematol oncol,
Vol.4
(10),
pp. 736-739.
Burns, S.
Travers, J.
Collins, I.
Rowlands, M.G.
Newbatt, Y.
Thompson, N.
Garrett, M.D.
Workman, P.
Aherne, W.
(2006). Identification of small-molecule inhibitors of protein kinase B (PKB/AKT) in an AlphaScreenâ„¢ high-throughput screen. Journal of biomolecular screening,
Vol.11
(7),
pp. 822-6.
Proisy, N.
Sharp, S.Y.
Boxall, K.
Connelly, S.
Roe, S.M.
Prodromou, C.
Slawin, A.M.
Pearl, L.H.
Workman, P.
Moody, C.J.
(2006). Inhibition of Hsp90 with synthetic macrolactones: synthesis and structural and biological evaluation of ring and conformational analogs of radicicol. Chem biol,
Vol.13
(11),
pp. 1203-1215.
show abstract
A series of benzo-macrolactones of varying ring size and conformation has been prepared by chemical synthesis and evaluated by structural and biological techniques. Thus, 12- to 16-membered lactones were obtained by concise routes, involving ring-closing metathesis as a key step. In enzyme assays, the 13-, 15-, and 16-membered analogs are good inhibitors, suggesting that they can adopt the required conformation to fit in the ATP-binding site. This was confirmed by cocrystallization of 13-, 14-, and 15-membered lactones with the N-terminal domain of yeast Hsp90, showing that they bind similarly to the "natural" 14-membered radicicol. The most active compounds in the ATPase assays also showed the greatest growth-inhibitory potency in HCT116 human colon cancer cells and the established molecular signature of Hsp90 inhibition, i.e., depletion of client proteins with upregulation of Hsp70..
Collins, I.
Workman, P.
(2006). New approaches to molecular cancer therapeutics. Nat chem biol,
Vol.2
(12),
pp. 689-700.
show abstract
Cancer drug development is leading the way in exploiting molecular biological and genetic information to develop "personalized" medicine. The new paradigm is to develop agents that target the precise molecular pathology driving the progression of individual cancers. Drug developers have benefited from decades of academic cancer research and from investment in genomics, genetics and automation; their success is exemplified by high-profile drugs such as Herceptin (trastuzumab), Gleevec (imatinib), Tarceva (erlotinib) and Avastin (bevacizumab). However, only 5% of cancer drugs entering clinical trials reach marketing approval. Cancer remains a high unmet medical need, and many potential cancer targets remain undrugged. In this review we assess the status of the discovery and development of small-molecule cancer therapeutics. We show how chemical biology approaches offer techniques for interconnecting elements of the traditional linear progression from gene to drug, thereby providing a basis for increasing speed and success in cancer drug discovery..
Powers, M.V.
Workman, P.
(2006). Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors. Endocr relat cancer,
Vol.13 Suppl 1,
pp. S125-S135.
show abstract
The last decade has seen the molecular chaperone heat shock protein 90 (HSP90) emerge as an exciting target for cancer therapy. This is because HSP90 is involved in maintaining the conformation, stability, activity and cellular localisation of several key oncogenic client proteins. These include, amongst others, ERBB2, C-RAF, CDK4, AKT/PKB, steroid hormone receptors, mutant p53, HIF-1alpha , survivin and telomerase hTERT. Therefore, modulation of this single drug target offers the prospect of simultaneously inhibiting all the multiple signalling pathways and biological processes that have been implicated in the development of the malignant phenotype. The chaperone function of HSP90 requires the formation of a multichaperone complex, which is dependent on the hydrolysis of ATP and ADP/ATP exchange. Most current inhibitors of HSP90 act as nucleotide mimetics, which block the intrinsic ATPase activity of this molecular chaperone. The first-in-class inhibitor to enter and complete phase I clinical trials was the geldanamycin analogue, 17-allylamino-17-demethoxygeldanamycin. The results of these trials have demonstrated that HSP90 is a valid drug target. Evidence of clinical activity has been seen in patients with melanoma, breast and prostate cancer. This article provides a personal perspective of the present efforts to increase our understanding of the molecular and cellular consequences of HSP90 inhibition, with examples from work in our own laboratory. We also review the discovery and development of novel small-molecule inhibitors and discuss alternative approaches to inhibit HSP90 activity, both of which offer exciting prospects for the future..
Collins, I.
Caldwell, J.
Fonseca, T.
Donald, A.
Bavetsias, V.
Hunter, L.-.
Garrett, M.D.
Rowlands, M.G.
Aherne, G.W.
Davies, T.G.
Berdini, V.
Woodhead, S.J.
Davis, D.
Seavers, L.C.
Wyatt, P.G.
Workman, P.
McDonald, E.
(2006). Structure-based design of isoquinoline-5-sulfonamide inhibitors of protein kinase B. Bioorg med chem,
Vol.14
(4),
pp. 1255-1273.
show abstract
Structure-based drug design of novel isoquinoline-5-sulfonamide inhibitors of PKB as potential antitumour agents was investigated. Constrained pyrrolidine analogues that mimicked the bound conformation of linear prototypes were identified and investigated by co-crystal structure determinations with the related protein PKA. Detailed variation in the binding modes between inhibitors with similar overall conformations was observed. Potent PKB inhibitors from this series inhibited GSK3beta phosphorylation in cellular assays, consistent with inhibition of PKB kinase activity in cells..
Howes, R.
Barril, X.
Dymock, B.W.
Grant, K.
Northfield, C.J.
Robertson, A.G.
Surgenor, A.
Wayne, J.
Wright, L.
James, K.
Matthews, T.
Cheung, K.M.
McDonald, E.
Workman, P.
Drysdale, M.J.
(2006). A fluorescence polarization assay for inhibitors of Hsp90. Analytical biochemistry,
Vol.350
(2),
pp. 202-12.
Hayakawa, M.
Kaizawa, H.
Moritomo, H.
Koizumi, T.
Ohishi, T.
Okada, M.
Ohta, M.
Tsukamoto, S.-.
Parker, P.
Workman, P.
Waterfield, M.
(2006). Synthesis and biological evaluation of 4-morpholino-2-phenylquinazolines and related derivatives as novel PI3 kinase p110α inhibitors. Bioorganic & medicinal chemistry,
Vol.14
(20),
pp. 6847-12.
Atrash, B.
Cooper, T.S.
Sheldrake, P.
Workman, P.
McDonald, E.
(2006). Development of synthetic routes to macrocyclic compounds based on the HSP90 inhibitor radicicol. Tetrahedron letters,
Vol.47
(13),
pp. 2237-4.
Cooper, T.S.
Atrash, B.
Sheldrake, P.
Workman, P.
McDonald, E.
(2006). Synthesis of resorcinylic macrocycles related to radicicol via ring-closing metathesis. Tetrahedron letters,
Vol.47
(13),
pp. 2241-3.
Knowles, H.J.
te Poele, R.T.
Workman, P.
Harris, A.L.
(2006). Niacin induces PPAR gamma expression and transcriptional activation in macrophages via HM74 and HM74a-mediated induction of prostaglandin synthesis pathways. Biochem pharmacol,
Vol.71
(5),
pp. 646-656.
show abstract
HM74 and HM74a have been identified as receptors for niacin. HM74a mediates the pharmacological anti-lipolytic effects of niacin in adipocytes by reducing intracellular cyclic AMP (cAMP) and inhibiting release of free fatty acids into the circulation. In macrophages, niacin induces peroxisome proliferator-activated receptor gamma (PPAR gamma)-dependent and cAMP-dependent expression of genes mediating reverse cholesterol transport, although via an unidentified receptor. We describe constitutive expression of HM74a mRNA and hypoxia-and IFN gamma-inducible expression of HM74 and HM74a in human monocytic cell lines and primary cells in culture. In U937 cells niacin-induced expression of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent endogenous ligand of PPAR gamma. Both niacin and the structurally distinct HM74/HM74a ligand acifran-induced nuclear expression of PPAR gamma protein and enhanced PPAR gamma transcriptional activity. Niacin-induced PPAR gamma transcriptional activity was pertussis toxin sensitive and required activity of phospholipase A(2) (EC 3.1.1.4), cyclo-oxygenase (EC 1.14.99.1) and prostaglandin D-2 synthase (EC 5.3.99.2). Niacin also induced PPAR gamma transcriptional activity in HM74 and HM74a CHO cell transfectants, although not in vector-only control cells. This was sensitive to pertussis toxin and to inhibition of phoshoplipase A(2) and cyclo-oxygenase activity. Additionally, niacin increased intracellular cAMP in U937 via a pertussis toxin and cyclo-oxygenase-sensitive mechanism. These results indicate that HM74 and HM74a can mediate macrophage responses to niacin via activation of the prostaglandin synthesis pathway and induction and activation of PPAR gamma. This suggests a novel mechanism(s) mediating the clinical effects of pharmacological doses of niacin. (c) 2005 Elsevier Inc. All rights reserved..
McDonald, E.
Workman, P.
Jones, K.
(2006). Inhibitors of the HSP90 molecular chaperone: attacking the master regulator in cancer. Curr top med chem,
Vol.6
(11),
pp. 1091-1107.
show abstract
The heat shock protein 90 (HSP90) chaperones represent some 1-2% of all cellular protein and are key players in protein quality control in cells. They are over expressed in many human cancers and the fact that many oncogenic proteins are clients has prompted much recent research on HSP90 inhibitors as new cancer therapeutics. A brief introduction is followed by a detailed review of the various classes of inhibitors, both natural product-based and synthetic, that have emerged over the last decade. The natural products geldanamycin, radicicol and novobiocin have provided the start points for new drugs in this area and their medicinal chemistry is reviewed, including the exciting recent results emerging from clinical trials using geldanamycin analogues. The detailed understanding of the binding mode of these compounds to HSP90 has been significantly enhanced by X-ray crystallography of HSP90 constructs co-crystallised with various ligands. Efforts to replace the natural product inhibitors with more drug-like synthetic compounds have mushroomed over the last 4 years. The purines and the 3,4-diarylpyrazoles have proven to be the most successful and their medicinal chemistry is reviewed with particular emphasis on structure-based design. Protein/ligand co-crystal structures have shown that conserved water molecules in the active site are a vital part of the hydrogen-bonding network established on binding both natural product and synthetic inhibitors. Medicinal chemists have used this information to develop high affinity lead compounds. Recent research provides the platform for exciting developments in the area of HSP90 inhibition over the next few years..
McDonald, E.
Jones, K.
Brough, P.A.
Drysdale, M.J.
Workman, P.
(2006). Discovery and development of pyrazole-scaffold Hsp90 inhibitors. Curr top med chem,
Vol.6
(11),
pp. 1193-1203.
show abstract
This review explains why the chaperone Hsp90 is an exciting protein target for the discovery of new drugs to treat cancer in the clinic, and summarises the properties of natural product derived inhibitors before relating the discovery and current state of development of synthetic pyrazole compounds. Blockade of Hsp90 results in reduced cellular levels of several proteins implicated in cancer including CDK4, ERBB2 and C-RAF, and causes simultaneous inhibition of cancer cell proliferation in culture and of tumor xenograft growth in vivo. Hsp90 has an ATPase domain that is necessary for its Hsp chaperone function, and X-ray crystallography has shown that natural product inhibitors (geldanamycin, radicicol) of Hsp90 function bind to this domain. High throughput assays focusing on the ATPase activity of Hsp90 were developed and used to discover novel chemical starting points for cancer drug discovery. The discovery, synthesis and SAR of 3,4-diaryl pyrazoles is described. X-Ray crystallography of protein-inhibitor complexes revealed important interactions involving the resorcinol substituent at C-3, and these X-ray structures strongly influenced subsequent medicinal chemistry research that has resulted in highly potent inhibitors with sub-micromolar activity in cells. SAR and X-ray data are summarised for analogues in which the 4-phenyl substituent is replaced by amides or piperazine derivatives. Prospects for the pyrazoles as they progress towards clinical development are discussed in relation to current Phase I trials with derivatives of geldanamycin..
Sharp, S.
Workman, P.
(2006). Inhibitors of the HSP90 molecular chaperone: current status. Adv cancer res,
Vol.95,
pp. 323-348.
show abstract
The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target for cancer therapy. It operates as part of a multichaperone complex and is essential for the conformation, stability, and function of several key oncogenic client proteins such as mutant p53, ERBB2, B-RAF, C-RAF, and CDK4. The HSP90-based chaperone machine is driven by the hydrolysis of ATP and ADP/ATP nucleotide exchange. Many of the inhibitors of HSP90 interrupt the intrinsic ATPase activity, causing degradation of the client proteins via the ubiquitin-proteasome pathway. The first-in-class HSP90 inhibitor in clinical trials is the geldanamycin analog, 17-allylamino, 17-demethoxygeldanamycin (17-AAG). The results that have emerged from these trials have been encouraging, with stable disease observed in two melanoma patients. Pharmacodynamic endpoints, such as induction of HSP70 and downregulation of C-RAF and CDK4 in peripheral blood mononuclear cells and tumor biopsies from treated patients, provided evidence of HSP90 inhibition at well-tolerated doses. The toxicity of 17-AAG has been mild. Several preclinical studies have shown that 17-AAG may enhance the efficacy of a variety of chemotherapeutic agents. Phase II clinical trials in various cancers have been initiated as well as Phase I trials of combined therapy with 17-AAG. However, there are several limitations with 17-AAG such as solubility, stability, and hepatotoxicity. Thus, it is not surprising that new HSP90 agents are under development against this novel target for cancer therapy and several show promise..
Workman, P.
(2006). Drugging the cancer kinome: progress and challenges in developing personalized molecular cancer therapeutics. Cold spring harb symp quant biol,
Vol.70,
pp. 499-515.
show abstract
A major goal of cancer research is to translate our understanding of the causation of malignancy at the level of the genome and biochemical pathways into the development of drugs with improved activity and cancer selectivity. This paper provides a personal perspective of the current status of efforts to achieve this goal, with a particular focus on drugging the cancer kinome. Remarkable progress has been made in this area, but many challenges remain. The value of cancer kinome sequencing is emphasized. Three projects in which the author's laboratory is involved are reviewed in detail. These involve the discovery and development of inhibitors of cyclin-dependent kinases, phosphoinositide 3-kinases, and the Hsp90 molecular chaperone..
Moreno-Farre, J.
Asad, Y.
Pacey, S.
Workman, P.
Raynaud, F.I.
(2006). Development and validation of a liquid chromatography/tandem mass spectrometry method for the determination of the novel anticancer agent 17-DMAG in human plasma. Rapid commun mass spectrom,
Vol.20
(19),
pp. 2845-2850.
show abstract
An accurate, sensitive, robust and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin hydrochloride (17-DMAG) in human plasma has been developed and validated. Plasma samples were prepared by liquid/liquid extraction with ethyl acetate. The chromatographic separation was achieved within 9 min on a Synergy Polar column with a linear gradient and a mobile phase consisting of methanol and 0.1% formic acid in water. Detection of 17-DMAG and the internal standard (IS), olomoucine, was achieved by MS/MS with electrospray ionisation in positive ion mode. The calibration curve, ranging from 1.89 to 1890 nM, was linear r > 0.994 using a 1/y2 weighted linear regression. The assay showed no significant interferences from endogenous compounds. The lower limit of quantitation (LLOQ) was 1.89 nM, using 250 microL of plasma, with inter-assay precision (%RSD) and accuracy (%RE) values of 11.6% and -5.8%, respectively. Intra-assay precision ranged from 7.8-13.6%. The method described here is being used to evaluate the pharmacokinetic profiles of 17-DMAG given as a once weekly infusion in patients with advanced solid tumours..
Moreno-Farre, J.
Workman, P.
Raynaud, F.I.
(2006). Analysis of potential drug-drug interactions for anticancer agents in human liver microsomes by high throughput liquid chromatography/mass spectrometry assay. Recent advances and research updates,
Vol.7,
p. 224.
Workman, P.
Clarke, P.A.
(2006). Hot off the press: Global gene expression signatures for global chemical probe and drug discovery. Mol biosyst,
Vol.2,
pp. 605-608.
Nutley, B.P.
Raynaud, F.I.
Wilson, S.C.
Fischer, P.M.
Hayes, A.
Goddard, P.M.
McClue, S.J.
Jarman, M.
Lane, D.P.
Workman, P.
(2005). Metabolism and pharmacokinetics of the cyclin-dependent kinase inhibitor R-roscovitine in the mouse. Mol cancer ther,
Vol.4
(1),
pp. 125-139.
show abstract
R-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials in patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of R-roscovitine at 10 microg/mL (28 micromol/L) for 60 minutes, 86.7% of the parent drug was metabolized and 60% of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in R-roscovitine. Identification was confirmed by chemical synthesis and comparison of an authentic sample of the R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor metabolites were identified as C8-oxo-R-roscovitine and N9-desisopropyl-R-roscovitine; these accounted for 4.9% and 2.6% of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC(infinity) for R-roscovitine (38 micromol/L/h) than for COOH-R-roscovitine (174 micromol/L/h). Excretion of R-roscovitine in the urine up to 24 hours post-dosing accounted for an average of only 0.02% of the administered dose of 50 mg/kg, whereas COOH-R-roscovitine represented 65% to 68% of the dose irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d9) was synthesized to investigate if formation of COOH-R-roscovitine could be inhibited by replacement of metabolically labile protons with deuterium. After 60 minutes of incubation of R-roscovitine-d9 or R-roscovitine with mouse liver microsomes, formation of COOH-R-roscovitine-d9 was decreased by approximately 24% compared with the production of COOH-R-roscovitine. In addition, the levels of R-roscovitine-d9 remaining were 33% higher than those of R-roscovitine. However, formation of several minor R-roscovitine metabolites was enhanced with R-roscovitine-d9, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-R-roscovitine and C8-oxo-R-roscovitine were tested in functional cyclin-dependent kinase assays and shown to be less active than R-roscovitine, confirming that these metabolic reactions are deactivation pathways..
Chau, N.-.
Rogers, P.
Aherne, W.
Carroll, V.
Collins, I.
McDonald, E.
Workman, P.
Ashcroft, M.
(2005). Identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1alpha induction in response to hypoxic stress and growth factors. Cancer res,
Vol.65
(11),
pp. 4918-4928.
show abstract
Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that is activated in response to hypoxia and growth factors. HIF-1 plays a central role in tumor progression, invasion, and metastasis. Overexpression of the HIF-1alpha subunit has been observed in many human cancers and is associated with a poor prognostic outcome with conventional treatments. Targeting HIF-1 using novel small molecule inhibitors is, therefore, an attractive strategy for therapeutic development. We have generated U2OS human osteosarcoma cells stably expressing a luciferase reporter construct under the control of a hypoxia response element (U2OS-HRE-luc). The U2OS-HRE-luc cells were robustly and reproducibly sensitive to hypoxic stress in a HIF-1-dependent manner. We developed an automated U2OS-HRE-luc cell-based assay that was used in a high-throughput screen to identify compounds that inhibited HIF-1 activity induced by treatment with the hypoxia mimetic, deferoxamine mesylate. We performed a pilot screen of the National Cancer Institute Diversity Set of 2,000 compounds. We identified eight hit compounds, six of these were also identified by Rapisarda et al. in an independent hypoxia screen. However, there were two novel hit compounds, NSC-134754 and NSC-643735, that did not significantly inhibit constitutive luciferase activity in U2OS cells (U2OS-luc). We showed that both NSC-134754 and NSC-643735 significantly inhibited HIF-1 activity and HIF-1alpha protein induced by deferoxamine mesylate. Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1alpha protein induced by hypoxia and significantly inhibited Glut-1 expression. Finally, we showed that both NCS-134754 and NCS-643735 inhibited HIF-1alpha protein induced by insulin-like growth factor-1. Our cell-based assay approach has successfully identified novel compounds that differentially target hypoxia and/or growth factor-mediated induction of HIF-1alpha..
Raynaud, F.I.
Whittaker, S.R.
Fischer, P.M.
McClue, S.
Walton, M.I.
Barrie, S.E.
Garrett, M.D.
Rogers, P.
Clarke, S.J.
Kelland, L.R.
Valenti, M.
Brunton, L.
Eccles, S.
Lane, D.P.
Workman, P.
(2005). In vitro and in vivo pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine and CYC202. Clin cancer res,
Vol.11
(13),
pp. 4875-4887.
show abstract
To investigate pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R-roscovitine; seliciclib) in the HCT116 human colon carcinoma model..
Brough, P.A.
Barril, X.
Beswick, M.
Dymock, B.W.
Drysdale, M.J.
Wright, L.
Grant, K.
Massey, A.
Surgenor, A.
Workman, P.
(2005). 3-(5-chloro-2,4-dihydroxyphenyl)-pyrazole-4-carboxamides as inhibitors of the Hsp90 molecular chaperone. Bioorganic & medicinal chemistry letters,
Vol.15
(23),
pp. 5197-5.
Banerji, U.
Walton, M.
Raynaud, F.
Grimshaw, R.
Kelland, L.
Valenti, M.
Judson, I.
Workman, P.
(2005). Pharmacokinetic-pharmacodynamic relationships for the heat shock protein 90 molecular chaperone inhibitor 17-allylamino, 17-demethoxygeldanamycin in human ovarian cancer xenograft models. Clin cancer res,
Vol.11
(19 Pt 1),
pp. 7023-7032.
show abstract
To establish the pharmacokinetic and pharmacodynamic profile of the heat shock protein 90 (HSP90) inhibitor 17-allylamino, 17-demethoxygeldanamycin (17-AAG) in ovarian cancer xenograft models..
Dias, S.D.
Friedlos, F.
Light, Y.
Springer, C.
Workman, P.
Marais, R.
(2005). Activated B-RAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin. Cancer research,
Vol.65
(23),
pp. 10686-6.
Workman, P.
(2005). Genomics and the second golden era of cancer drug development. Mol biosyst,
Vol.1
(1),
pp. 17-26.
show abstract
The first golden era of cancer drug development was initiated in the 1940s and gave rise to the cytotoxic agents that dominate current cancer medicine. The second golden era is now underway in which cancer genomics will direct drug development..
Hardcastle, A.
Boxall, K.
Richards, J.
Tomlin, P.
Sharp, S.
Clarke, P.
Workman, P.
Aherne, W.
(2005). Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90. Assay drug dev technol,
Vol.3
(3),
pp. 273-285.
show abstract
High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis..
Workman, P.
(2005). New cancer drugs on the horizon. Future oncol,
Vol.1
(3),
pp. 315-318.
Leach, M.O.
Brindle, K.M.
Evelhoch, J.L.
Griffiths, J.R.
Horsman, M.R.
Jackson, A.
Jayson, G.C.
Judson, I.R.
Knopp, M.V.
Maxwell, R.J.
McIntyre, D.
Padhani, A.R.
Price, P.
Rathbone, R.
Rustin, G.J.
Tofts, P.S.
Tozer, G.M.
Vennart, W.
Waterton, J.C.
Williams, S.R.
Workman, P.
Pharmacodynamic/Pharmacokinetic Technologies Advisory Committee, Drug Development Office, Cancer Research UK,
(2005). The assessment of antiangiogenic and antivascular therapies in early-stage clinical trials using magnetic resonance imaging: issues and recommendations. Br j cancer,
Vol.92
(9),
pp. 1599-1610.
show abstract
Vascular and angiogenic processes provide an important target for novel cancer therapeutics. Dynamic contrast-enhanced magnetic resonance imaging is being used increasingly to noninvasively monitor the action of these therapeutics in early-stage clinical trials. This publication reports the outcome of a workshop that considered the methodology and design of magnetic resonance studies, recommending how this new tool might best be used..
Walton, M.I.
Wilson, S.C.
Hardcastle, I.R.
Mirza, A.R.
Workman, P.
(2005). An evaluation of the ability of pifithrin-alpha and -beta to inhibit p53 function in two wild-type p53 human tumor cell lines. Mol cancer ther,
Vol.4
(9),
pp. 1369-1377.
show abstract
The small-molecule compound pifithrin-alpha (PFT-alpha) has been reported to inhibit p53 function and protect against a variety of genotoxic agents. We show here that PFT-alpha is unstable in tissue culture medium and is rapidly converted to its condensation product PFT-beta. Both compounds showed limited solubility with PFT-alpha precipitating out of tissue culture medium at concentrations >30 micromol/L. PFT-alpha and -beta exhibited cytotoxic effects in vitro towards two human wild-type p53-expressing tumor cell lines, A2780 ovarian and HCT116 colon (IC(50) values for both cell lines were 21.3 +/- 8.1 micromol/L for PFT-alpha and 90.3 +/- 15.5 micromol/L for PFT-beta, mean +/- SD, n = 4). There was no evidence of protection by clonogenic assay with either compound in combination with ionizing radiation. Indeed, there was some evidence that PFT-alpha enhanced cytotoxicity, particularly at higher concentrations of PFT-alpha. Neither compound had any effect on p53, p21, or MDM-2 protein expression following ionizing radiation exposure and there was no evidence of any abrogation of p53-dependent, ionizing radiation-induced cell cycle arrest. Similarly, there was no evidence of cellular protection, or of effects on p53-dependent gene transcription, or on translation of MDM-2 or p21 following UV treatment of these human tumor cell lines. In addition, there was no effect on p53 or p21 gene transactivation or p38 phosphorylation after UV irradiation of NIH-3T3 mouse fibroblasts. In conclusion, neither PFT-alpha nor -beta can be regarded as a ubiquitous inhibitor of p53 function, and caution should be exercised in the use of these agents as specific p53 inhibitors..
Stimson, L.
Rowlands, M.G.
Newbatt, Y.M.
Smith, N.F.
Raynaud, F.I.
Rogers, P.
Bavetsias, V.
Gorsuch, S.
Jarman, M.
Bannister, A.
Kouzarides, T.
McDonald, E.
Workman, P.
Aherne, G.W.
(2005). Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity. Mol cancer ther,
Vol.4
(10),
pp. 1521-1532.
show abstract
Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF) and p300 (1 to >50 micromol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 micromol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2-48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of alpha-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition..
Workman, P.
Johnston, P.G.
(2005). Genomic profiling of cancer: What next?. J clin oncol,
Vol.23
(29),
pp. 7253-7256.
Beloueche-Babari, M.
Jackson, L.E.
Al-Saffar, N.M.
Workman, P.
Leach, M.O.
Ronen, S.M.
(2005). Magnetic resonance spectroscopy monitoring of mitogen-activated protein kinase signaling inhibition. Cancer res,
Vol.65
(8),
pp. 3356-3363.
show abstract
Several mitogen-activated protein kinase (MAPK) signaling inhibitors are currently undergoing clinical trial as part of novel mechanism-based anticancer treatment strategies. This study was aimed at detecting biomarkers of MAPK signaling inhibition in human breast and colon carcinoma cells using magnetic resonance spectroscopy. We investigated the effect of the prototype MAPK kinase inhibitor U0126 on the (31)P-MR spectra of MDA-MB-231, MCF-7 and Hs578T breast, and HCT116 colon carcinoma cells. Treatment of MDA-MB-231 cells with 50 micromol/L U0126 for 2, 4, 8, 16, 24, 32, and 40 hours caused inhibition of extracellular signal-regulated kinases (ERK1/2) phosphorylation from 2 hours onwards. (31)P-MR spectra of extracted cells indicated that this was associated with a significant drop in phosphocholine levels to 78 +/- 8% at 8 hours, 74 +/- 8% at 16 hours, 66 +/- 7% at 24 hours, 71 +/- 10% at 32 hours, and 65 +/- 10% at 40 hours post-treatment. In contrast, the lower concentration of 10 micromol/L U0126 for 40 hours had no significant effect on either P-ERK1/ 2 or phosphocholine levels in MDA-MB-231 cells. Depletion of P-ERK1/2 in MCF-7 and Hs578T cells with 50 micromol/L U0126 also produced a drop in phosphocholine levels to 51 +/- 17% at 40 hours and 23 +/- 12% at 48 hours, respectively. Similarly, in HCT116 cells, inhibition with 30 micromol/L U0126 caused depletion of P-ERK1/2 and a decrease in phosphocholine levels to 80 +/- 9% at 16 hours and 61 +/- 4% at 24 hours post-treatment. The reduction in phosphocholine in MDA-MB-231 and HCT116 cells correlated positively with the drop in P-ERK1/2 levels. Our results show that MAPK signaling inhibition with U0126 is associated with a time-dependent decrease in cellular phosphocholine levels. Thus, phosphocholine has potential as a noninvasive pharmacodynamic marker for monitoring MAPK signaling blockade..
Cheung, K.M.
Matthews, T.P.
James, K.
Rowlands, M.G.
Boxall, K.J.
Sharp, S.Y.
Maloney, A.
Roe, S.M.
Prodromou, C.
Pearl, L.H.
Aherne, G.W.
McDonald, E.
Workman, P.
(2005). The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors. Bioorg med chem lett,
Vol.15
(14),
pp. 3338-3343.
show abstract
High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties. Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90..
O'Donnell, A.
Padhani, A.
Hayes, C.
Kakkar, A.J.
Leach, M.
Trigo, J.M.
Scurr, M.
Raynaud, F.
Phillips, S.
Aherne, W.
Hardcastle, A.
Workman, P.
Hannah, A.
Judson, I.
(2005). A phase I study of the angiogenesis inhibitor SU5416 (semaxanib) in solid tumours, incorporating dynamic contrast MR pharmacodynamic end points. British journal of cancer,
Vol.93
(8),
pp. 876-8.
Banerji, U.
O'Donnell, A.
Scurr, M.
Pacey, S.
Stapleton, S.
Asad, Y.
Simmons, L.
Maloney, A.
Raynaud, F.
Campbell, M.
Walton, M.
Lakhani, S.
Kaye, S.
Workman, P.
Judson, I.
(2005). Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies. J clin oncol,
Vol.23
(18),
pp. 4152-4161.
show abstract
To study the toxicity and pharmacokinetic-pharmacodynamic profile of 17-allylamino, 17- demethoxygeldanamycin (17-AAG) and to recommend a dose for phase II trials..
Wood, N.
Gibbs, D.D.
Jackman, A.L.
Henley, A.
Workman, P.
Raynaud, F.
(2005). A liquid chromatographic-tandem mass spectrometric method for the determination of two selective thymidylate synthase inhibitors, BGC945 and BGC638, in mouse plasma. J chromatogr b analyt technol biomed life sci,
Vol.824
(1-2),
pp. 181-188.
show abstract
A LC-tandem mass spectrometry method to quantify the quinazoline-based thymidylate synthase inhibitors BGC945 and BGC638 in mouse plasma was developed. BGC945 and BGC638 were extracted from mouse plasma using protein precipitation with acetonitrile. Chromatography was performed on a Fluophase RP 5 microm, 100 mmx2.0mm i.d. column using a gradient of ammonium acetate and acetonitrile as a mobile phase with a flow rate of 0.2 mLmin(-1). The injection volume for each sample was 20 microL with a total run time of 7.5 min. This method was validated in the range 25-4000 nM (r2=0.99). The analytical assay performance showed that the method was accurate (mean intra- and inter-day assay R.E. were below 12% and 11%, respectively), reproducible (mean intra- and inter-day R.S.D. were less than 13% and 5% for all quality control levels, respectively) and sensitive (lower limit of quantification was 25 nM) in the range studied. This validated method has been used to define the first pharmacokinetic report of BGC945 and BGC638 in mice..
Dymock, B.W.
Barril, X.
Brough, P.A.
Cansfield, J.E.
Massey, A.
McDonald, E.
Hubbard, R.E.
Surgenor, A.
Roughley, S.D.
Webb, P.
Workman, P.
Wright, L.
Drysdale, M.J.
(2005). Novel, potent small-molecule inhibitors of the molecular chaperone Hsp90 discovered through structure-based design. Journal of medicinal chemistry,
Vol.48
(13),
pp. 4212-4.
Nutley, B.P.
Smith, N.F.
Hayes, A.
Kelland, L.R.
Brunton, L.
Golding, B.T.
Smith, G.C.
Martin, N.M.
Workman, P.
Raynaud, F.I.
(2005). Preclinical pharmacokinetics and metabolism of a novel prototype DNA-PK inhibitor NU7026. Br j cancer,
Vol.93
(9),
pp. 1011-1018.
show abstract
In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 muM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg(-1), NU7026 underwent rapid plasma clearance (0.108 l h(-1)) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg(-1) was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg(-1) i.p. in order to obtain the drug exposure required for radiosensitisation..
Whittaker, S.R.
Walton, M.I.
Garrett, M.D.
Workman, P.
(2004). The Cyclin-dependent kinase inhibitor CYC202 (R-roscovitine) inhibits retinoblastoma protein phosphorylation, causes loss of Cyclin D1, and activates the mitogen-activated protein kinase pathway. Cancer res,
Vol.64
(1),
pp. 262-272.
show abstract
Deregulation of the cell cycle commonly occurs during tumorigenesis, resulting in unrestricted cell proliferation and independence from mitogens. Cyclin-dependent kinase inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells. CYC202 (R-roscovitine) is a potent inhibitor of CDK2/cyclin E that is undergoing clinical trials. Drugs selected to act on a particular molecular target may exert additional or alternative effects in intact cells. We therefore studied the molecular pharmacology of CYC202 in human colon cancer cells. Treatment of HT29 and KM12 colon carcinoma cell lines with CYC202 decreased both retinoblastoma protein phosphorylation and total retinoblastoma protein. In addition, an increase in the phosphorylation of extracellular signal-regulated kinases 1/2 was observed. As a result, downstream activation of the mitogen-activated protein kinase pathway occurred, as demonstrated by an increase in ELK-1 phosphorylation and in c-FOS expression. Use of mitogen-activated protein kinase kinases 1/2 inhibitors showed that the CYC202-induced extracellular signal-regulated kinases 1/2 phosphorylation was mitogen-activated protein kinase kinases 1/2 dependent but did not contribute to the cell cycle effects of the drug, which included a reduction of cells in G(1), inhibition of bromodeoxyuridine incorporation during S-phase, and a moderate increase in G(2)-M phase. Despite activation of the mitogen-activated protein kinase pathway, cyclin D1 protein levels were decreased by CYC202, an effect that occurred simultaneously with loss of retinoblastoma protein phosphorylation and inhibition of cell cycle progression. The reduced expression of cyclin D1 protein was independent of the p38(SAPK) and phosphatidylinositol 3-kinase pathways, which are known regulators of cyclin D1 protein. Interestingly, CYC202 caused a clear reduction in cyclins D1, A, and B1 mRNA, whereas c-FOS mRNA increased by 2-fold. This was accompanied by a loss of RNA polymerase II phosphorylation and total RNA polymerase II protein, suggesting that CYC202 was inhibiting transcription, possibly via inhibition of CDK7 and CDK9 complexes. It can be concluded that although CYC202 can act as a CDK2 inhibitor, it also has the potential to inhibit CDK4 and CDK1 activities in cancer cells through the down-regulation of the corresponding cyclin partners. This provides a possible mechanism by which CYC202 can cause a reduction in retinoblastoma protein phosphorylation at multiple sites and cell cycle arrest in G(1), S, and G(2)-M phases. In addition to providing useful insights into the molecular pharmacology of CYC202 in human cancer cells, the results also suggest potential pharmacodynamic end points for use in clinical trials with the drug..
Kote-Jarai, Z.
Williams, R.D.
Cattini, N.
Copeland, M.
Giddings, I.
Wooster, R.
tePoele, R.H.
Workman, P.
Gusterson, B.
Peacock, J.
Gui, G.
Campbell, C.
Eeles, R.
(2004). Gene expression profiling after radiation-induced DNA damage is strongly predictive of BRCA1 mutation carrier status. Clin cancer res,
Vol.10
(3),
pp. 958-963.
show abstract
The impact of the presence of a germ-line BRCA1 mutation on gene expression in normal breast fibroblasts after radiation-induced DNA damage has been investigated..
Barthel, H.
Wilson, H.
Collingridge, D.R.
Brown, G.
Osman, S.
Luthra, S.K.
Brady, F.
Workman, P.
Price, P.M.
Aboagye, E.O.
(2004). In vivo evaluation of [F-18]fluoroetanidazole as a new marker for imaging tumour hypoxia with positron emission tomography. British journal of cancer,
Vol.90
(11),
pp. 2232-11.
Workman, P.
(2004). Altered states: selectively drugging the Hsp90 cancer chaperone. Trends mol med,
Vol.10
(2),
pp. 47-51.
show abstract
The molecular chaperone Hsp90 is an exciting cancer drug target. The first Hsp90 inhibitor to enter clinical trials--the geldanamycin derivative 17AAG--has recently demonstrated proof-of-concept for successful target modulation, with sighs of therapeutic benefit. An important property of Hsp90 inhibitors is their ability to cause simultaneous, combinatorial blockade of multiple cancer-causing pathways by promoting the degradation of many oncogenic client proteins. However, the reason for therapeutic selectivity in cancer cells versus normal cells is unclear. New research now shows that Hsp90 exists in cancer cells in a heightened, activated state that is highly susceptible to inhibition by 17AAG..
Raynaud, F.I.
Fischer, P.M.
Nutley, B.P.
Goddard, P.M.
Lane, D.P.
Workman, P.
(2004). Cassette dosing pharmacokinetics of a library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 inhibitors prepared by parallel synthesis. Mol cancer ther,
Vol.3
(3),
pp. 353-362.
show abstract
Determination of pharmacokinetic properties in the intact animal remains a major bottleneck in drug discovery. Cassette dosing involves administration of a cocktail of drugs to individual animals. Here we describe the cassette dosing properties of a 107-membered library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 (CDK2) inhibitors. A three-step parallel synthesis approach produced compounds with purity ranging from 63% to 100%. Cassette dosing was validated by comparing the pharmacokinetic parameters obtained following i.v. administration of a mixture of olomoucine, R-roscovitine (CYC202), and bohemine, each at 16.6 mg/kg, with results for administration of single agents at 50 mg/kg. No significant difference was observed between the pharmacokinetic parameters of agents when dosed in combination compared with those of individual compounds. CYC202 showed the highest area under the curve (AUC) and the longest elimination half-life (t(1/2)). Further cassettes evaluated the library of trisubstituted purines with CYC202 and purvalanol A included as pharmacokinetic standards in a validated limited sampling strategy. The ratios of pharmacokinetic parameters to that of CYC202 [AUC, maximum concentration (C(max)), and t(1/2)] remained similar when compounds were tested in two different cassettes or as individual compounds. Following dosing of the same cassette on three different days, there was less than 20% variation in pharmacokinetic parameters between days. The structure-pharmacokinetics relationship showed that the favored purine substituents are benzylamine and veratrylamine at position 6, amino-2 propanol at position 2, and methylpropyl or hydroxyethyl at position 9. Without cassette dosing, this study would have used 3 times as many animals and would have taken 4 times longer, illustrating the power of this method in lead optimization..
Workman, P.
(2004). Inhibiting the phosphoinositide 3-kinase pathway for cancer treatment. Biochem soc trans,
Vol.32
(Pt 2),
pp. 393-396.
show abstract
There is extensive evidence from the molecular and genomic analysis of human cancers that the PI 3-kinase (phosphoinositide 3-kinase)-Akt/PKB (protein kinase B) pathway is deregulated in malignant progression. Furthermore, the causal involvement of PI 3-kinase is supported by gene-knockout mouse models. Prototype inhibitors show evidence of anticancer activity in vitro and in vivo animal models. The recent development of isoform-selective inhibitors shows considerable promise for cancer treatment..
Kristeleit, R.
Stimson, L.
Workman, P.
Aherne, W.
(2004). Histone modification enzymes: novel targets for cancer drugs. Expert opin emerg drugs,
Vol.9
(1),
pp. 135-154.
show abstract
In eukaryotes, genomic DNA is packaged with histone proteins into the cell nucleus as chromatin, condensing the DNA > 10,000-fold. Chromatin is highly dynamic and exerts profound control on gene expression. Localised chromatin decondensation facilitates access of nuclear machinery. Chromatin displays epigenetic inheritance, in that changes in its structure can pass to the next generation independently of the DNA sequence itself. It is now clear that the post-translational modification of histones, for example, acetylation, methylation and phosphorylation, plays a crucial role in the regulation of nuclear function through the 'histone code'. There has been significant progress in identifying and understanding the enzymes that control these complex processes, in particular histone acetyltransferases and histone deacetylases. The exciting discovery that compounds inhibiting histone deacetylase activity also have antitumour properties has focused attention on their use as anticancer drugs. As a consequence, there is ongoing evaluation of several histone deacetylase inhibitor compounds in Phase I and II clinical trials with promising early results. It is likely that many of the enzymes involved in the control of histone modification will provide therapeutic opportunities for the treatment of cancer, including histone methyltransferases and Aurora kinases..
Wright, L.
Barril, X.
Dymock, B.
Sheridan, L.
Surgenor, A.
Beswick, M.
Drysdale, M.
Collier, A.
Massey, A.
Davies, N.
Fink, A.
Fromont, C.
Aherne, W.
Boxall, K.
Sharp, S.
Workman, P.
Hubbard, R.E.
(2004). Structure-activity relationships in purine-based inhibitor binding to HSP90 isoforms. Chemistry & biology,
Vol.11
(6),
pp. 775-11.
Dymock, B.W.
Drysdale, M.J.
McDonald, E.
Workman, P.
(2004). Inhibitors of HSP90 and other chaperones for the treatment of cancer. Expert opinion on therapeutic patents,
Vol.14
(6),
pp. 837-11.
Workman, P.
(2004). Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone. Cancer lett,
Vol.206
(2),
pp. 149-157.
show abstract
The molecular chaperone Hsp90 is not only of major current interest in fundamental biological research but also recognised as an exciting new target for the treatment of cancer and other diseases. In addition to playing an important role in response to proteotoxic heat shock and others stresses, Hsp90 is also critical for maintaining normal cellular homeostasis. Hsp90 is responsible for ensuring the conformational stability, shape and function of a selected range of key proteins, including many kinases and transcription factors. Furthermore, recent studies show that Hsp90 plays a key role in development and evolution. Hsp90 is overexpressed in cancer cells and is thought to be involved in dealing with the cellular stress associated with malignancy, as well as being essential for a range of key oncogenic proteins, including ErbB2, Raf-1, Akt/PKB, mutant p53 and many others. A major attraction of Hsp90 inhibitors is their potential to inhibit a range of 'mission critical' cancer pathways, thereby blocking all of the 'hallmark traits' of malignancy and exhibiting broad-spectrum antitumour activity. The first-in-class Hsp90 inhibitor 17AAG has entered clinical trials with promising early results and a range of other agents is under investigation and preclinical development. This article reviews the current status and future prospects for the exploitation of Hsp90 as a new molecular target for cancer treatment..
Sun, C.
Newbatt, Y.
Douglas, L.
Workman, P.
Aherne, W.
Linardopoulos, S.
(2004). High-throughput screening assay for identification of small molecule inhibitors of Aurora2/STK15 kinase. J biomol screen,
Vol.9
(5),
pp. 391-397.
show abstract
STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, gamma-(33)P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate(R) to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z' factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity..
Renshaw, J.
Orr, R.M.
Walton, M.I.
Te Poele, R.
Williams, R.D.
Wancewicz, E.V.
Monia, B.P.
Workman, P.
Pritchard-Jones, K.
(2004). Disruption of WT1 gene expression and exon 5 splicing following cytotoxic drug treatment: antisense down-regulation of exon 5 alters target gene expression and inhibits cell survival. Mol cancer ther,
Vol.3
(11),
pp. 1467-1484.
show abstract
Deregulated expression of the Wilms' tumor gene (WT1) has been implicated in the maintenance of a malignant phenotype in leukemias and a wide range of solid tumors through interference with normal signaling in differentiation and apoptotic pathways. Expression of high levels of WT1 is associated with poor prognosis in leukemias and breast cancer. Using real-time (Taqman) reverse transcription-PCR and RNase protection assay, we have shown up-regulation of WT1 expression following cytotoxic treatment of cells exhibiting drug resistance, a phenomenon not seen in sensitive cells. WT1 is subject to alternative splicing involving exon 5 and three amino acids (KTS) at the end of exon 9, producing four major isoforms. Exon 5 splicing was disrupted in all cell lines studied following a cytotoxic insult probably due to increased exon 5 skipping. Disruption of exon 5 splicing may be a proapoptotic signal because specific targeting of WT1 exon 5-containing transcripts using a nuclease-resistant antisense oligonucleotide (ASO) killed HL60 leukemia cells, which were resistant to an ASO targeting all four alternatively spliced transcripts simultaneously. K562 cells were sensitive to both target-specific ASOs. Gene expression profiling following treatment with WT1 exon 5-targeted antisense showed up-regulation of the known WT1 target gene, thrombospondin 1, in HL60 cells, which correlated with cell death. In addition, novel potential WT1 target genes were identified in each cell line. These studies highlight a new layer of complexity in the regulation and function of the WT1 gene product and suggest that antisense directed to WT1 exon 5 might have therapeutic potential..
Renshaw, J.
Orr, R.M.
Walton, M.I.
Poele, R.T.
Williams, R.D.
Wancewicz, E.V.
Monia, B.P.
Workman, P.
Pritchard-Jones, K.
(2004). Disruption of WT1 gene expression and exon 5 splicing following cytotoxic drug treatment:: Antisense down-regulation of exon 5 alters target gene expression and inhibits cell survival. Molecular cancer therapeutics,
Vol.3
(11),
pp. 1467-17.
Clarke, P.A.
te Poele, R.
Workman, P.
(2004). Gene expression microarray technologies in the development of new therapeutic agents. Eur j cancer,
Vol.40
(17),
pp. 2560-2591.
show abstract
We review in detail how gene expression microarray technology is benefiting all phases of the discovery, development and subsequent use of new cancer therapeutics. Global gene expression profiling is valuable in cancer classification, elucidation of biochemical pathways and the identification of potential targets for novel molecular therapeutics. We exemplify the value in tissue culture and animal models of cancer, as well as in clinical studies. The power of expression profiling alongside gene knockout or knockdown methods such as RNA interference is illustrated. The use of basal or constitutive gene expression profiling to understand and predict drug sensitivity or resistance is described. The ability of expression profiling to define detailed molecular signatures of drug action is emphasised. The approach can identify on-target and off-target effects. It can be used to identify molecular biomarkers for proof of concept studies, pharmacodynamic endpoints and prognostic markers for predicting outcome and patient selection..
Workman, P.
(2004). Drug discovery strategies: technologies to accelerate translation from target to drug. J chemother,
Vol.16 Suppl 4,
pp. 13-15.
show abstract
The development of new molecular therapeutics for cancer treatment is based on the identification of molecular abnormalities that drive the malignant process. The discovery of novel molecular targets is made quicker and more efficient by use of powerful high throughput technologies such as genome sequencing and gene expression microarrays. Similarly, the drug discovery process is being accelerated by a range of other valuable technologies. Using these approaches, we can expect to see a range of personalised molecular therapeutics becoming available for cancer treatment..
Smith, N.F.
Hayes, A.
Nutley, B.P.
Raynaud, F.I.
Workman, P.
(2004). Evaluation of the cassette dosing approach for assessing the pharmacokinetics of geldanamycin analogues in mice. Cancer chemother pharmacol,
Vol.54
(6),
pp. 475-486.
show abstract
There is currently much interest in developing analogues of the benzoquinone ansamycin geldanamycin that may overcome the limitations of 17-(allylamino)-17-demethoxygeldanamycin (17AAG), which is the first known inhibitor of heat shock protein 90 (Hsp90) to enter clinical trials. Studies were performed to assess whether cassette dosing, the coadministration of several compounds to a single animal, is a suitable approach to evaluate the preclinical pharmacokinetics of geldanamycin analogues in high throughput..
Rowlands, M.G.
Newbatt, Y.M.
Prodromou, C.
Pearl, L.H.
Workman, P.
Aherne, W.
(2004). High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Anal biochem,
Vol.327
(2),
pp. 176-183.
show abstract
The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8..
Clarke, P.A.
Pestell, K.E.
Di Stefano, F.
Workman, P.
Walton, M.I.
(2004). Characterisation of molecular events following cisplatin treatment of two curable ovarian cancer models: contrasting role for p53 induction and apoptosis in vivo. Br j cancer,
Vol.91
(8),
pp. 1614-1623.
show abstract
The detailed molecular basis and determinants of in vivo tumour sensitivity to conventional anticancer agents remain unclear. We examined the cellular and molecular consequences of cisplatin treatment using two ovarian tumour xenograft models that had not been previously adapted to culture in vitro. Both xenografts were curable with clinically relevant multiple doses of cisplatin. Following a single dose of cisplatin (6 mg kg(-1) i.p.) growth delays of 25 and 75 days were obtained for pxn100 and pxn65, respectively. This difference in response was not due to differences in DNA damage. Pxn100 tumours had a functional p53 response and a wild-type p53 sequence, whereas pxn65 harboured a mutant p53 and lacked a functional p53 response. Microarray analysis revealed the induction of p53-regulated genes and regulators of checkpoint control and apoptosis in pxn100 tumours following cisplatin-treatment. By contrast, there was no p53-dependent response and only limited changes in gene expression were detected in the pxn65 tumours. TUNEL analysis demonstrated high levels of apoptosis in the pxn100 tumours following cisplatin treatment, but there was no detectable apoptosis in the pxn65 tumours. Our observations show that a marked in vivo response to cisplatin can occur via p53-dependent apoptosis or independently of p53 status in human ovarian xenografts..
Griffiths, G.J.
Koh, M.Y.
Brunton, V.G.
Cawthorne, C.
Reeves, N.A.
Greaves, M.
Tilby, M.J.
Pearson, D.G.
Ottley, C.J.
Workman, P.
Frame, M.C.
Dive, C.
(2004). Expression of kinase-defective mutants of c-Src in human metastatic colon cancer cells decreases Bcl-x(L) and increases oxaliplatin- and Fas-induced apoptosis. Journal of biological chemistry,
Vol.279
(44),
pp. 46113-9.
Seddon, B.
Kelland, L.R.
Workman, P.
(2004). Bioreductive prodrugs for cancer therapy. Methods mol med,
Vol.90,
pp. 515-542.
Jackman, A.L.
Workman, P.
(2004). The combination of cycotoxic and molecularly targeted therapies - can it be done?. Drug discovery today,
Vol.4,
pp. 445-454.
Leach, M.O.
Brindl, K.M.
Evelhoch, J.L.
Griffiths, J.R.
Horsman, M.R.
Jackson, A.
Hayson, G.
Judson, I.R.
Knopp, M.V.
Maxwell, R.J.
McIntyre, D.
Padhani, A.R.
Price, P.
Rathbone, R.
Rustin, G.
Tofts, P.S.
Tozer, G.M.
Vennart, W.
Waterton, J.C.
Williams, S.R.
Workman, P.
(2004). Assessment of antiangiogenic and antivascular therapeutics using MRI: Recommendations for appropriate methodology for clinical trials. Br j radiol,
Vol.76,
pp. S87-S91.
Barrie, S.E.
Eno-Amooquaye, E.
Hardcastle, A.
Platt, G.
Richards, J.
Bedford, D.
Workman, P.
Aherne, W.
Mittnacht, S.
Garrett, M.D.
(2003). High-throughput screening for the identification of small-molecule inhibitors of retinoblastoma protein phosphorylation in cells. Anal biochem,
Vol.320
(1),
pp. 66-74.
show abstract
The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK inhibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on pRb, a known site of CDK phosphorylation, and a Europium-labeled secondary antibody for signal detection. The assay is reproducible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules that will block pRb phosphorylation..
Seddon, B.M.
Payne, G.S.
Simmons, L.
Ruddle, R.
Grimshaw, R.
Tan, S.
Turner, A.
Raynaud, F.
Halbert, G.
Leach, M.O.
Judson, I.
Workman, P.
(2003). A phase I study of SR-4554 via intravenous administration for noninvasive investigation of tumor hypoxia by magnetic resonance spectroscopy in patients with malignancy. Clin cancer res,
Vol.9
(14),
pp. 5101-5112.
show abstract
To perform a Phase I study of SR-4554, a fluorinated 2-nitroimidazole noninvasive probe of tumor hypoxia detected by (19)F magnetic resonance spectroscopy (MRS)..
Pestell, K.E.
Workman, P.
(2003). Paul Workman on the challenges of cancer drug development. Drug discov today,
Vol.8
(17),
pp. 775-777.
Workman, P.
(2003). Auditing the pharmacological accounts for Hsp90 molecular chaperone inhibitors: unfolding the relationship between pharmacokinetics and pharmacodynamics. Mol cancer ther,
Vol.2
(2),
pp. 131-138.
Chung, Y.L.
Troy, H.
Banerji, U.
Jackson, L.E.
Walton, M.I.
Stubbs, M.
Griffiths, J.R.
Judson, I.R.
Leach, M.O.
Workman, P.
Ronen, S.M.
(2003). Magnetic resonance spectroscopic pharmacodynamic markers of the heat shock protein 90 inhibitor 17-allylamino,17-demethoxygeldanamycin (17AAG) in human colon cancer models. J natl cancer inst,
Vol.95
(21),
pp. 1624-1633.
show abstract
17-allylamino,17-demethoxygeldanamycin (17AAG) is a novel anticancer drug that inhibits heat shock protein 90 (Hsp90), resulting in proteasomal degradation of several oncogenic proteins. We used phosphorus magnetic resonance spectroscopy (31P-MRS) to determine whether 17AAG treatment leads to alterations in phospholipids that could serve as pharmacodynamic markers for tumor response to 17AAG..
Collingridge, D.R.
Glaser, M.
Osman, S.
Barthel, H.
Hutchinson, O.C.
Luthra, S.K.
Brady, F.
Bouchier-Hayes, L.
Martin, S.J.
Workman, P.
Price, P.
Aboagye, E.O.
(2003). In vitro selectivity, in vivo biodistribution and tumour uptake of annexin V radiolabelled with a positron emitting radioisotope. British journal of cancer,
Vol.89
(7),
pp. 1327-7.
Workman, P.
(2003). The opportunities and challenges of personalized genome-based molecular therapies for cancer: targets, technologies, and molecular chaperones. Cancer chemother pharmacol,
Vol.52 Suppl 1,
pp. S45-S56.
show abstract
There are now unprecedented opportunities for the development of improved drugs for cancer treatment. Following on from the Human Genome Project, the Cancer Genome Project and related activities will define most of the genes in the majority of common human cancers over the next 5 years. This will provide the opportunity to develop a range of drugs targeted to the precise molecular abnormalities that drive various human cancers and opens up the possibility of personalized therapies targeted to the molecular pathology and genomics of individual patients and their malignancies. The new molecular therapies should be more effective and have less-severe side effects than cytotoxic agents. To develop the new generation of molecular cancer therapeutics as rapidly as possible, it is essential to harness the power of a range of new technologies. These include: genomic and proteomic methodologies (particularly gene expression microarrays); robotic high-throughput screening of diverse compound collections, together with in silico and fragment-based screening techniques; new structural biology methods for rational drug design (especially high-throughput X-ray crystallography and nuclear magnetic resonance); and advanced chemical technologies, including combinatorial and parallel synthesis. Two major challenges to cancer drug discovery are: (1) the ability to convert potent and selective lead compounds with activity by the desired mechanism on tumor cells in culture into agents with robust, drug-like properties, particularly in terms of pharmacokinetic and metabolic properties; and (2) the development of validated pharmacodynamic endpoints and molecular markers of drug response, ideally using noninvasive imaging technologies. The use of various new technologies will be exemplified. A major conceptual and practical issue facing the development and use of the new molecular cancer therapeutics is whether a single drug that targets one of a series of key molecular abnormalities in a particular cancer (e.g. BRAF) will be sufficient on its own to deliver clinical benefit ("house of cards" and tumor addiction models). The alternative scenario is that it will require either a combination of agents or a class of drug that has downstream effects on a range of oncogenic targets. Inhibitors of the heat-shock protein (HSP) 90 molecular chaperone are of particular interest in the latter regard, because they offer the potential of inhibiting multiple oncogenic pathways and simultaneous blockade of all six "hallmark traits" of cancer through direct interaction with a single molecular drug target. The first-in-class HSP90 inhibitor 17AAG exhibited good activity in animal models and is now showing evidence of molecular and clinical activity in ongoing clinical trials. Novel HSP90 inhibitors are also being sought. The development of HSP90 inhibitors is used to exemplify the application of new technologies in drug discovery against a novel molecular target, and in particular the need for innovative pharmacodynamic endpoints is emphasized as an essential component of hypothesis-testing clinical trials..
Maloney, A.
Clarke, P.A.
Workman, P.
(2003). Genes and proteins governing the cellular sensitivity to HSP90 inhibitors: a mechanistic perspective. Curr cancer drug targets,
Vol.3
(5),
pp. 331-341.
show abstract
HSP90 inhibitors such as 17AAG have the major therapeutic advantage that they exert downstream inhibitory effects on multiple oncogenic client proteins. They therefore block several mission critical cancer-causing pathways and have the potential to modulate all of the hallmark biological features of malignancy. Consistent with this combinatorial anti-oncogenic profile, 17AAG exhibits broad-spectrum antitumour activity against cultured cancer cell lines and in vivo animal models. However, there are clear differences in sensitivity between various cancer cell lines and it is quite possible that some tumour types or individual patients will be more responsive in the clinic than others. We describe the methods used to investigate the genes and proteins involved in the mechanism of action of HSP90 inhibitors and discuss the significance of these for cellular sensitivity. Methods used involve the conventional cell and molecular biology techniques, together with the more recent application of high throughput global technologies such as gene expression microarrays and proteomics. Selected examples that seem to play a role in sensitivity to HSP90 inhibitors are highlighted and the potential relevance to the response of cancer patients is discussed. Important determinants of response include: 1) Dependence upon key HSP90 client proteins such as ERBB2, steroid hormone receptors and AKT/PKB; 2) Levels of HSP90 family members and co-chaperones, such as HSP70 and AHA1; and 3) expression of various cell cycle and apoptotic regulators. In the case of 17AAG, metabolic enzymes such as NQO1 and membrane efflux pumps are also important for sensitivity..
Banerji, U.
Judson, I.
Workman, P.
(2003). The clinical applications of heat shock protein inhibitors in cancer - present and future. Curr cancer drug targets,
Vol.3
(5),
pp. 385-390.
show abstract
The potential clinical applications of the prototype first-in-class Hsp90 inhibitor 17AAG and other emerging Hsp90 drugs are very exciting. Rigorously planned and executed clinical trials, incorporating measurement of appropriate biomarkers and pharmocodynamic endpoints are critical for selecting the optimal dose and schedule. A detailed understanding of the molecular mode of action of Hsp90 inhibitors alongside the elucidation of the molecular pathology of individual cancers will help us to identify tumour types and individual patients that will benefit most from treatment. Careful in vitro and in vivo experiments are needed to choose the most potentially advantageous combination studies. It is important to construct a pharmacologic audit trail linking molecular biomarkers and pharmacokinetic and pharmacodynamic parameters to tumour response endpoints. Phase I clinical studies with 17AAG have shown that the drug can be given at does that are well tolerated and that also achieve active pharmacokinetic exposures and elicit molecular signatures of gene and protein expression that are indicative of Hsp90 inhibition. Furthermore, examples of disease stabilisation have been documented, consistent with the generally cytostatic responses that are seen in animal models. Selecting tumour types for Phase II clinical trials must involve balancing 1) our knowledge of molecular response determinants, such as the expression of and dependence upon key client proteins and 2) more pragmatic evidence of antitumour activity in the relevant preclinical models. Examples of likely disease targets include chronic myeloid leukaemia, melanoma, breast, ovarian, brain, thyroid, colorectal and prostate cancer..
Workman, P.
(2003). Overview: translating Hsp90 biology into Hsp90 drugs. Curr cancer drug targets,
Vol.3
(5),
pp. 297-300.
show abstract
The Hsp90 molecular chaperone has emerged as one of the most exciting targets for cancer drug development. Hsp90 is overexpressed in many malignancies, very likely as a result of the stress that is induced both by the hostile cancer microenvironment and also by the mutation and abberant expression of oncoproteins. A particularly attractive feature of Hsp90 as a cancer drug target is that it is required for the conformational stability and function of a wide range of oncogenic 'client' proteins, including c-Raf-1, Cdk4, ErbB2, mutant p53, c-Met, Polo-1 and telomerase hTERT. Inhibition of Hsp90 should therefore block multiple mission critical oncogenic pathways in the cancer cell, leading to inhibition of all the hallmark traits of malignancy. This combinatorial blockade of oncogenic targets should give rise to board spectrum antitumour activity across multiple cancer types. The 'druggability' of Hsp90 was confirmed by the discovery that the natural products geldanamycin and radicicol, which have anticancer activity, exert their biological effects by inhibiting the essential ATPase activity associated with the N-terminal domain of the protein. The first-in-class Hsp90 inhibitor has entered clinical trial and provided proof of concept that Hsp90 can be inhibited and clinical benefit seen at non-toxic doses. Further development is underway and a related analogue 17DMAG also shows promise in preclinical models. In addition, novel Hsp90 inhibitors have been identified using methods such as high throughput screening and x-ray crystallography. The opportunities and challenges involved in translating the fast moving biology of Hsp90 into patient benefit is discussed..
Workman, P.
(2003). Strategies for treating cancers caused by multiple genome abnormalities: from concepts to cures?. Curr opin investig drugs,
Vol.4
(12),
pp. 1410-1415.
show abstract
The epidemiology, genetics/genomics and molecular biology of cancer all point to the involvement of a large number of genes in the malignant progression of the vast majority of human cancers. Our current conceptual models of cancer are discussed here and are integrated with an assessment of the strategies required for treating and potentially curing human cancers driven by multiple genome abnormalities. There are settings in which excellent responses will be seen in cancers driven primarily by single genomic abnormalities, e.g., imatinib in chronic myeloid leukemia and gastrointestinal stromal tumors. Other multigenic cancers will require drug cocktails or single drugs acting on multiple downstream targets..
Clarke, P.A.
George, M.L.
Easdale, S.
Cunningham, D.
Swift, R.I.
Hill, M.E.
Tait, D.M.
Workman, P.
(2003). Molecular pharmacology of cancer therapy in human colorectal cancer by gene expression profiling. Cancer res,
Vol.63
(20),
pp. 6855-6863.
show abstract
Global gene expression profiling has potential for elucidating the complex cellular effects and mechanisms of action of novel targeted anticancer agents or existing chemotherapeutics for which the precise molecular mechanism of action may be unclear. In this study, decreased expression of genes required for RNA and protein synthesis, and for metabolism were detected in rectal cancer biopsies taken from patients during a 5-fluorouracil infusion. Our observations demonstrate that this approach is feasible and can detect responses that may have otherwise been missed by conventional methods. The results suggested new mechanism-based combination treatments for colorectal cancer and demonstrated that expression profiling could provide valuable information on the molecular pharmacology of established and novel drugs..
Workman, P.
(2003). How much gets there and what does it do?: The need for better pharmacokinetic and pharmacodynamic endpoints in contemporary drug discovery and development. Curr pharm des,
Vol.9
(11),
pp. 891-902.
show abstract
Drug discovery is an expensive, slow and high risk enterprise. Only one in ten of the agents that enter clinical development is successful, with an average cost of US dollars 500-800 million and a typical time-scale of 10-15 years from preclinical discovery research to regulatory approval. On the other hand, many new targets are emerging from genome sequencing and the improved understanding of molecular pathology. Also, new technologies are increasing the speed and improving the efficiency of drug discovery. These new advances should facilitate progress towards the development of personalised therapies that are targeted to the genetics and molecular pathology of individual patients. The availability of pharmacokinetic (PK) and pharmacodynamic (PD) endpoints is absolutely critical to modern drug development. They allow us to understand how much of the drug gets there (into the body and ideally to the target cells) and what it does (with respect to modulation of the molecular target and the cognate biochemical pathways and downstream biological effects). PK and PD endpoints allow us to construct a pharmacological audit trail, so that all of the successive stages from drug administration through to biological effects and clinical outcome can be monitored and interpreted. This in turn provides a rational basis for decision making, e.g. stop/go, during development. An understanding of PK/PD relationships also gives us s basis for selecting the optimal drug dose and schedule. Better, less invasive methods are required. Developments in molecular/functional imaging show promise and current examples are provided..
Aherne, W.
Maloney, A.
Prodromou, C.
Rowlands, M.G.
Hardcastle, A.
Boxall, K.
Clarke, P.
Walton, M.I.
Pearl, L.
Workman, P.
(2003). Assays for HSP90 and inhibitors. Methods mol med,
Vol.85,
pp. 149-161.
Garrett, M.D.
Walton, M.I.
McDonald, E.
Judson, I.
Workman, P.
(2003). The contemporary drug development process: advances and challenges in preclinical and clinical development. Prog cell cycle res,
Vol.5,
pp. 145-158.
show abstract
We are in a new era of drug discovery, in which it is feasible to develop therapeutic agents targeted at a particular protein or biological activity in a living cell. This has been made possible by major advances in our understanding of cell and molecular biology, epitomized by the 2001 Nobel prize award for Physiology or Medicine to Lee Hartwell, Tim Hunt and Paul Nurse, who were recognised for their work on key regulators of the cell cycle. Technological advances have also played a decisive role, leading to the sequencing of the human genome and increased throughput at many stages of the drug discovery and development process. For example, developments in high throughput screening, structural biology and microarray technology are increasing the speed of drug discovery. In this chapter we focus on the long, and often difficult, pathway which leads from identification of a hit in a screen to regulatory approval of a drug for disease treatment. The emphasis in this chapter is on the development of anticancer drugs, as this is our own area of expertise and also because cancer is a disease in which the cell cycle is already a major target for therapeutic intervention. However, many of the concepts, approaches and issues are generally common to other therapeutic areas..
Seddon, B.M.
Workman, P.
(2003). The role of functional and molecular imaging in cancer drug discovery and development. Br j radiol,
Vol.76 Spec No 2,
pp. S128-S138.
show abstract
Studies of pharmacokinetics (which is what the body does to the drug) and pharmacodynamics (which is what the drug does to the body) are essential components of the modern process of cancer drug discovery and development. Defining the precise relationship between pharmacokinetics and pharmacodynamics is critical. It is especially important to establish a well understood pharmacological "audit trail" that links together all of the essential parameters of drug action, from the molecular target to the clinical effects. The pharmacological audit trail allows us to answer two absolutely crucial questions: (1) how much gets there; and (2) what does it do? During the pre-clinical drug discovery phase, it is essential that pharmacokinetic/pharmacodynamic (PK/PD) properties are optimized, so that the best candidate can be selected for clinical development. As part of contemporary mechanistic, hypothesis-testing clinical trials, construction of the pharmacological PK/PD audit trail facilitates rational decision-making. However, PK/PD endpoints frequently require invasive sampling of body fluids and tissues. Non-invasive molecular measurements, e.g. using MRI or spectroscopy, or positron emission tomography, are therefore very attractive. This review highlights the need for PK/PD endpoints in modern drug design and development, illustrates the value of PK/PD endpoints, and emphasises the importance of non-invasive molecular imaging in drug development. Examples cited include the use of PK/PD endpoints in the development of molecular therapeutic drugs such as the Hsp90 molecular chaperone inhibitor 17AAG, as well as the development of SR-4554 as a non-invasive probe for the detection of tumour hypoxia..
Leach, M.O.
Brindle, K.M.
Evelhoch, J.L.
Griffiths, J.R.
Horsman, M.R.
Jackson, A.
Jayson, G.
Judson, I.R.
Knopp, M.V.
Maxwell, R.J.
McIntyre, D.
Padhani, A.R.
Price, P.
Rathbone, R.
Rustin, G.
Tofts, P.S.
Tozer, G.M.
Vennart, W.
Waterton, J.C.
Williams, S.R.
Workman, P.
(2003). Assessment of antiangiogenic and antivascular therapeutics using MRI: recommendations for appropriate methodology for clinical trials. Brit j radiol,
Vol.76,
pp. S87-S91.
Aherne, W.
Workman, P.
(2003). Screening for novel anticancer drugs i an academic environment: The right time and the right place?. Trends in drug discovery,
Vol.4,
pp. 44-47.
Workman, P.
(2003). Preface. Current cancer drug targets,
Vol.3,
p. i.
Workman, P.
(2003). Hsp90 molecular chaperone inhibitors: Opportunities and challenges. Current cancer drug targets,
Vol.3,
pp. 297-300.
Workman, P.
Pestell, K.E.
(2003). Interview by Pestell KE: Paul Workman on the challenges of cancer drug development. Drug discovery today,
Vol.8,
pp. 775-777.
Asad, Y.
Cropp, G.
Adams, A.
O'Donnell, A.
Raynaud, F.
Judson, I.
Workman, P.
(2003). Validation of a liquid chromatography assay for the quantitation of (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl[]propionic acid (SU006668) in human plasma and its application to a phase I clinical trial. J chromatogr b,
Vol.785,
pp. 175-186.
Workman, P.
(2003). Hot topic: Hsp90 Molecular Chaperone Inhibitors: Opportunities and Challenges Preface. Current cancer drug targets,
Vol.3
(5),
pp. I-I.
Maloney, A.
Workman, P.
(2002). HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert opin biol ther,
Vol.2
(1),
pp. 3-24.
show abstract
Current anticancer drug development strategies involve identifying novel molecular targets which are crucial for tumourigenesis. The molecular chaperone heat shock protein (HSP) 90 is of interest as an anticancer drug target because of its importance in maintaining the conformation, stability and function of key oncogenic client proteins involved in signal transduction pathways leading to proliferation, cell cycle progression and apoptosis, as well as other features of the malignant phenotype such as invasion, angiogenesis and metastasis. The natural product HSP90 inhibitors geldanamycin and radicicol exert their antitumour effect by inhibiting the intrinsic ATPase activity of HSP90, resulting in degradation of HSP90 client proteins via the ubiquitin proteosome pathway. Anticancer selectivity may derive from the simultaneous combinatorial effects of HSP90 inhibitors on multiple cancer targets and pathways. 17-allylamino, 17-demethoxygeldanamycin (17AAG), a geldanamycin derivative, showed good activity and cancer selectivity in preclinical models and has now progressed to Phase I clinical trial in cancer patients with encouraging initial results. Phase II trials including combination studies with cytotoxic agents are now being planned and these should allow the therapeutic activity of 17AAG to be determined. Second generation HSP90 inhibitors may be designed to overcome some of the drawbacks of 17AAG, including limited oral bioavailability and solubility. They could also be engineered to target specific functions of HSP90, which may not only provide greater molecular selectivity and clinical benefit but may also increase understanding of the complex functions of this molecular chaperone. HSP90 inhibitors provide proof of concept for drugs directed at HSP90 and protein folding and this principle may be applicable to other medical conditions involving protein aggregation and stability..
Workman, P.
(2002). Postgenomic approaches to new drug discovery. International journal of cancer,
,
pp. 62-1.
Stimson, L.
Newbatt, Y.
Hardcastle, A.
Boxall, K.
Workman, P.
Aherne, G.W.
(2002). A cell-based ELISA is a useful pharmacodynamic and screening tool to evaluate changes in cellular acetylation. British journal of cancer,
Vol.86,
pp. S79-1.
Brader, S.
Court, W.
Box, G.
Eccles, S.A.
Workman, P.
(2002). Effects of signalling inhibitors on endothelial cell functions required for neoangiogenesis. British journal of cancer,
Vol.86,
pp. S96-1.
Smith, N.F.
Raynaud, F.I.
Griffin, R.J.
Hardcastle, I.R.
Rigoreau, L.
Stockley, M.L.
Leahy, J.J.
Golding, B.T.
Martin, N.
Smith, G.C.
Workman, P.
(2002). High throughput pharmacokinetics of novel DNA-dependent protein kinase inhibitors. British journal of cancer,
Vol.86,
pp. S111-1.
Smith, V.
Rowlands, M.G.
Barrie, E.
Workman, P.
Kelland, L.R.
(2002). Establishment and characterization of acquired resistance to the farnesyl protein transferase inhibitor R115777 in a human colon cancer cell line. Clinical cancer research,
Vol.8
(6),
pp. 2002-8.
Aherne, G.W.
Rowlands, M.G.
Stimson, L.
Workman, P.
(2002). Assays for the identification and evaluation of histone acetyltransferase inhibitors. Methods,
Vol.26
(3),
pp. 245-253.
show abstract
There is presently enormous interest in the function and regulatory roles of histone acetyltransferase enzymes. Along with deacetylases it is now evident that these enzymes play a key role in many cellular processes including chromatin remodeling and gene transcription. As such, effective small molecule enzyme inhibitors would be useful tools for molecular pharmacology and may also be suitable for further development into agents for the treatment of diseases such as cancer, A high-throughput assay based on the use of scintillating microplates (FlashPlates) suitable for screening libraries of compounds for inhibitors of acetylase activity is described here. Confirmation of activity of selected compounds is achieved with a conventional filter assay, the details of which are also described. In addition, an assay suitable for confirming that cellular protein acetylation has been altered by inhibition of acetylases or deacetylases is also presented. On the same plate, cells are grown, exposed to compound, fixed, and permeabilized, and protein acetylation is determined using standard ELISA methodology and a europium-labeled second antibody. This latter method provides a medium-throughput alternative to the use of immunoblotting for mechanistic studies. (C) 2002 Elsevier Science (USA). All rights reserved..
Easdale, S.
Clarke, P.
Titley, J.
Wooster, R.
Workman, P.
(2002). Gene expression and cell cycle analysis of colorectal cancer cell lines exposed to 5FU and 5FDURD. Brit j cancer,
Vol.86,
pp. S49-S49.
Seddon, B.M.
Maxwell, R.J.
Honess, D.J.
Grimshaw, R.
Raynaud, F.
Tozer, G.M.
Workman, P.
(2002). Validation of the fluorinated 2-nitroimidazole SR-4554 as a noninvasive hypoxia marker detected by magnetic resonance spectroscopy. Clin cancer res,
Vol.8
(7),
pp. 2323-2335.
show abstract
Tumor hypoxia is associated with poor prognosis and a more malignant tumor phenotype. SR-4554, a fluorinated 2-nitroimidazole, is selectively bioreduced and bound in hypoxic cells. We present validation studies of SR-4554 as a noninvasive hypoxia marker detected by fluorine-19 magnetic resonance spectroscopy ((19)F MRS) in the P22 carcinosarcoma, a tumor with clinically relevant hypoxia levels..
Smith, V.
Hobbs, S.
Court, W.
Eccles, S.
Workman, P.
Kelland, L.R.
(2002). ErbB2 overexpression in an ovarian cancer cell line confers sensitivity to the HSP90 inhibitor geldanamycin. Anticancer res,
Vol.22
(4),
pp. 1993-1999.
show abstract
ErbB2 is overexpressed in 25-30% of breast and ovarian cancers, correlates with poor prognosis and lower survival and has also been associated with chemoresistance. We have established an isogenic pair of human ovarian cells that differ only in the expression of erbB2 protein in order to elucidate the role of the protein in determining cellular sensitivity to various drugs and agents. These included cisplatin and paclitaxel, the main drugs used in the treatment of ovarian cancer, and also various signal transduction inhibitors affecting the ras and PI3K pathways. Transfection of erbB2 resulted in cells stably overexpressing the protein and showing increased motility compared to the empty vector control cells. In cells overexpressing erbB2, the most notable effect on chemosensitivity was that of significantly increased (5-fold) sensitivity to the heat shock protein 90 (HSP90) molecular chaperone inhibitor geldanamycin. In contrast, erbB2-overexpressing cells showed statistically significant resistance to cisplatin, the PI3K inhibitor LY294002 and the tyrosine kinase inhibitor emodin. No significant difference in growth inhibition was observed after exposure to paclitaxel, two additional HSP90 inhibitors radicicol and 17AAG, the cyclin-dependent kinase inhibitor flavopiridol, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor PD153035, the mek inhibitor UO126 or the farnesyl transferase inhibitor R115777. Exposure of cells to geldanamycin, 17AAG, emodin, LY294002 and cisplatin led to depletion of erbB2 in the transfected cells. These data suggest that erbB2 status in ovarian cancr may contribute to chemosensitivity, in some cases leading to increased sensitivity (as with geldanamycin) but in other cases leading to resistance (as with cisplatin)..
Workman, P.
(2002). Challenges of PK/PD measurements in modern drug development. Eur j cancer,
Vol.38
(16),
pp. 2189-2193.
Panaretou, B.
Siligardi, G.
Meyer, P.
Maloney, A.
Sullivan, J.K.
Singh, S.
Millson, S.H.
Clarke, P.A.
Naaby-Hansen, S.
Stein, R.
Cramer, R.
Mollapour, M.
Workman, P.
Piper, P.W.
Pearl, L.H.
Prodromou, C.
(2002). Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1. Mol cell,
Vol.10
(6),
pp. 1307-1318.
show abstract
Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle..
Liu, D.
Hutchinson, O.C.
Osman, S.
Price, P.
Workman, P.
Aboagye, E.O.
(2002). Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin. British journal of cancer,
Vol.87
(7),
pp. 783-7.
Workman, P.
Kaye, S.B.
(2002). Translating basic cancer research into new cancer therapeutics. Trends mol med,
Vol.8
(4 Suppl),
pp. S1-S9.
Aherne, G.W.
McDonald, E.
Workman, P.
(2002). Finding the needle in the haystack: why high-throughput screening is good for your health. Breast cancer res,
Vol.4
(4),
pp. 148-154.
show abstract
High-throughput screening is an essential component of the toolbox of modern technologies that improve speed and efficiency in contemporary cancer drug development. This is particularly important as we seek to exploit, for maximum therapeutic benefit, the large number of new molecular targets emerging from the Human Genome Project and cancer genomics. Screening of diverse collections of low molecular weight compounds plays a key role in providing chemical starting points for iterative optimisation by medicinal chemistry. Examples of successful drug discovery programmes based on high-throughput screening are described, and these offer potential in the treatment of breast cancer and other malignancies..
Workman, P.
(2002). Pharmacogenomics in cancer drug discovery and development: inhibitors of the Hsp90 molecular chaperone. Cancer detect prev,
Vol.26
(6),
pp. 405-410.
show abstract
Drug discovery is being revolutionised by a number of technological developments. These include high throughput screening, combinatorial chemistry and genomics. The impact of the new technologies is to accelerate the pace of anticancer discovery. The completion of the Human Genome Project and the ongoing high throughput sequencing of cancer genomes will facilitate the identification of a range of new molecular targets for drug discovery. Over the next few years we will have a complete molecular understanding of the various combinations of genes and cognate pathways that drive the malignant phenotype and tumour progression. The vision for postgenomic cancer drug discovery must be to identify therapeutic agents that correct or exploit each of these molecular abnormalities. In this way, it will be possible to develop personalised drug combinations that are targeted to the molecular make up of individual tumours. It is anticipated that these therapies will be more effective and less toxic than current approaches, although combinations of novel agents with existing cytotoxic therapies are likely to continue for some time. Examples of postgenomic, mechanism-based drugs include Glivec, Herceptin and Iressa, with many more agents undergoing preclinical and clinical development. An interesting new approach involves the development of inhibitors of heat shock protein (Hsp90) molecular chaperone. Because Hsp90 is required for the correct folding, stability and function of a range of oncoproteins that are mutated or over expressed in cancer, Hsp90 inhibitors have the potential to provide a simultaneous, combinatorial attack on multiple oncogenic pathways. By depleting the levels of multiple oncoproteins in cancer cells and blocking a wide range of oncogenic pathways, Hsp90 inhibitors have the potential to inhibit all of the hallmark characteristics of cancer cells. Progress in the preclinical and clinical development of Hsp90 inhibitors will be described, including an update on clinical studies with the first-in-class agent 17AAG. The use of the postgenomic technology of gene expression microarrays in cancer pharmacology and drug development will be exemplified..
Workman, P.
(2002). Cancer genome targets: Raf-ing up tumour cell to overcome oncogene addiction. Expert rev anticancer ther,
Vol.2,
pp. 611-614.
Workman, P.
(2002). The impact of genomic and protoeomic technologies on the development of new cancer drugs. Ann oncolo,
Vol.13,
pp. 115-124.
Workman, P.
(2002). Priorities for cancer research in the 21st century: Translating the cancer genome into new cancer therapies. The vista de oncologia,
Vol.4,
p. 4.
Workman, P.
(2002). Cancer - 21st century solutions. Biotechnology investment today,
,
p. 1.
Workman, P.
(2002). From cancer genomics to new cancer therapeutics. Eur j hum genet,
Vol.10,
p. 56.
Turlais, F.
Hardcastle, A.
Rowlands, M.
Newbatt, Y.
Bannister, A.
Kouzarides, T.
Workman, P.
Aherne, G.W.
(2001). High-throughput screening for identification of small molecule inhibitors of histone acetyltransferases using scintillating microplates (FlashPlate). Anal biochem,
Vol.298
(1),
pp. 62-68.
show abstract
The role of histone acetyltransferases (HATs) in the regulation of crucial cellular functions, e.g., gene transcription, differentiation, and proliferation, has recently been documented and there is increasing evidence that aberrant expression of these enzymes may have a role to play in the development of the malignant phenotype. The availability of potent and selective small molecule inhibitors of HATs would provide useful proof of principle probes for further validation of these enzymes as drug discovery targets and may also provide lead molecules for clinical drug development. We have developed a microplate assay for HAT activity suitable for high-throughput screening. In the assay, following incubation of histone H3, [3H]acetylCoA, and enzyme (recombinant p300/CBP-associated factor expressed as a glutathione S-transferase fusion protein), radiolabeled histone was captured onto the walls of a scintillating microplate (FlashPlate) generating a scintillation signal. The assay was reproducible, amenable to automation, and generated a wide signal to noise ratio. Although antiacetylated histone antibodies were initially used to capture the radiolabeled product, it was subsequently shown that a signal was effectively produced by histone passively binding to the walls of the FlashPlate. This resulted in a simple "mix and measure" assay that is currently being used for the identification of HAT inhibitors..
Workman, P.
(2001). Gasping for breath: Development of therapeutic and imaging agents targeting tumour hypoxia and bioreductive enzymology. British journal of cancer,
Vol.85,
pp. 4-1.
Hutchinson, O.C.
Liu, D.
Osman, S.
Price, P.
Workman, P.
Aboagye, E.O.
(2001). Use of C-14 methyl choline to monitor inhibition of the extracellular signal-regulated kinase cascade: A novel approach towards imaging of signal-transduction inhibition. British journal of cancer,
Vol.85,
pp. 89-1.
Smith, N.F.
Raynaud, F.L.
Goddard, P.M.
Workman, P.
(2001). Comparison of the plasma pharmacokinetics of 17-allylamino-17-demethoxygeldamycin analogues following cassette and single dosing. British journal of cancer,
Vol.85,
pp. 91-1.
Smith, V.
Hobbs, S.
Workman, P.
Kelland, L.R.
(2001). The effect of overexpression of C-ERBB2 on the chemosensitivity of a human ovarian carcinoma cell line. British journal of cancer,
Vol.85,
pp. 100-1.
Seddon, B.
Simmons, L.
Grimshaw, R.
Tan, S.
Raynaud, F.
Judson, I.
Workman, P.
Payne, G.
Leach, M.
(2001). Phase I pharmacokinetic and spectroscopic study of SR 4554: A new nitroimidazole used as a non-invasive probe of tumor hypoxia detected by F-19 magnetic resonance spectroscopy. Clinical cancer research,
Vol.7
(11),
pp. 3700S-1.
Smith, N.F.
Goddard, P.M.
Clark, S.J.
Raynaud, F.I.
Workman, P.
(2001). Plasma and tissue pharmacokinetics of 17-allylamino-17-demethoxygeldanamycin (17AAG) analogues following cassette and single dosing. Clinical cancer research,
Vol.7
(11),
pp. 3734S-2.
Rowlands, M.G.
Newbatt, Y.M.
Turlais, F.
Prodromou, C.
Pearl, L.
Workman, P.
Aherne, W.
(2001). High throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Clinical cancer research,
Vol.7
(11),
pp. 3749S-1.
Workman, P.
(2001). Mechanism-based screening, combinatorial chemistry and microarrays in antisignalling drug development. Clinical cancer research,
Vol.7
(11),
pp. 3821S-1.
Bonn, D.
Workman, P.
(2001). Paul Workman - at the cutting edge of drug discovery. Lancet oncology,
Vol.2
(2),
pp. 113-118.
Workman, P.
(2001). New drug targets for genomic cancer therapy: successes, limitations, opportunities and future challenges. Curr cancer drug targets,
Vol.1
(1),
pp. 33-47.
show abstract
Cancer drug therapy is undergoing a major transition from the previous pregenomic cytotoxic era to the new postgenomic era. Future mechanism-based therapeutic agents will increasingly be designed to act on molecular targets that are causally involved in the malignant progression of human cancers. Such agents are predicted to show greater therapeutic selectivity for cancer versus normal cells. New cancer drug targets are identified and validated in various ways. The determination of the normal human genome sequence, followed by that of multiple cancer genomes, is accelerating target discovery. Other new technologies, particularly high throughput screening, combinatorial chemistry and gene expression microarrays, are increasing the speed and efficiency of drug development. Examples of new molecular therapeutics showing promising activity in the clinic include Herceptin, Glivec and Iressa. However, many challenges remain as we test the vision of individualised combinatorial genome-based therapy, using drugs targeted to every significant molecular abnormality in cancer..
Seddon, B.M.
Honess, D.J.
Vojnovic, B.
Tozer, G.M.
Workman, P.
(2001). Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic electrode in the p22 tumor. Radiat res,
Vol.155
(6),
pp. 837-846.
show abstract
Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO(2) readings lay in a range over twice that of sensor pO(2(5min)) values (-3.2 to 80 mm Hg and -0.1 to 34.8 mm Hg, respectively). However, there was no significant difference between the means +/- 2 SE of the median pO(2) values recorded by each instrument (11.0 +/- 3.3 and 8.1 +/- 1.9 mm Hg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means +/- 2 SE of the median pO(2) values reported by the electrode and sensor (1.7 +/- 0.9 and 2.9 +/- 0.7 mm Hg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO(2) values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO(2) values of 0.6 to 28.1 mm Hg..
Workman, P.
Clarke, P.A.
(2001). Innovative cancer drug targets: genomics, transcriptomics and clinomics. Expert opin pharmacother,
Vol.2
(6),
pp. 911-915.
Workman, P.
(2001). Scoring a bull's-eye against cancer genome targets. Curr opin pharmacol,
Vol.1
(4),
pp. 342-352.
show abstract
New anticancer drugs are increasingly targeted to specific abnormalities in the sequence and expression of a series of genes that operate in a stepwise, combinatorial manner to drive the progression of human cancer. These new-generation molecular therapeutics are expected to be more effective and less toxic than the broadly antiproliferative cytotoxic drugs of the previous era, which still dominate medical treatment of cancer today. Molecular and genomic technologies, particularly the availability of the human genome sequence and the ongoing sequencing of cancer genomes, are now having a major impact on target discovery and validation. Over the past year, the progress of three novel anticancer agents in particular (Herceptin, Glivec and Iressa) has exemplified the potential utility of innovative molecular therapeutics in the clinic. Drugs acting on a range of new genome-based molecular targets are now in preclinical and clinical development..
Workman, P.
(2001). Overview: changing times: developing cancer drugs in genomeland. Curr opin investig drugs,
Vol.2
(8),
pp. 1128-1135.
Kelland, L.R.
Smith, V.
Valenti, M.
Patterson, L.
Clarke, P.A.
Detre, S.
End, D.
Howes, A.J.
Dowsett, M.
Workman, P.
Johnston, S.R.
(2001). Preclinical antitumor activity and pharmacodynamic studies with the farnesyl protein transferase inhibitor R115777 in human breast cancer. Clin cancer res,
Vol.7
(11),
pp. 3544-3550.
show abstract
Antitumor and pharmacodynamic studies were performed in MCF-7 human breast cancer cells and companion xenografts with the farnesyl protein transferase inhibitor, R115777, presently undergoing Phase II clinical trials, including in breast cancer. R115777 inhibited growth of MCF-7 cells in vitro with an IC(50) of 0.31 +/- 0.25 microM. Exposure of MCF-7 cells to increasing concentrations of R115777 for 24 h resulted in the inhibition of protein farnesylation, as indicated by the appearance of prelamin A at concentrations >1 microM. After continuous exposure to 2 microM R115777, prelamin A levels peaked at 2 h post drug exposure and remained high for up to 72 h. R115777 administered p.o. twice daily for 10 consecutive days to mice bearing established s.c. MCF-7 xenografts induced tumor inhibition at a dose of 25 mg/kg [percentage of treated versus control (% T/C) = 63% at day 21]. Greater inhibition was observed at doses of 50 mg/kg (% T/C at day 21 = 38%) or 100 mg/kg (% T/C at day 21 = 43%). The antitumor effect appeared to be mainly cytostatic with little evidence of tumor shrinkage to less than the starting volume. Tumor response correlated with an increase in the appearance of prelamin A, but no changes in the prenylation of lamin B, heat shock protein 40, or N-Ras were detectable. In addition, significant increases in apoptotic index and p21(WAF1/CIP1) expression were observed, concomitant with a decrease in proliferation as measured by Ki-67 staining. An increase in prelamin A was also observed in peripheral blood lymphocytes in a breast cancer patient who responded to R115777. These data show that R115777 possesses preclinical antitumor activity against human breast cancer and that the appearance of prelamin A may provide a sensitive and convenient pharmacodynamic marker of inhibition of prenylation and/or response..
Smith, V.
Raynaud, F.
Workman, P.
Kelland, L.R.
(2001). Characterization of a human colorectal carcinoma cell line with acquired resistance to flavopiridol. Mol pharmacol,
Vol.60
(5),
pp. 885-893.
show abstract
Flavopiridol is a broad-spectrum inhibitor of cyclin-dependent kinases (cdks) and represents the first in this anticancer class to enter clinical trials. In anticipation of the likelihood that, as with other cancer drugs, acquired resistance may limit the drug's efficacy, an acquired resistance model has been established by in vitro drug exposure of the human colon carcinoma cell line HCT116. This stably resistant line, possessing 8-fold resistance to flavopiridol, showed a lack of cross-resistance to the anticancer agents etoposide, doxorubicin, paclitaxel, topotecan, and cisplatin, and notably to other chemical classes of cdk inhibitors: the aminopurines roscovitine and purvalanol A, 9-nitropaullone, and hymenialdisine. Resistance did not seem to be related to differences in the levels of multidrug resistance drug efflux proteins, P-glycoprotein, and MRP1. Moreover, there were no changes in overall drug accumulation between the resistant and sensitive cell lines. Flavopiridol induced cell cycle arrest, apoptosis, and inhibition of retinoblastoma gene product phosphorylation on serine 780 in both parental and resistant lines, but the latter required 8-fold higher concentrations to achieve these effects. Cyclin E protein levels and cyclin E-associated kinase activity were increased in the resistant line, suggesting that overexpression of cyclin E may be the mechanism of resistance to flavopiridol. However, transfection of cyclin E to increase expression of this protein did not result in an increase in resistance to flavopiridol. Thus, up-regulation of cyclin E alone does not seem to cause resistance to this cdk inhibitor..
Ross, P.J.
George, M.
Cunningham, D.
DiStefano, F.
Andreyev, H.J.
Workman, P.
Clarke, P.A.
(2001). Inhibition of Kirsten-ras expression in human colorectal cancer using rationally selected Kirsten-ras antisense oligonucleotides. Mol cancer ther,
Vol.1
(1),
pp. 29-41.
show abstract
Kirsten-ras is frequently mutated in colorectal cancers and may be an important therapeutic target, particularly because we have previously shown that acquisition of a mutation is associated with a poorer outcome. Understanding the role of Kirsten-ras and the consequences of inhibiting its activity or expression will contribute to our comprehension of colorectal cancer biology and may help to rationalize the choice of molecular targets suitable for therapeutic manipulation. Therefore we undertook a simple screen, incubating a library of oligonucleotides with Kirsten-ras mRNA and RNase H to identify an antisense oligonucleotide that effectively inhibited Kirsten-ras expression. We show for the first time in a human colon cancer cell line that inhibition of Kirsten-ras expression inhibits constitutive phosphorylation of Erk1/2, but not c-Akt, suggesting that in these cells constitutive phosphorylation of Erk 1/2 is dependent upon Kirsten-ras. Successful inhibition of Kirsten-ras had little effect on cell number or cell death and there was no evidence for accumulation of cells in any particular phase of the cell cycle. Kirsten-ras inhibition significantly reduced secretion of VEGF-A165 into the culture medium. Gene expression profiling by microarray detected altered expression of a number of genes. Of particular interest for future studies was the altered expression of genes encoding products involved in protein trafficking and the potential effects of these changes on cell adhesion. Our results suggest that, at least in this model, Kirsten-ras may contribute to malignancy predominantly through effects on angiogenesis, invasion, and metastasis, and that therapies directed at Kirsten-ras, including antisense approaches, may have particular utility through these mechanisms..
Hostein, I.
Robertson, D.
DiStefano, F.
Workman, P.
Clarke, P.A.
(2001). Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer res,
Vol.61
(10),
pp. 4003-4009.
show abstract
17-Allylamino-17-demethoxygeldanamycin (17AAG) is a first-in-class heat shock protein 90 (Hsp90) molecular chaperone inhibitor to enter clinical trials. The downstream molecular and cellular consequences of Hsp90 inhibition are not well defined. 17AAG has shown activity against human colon cancer in cell culture and xenograft models. In this study, we demonstrated that in addition to depleting c-Raf-1 and inhibiting ERK-1/2 phosphorylation in human colon adenocarcinoma cells, 17AAG also depleted N-ras, Ki-ras, and c-Akt and inhibited phosphorylation of c-AKT: A consequence of these events was the induction of cell line-dependent cytostasis and apoptosis, although the latter did not result from dephosphorylation of proapoptotic BAD: One cell line, KM12, did not exhibit apoptosis and in contrast to the other cell lines overexpressed Bag-1, but did not express BAX: Taken together with other determinants of 17AAG sensitivity, these results should contribute to a more complete understanding of the molecular pharmacology of 17AAG, which in turn should aid the future rational clinical development and use of the drug in colon and other tumor types..
Clarke, P.A.
te Poele, R.
Wooster, R.
Workman, P.
(2001). Gene expression microarray analysis in cancer biology, pharmacology, and drug development: progress and potential. Biochem pharmacol,
Vol.62
(10),
pp. 1311-1336.
show abstract
With the imminent completion of the Human Genome Project, biomedical research is being revolutionised by the ability to carry out investigations on a genome wide scale. This is particularly important in cancer, a disease that is caused by accumulating abnormalities in the sequence and expression of a number of critical genes. Gene expression microarray technology is gaining increasingly widespread use as a means to determine the expression of potentially all human genes at the level of messenger RNA. In this commentary, we review developments in gene expression microarray technology and illustrate the progress and potential of the methodology in cancer biology, pharmacology, and drug development. Important applications include: (a) development of a more global understanding of the gene expression abnormalities that contribute to malignant progression; (b) discovery of new diagnostic and prognostic indicators and biomarkers of therapeutic response; (c) identification and validation of new molecular targets for drug development; (d) provision of an improved understanding of the molecular mode of action during lead identification and optimisation, including structure-activity relationships for on-target versus off-target effects; (e) prediction of potential side-effects during preclinical development and toxicology studies; (f) confirmation of a molecular mode of action during hypothesis-testing clinical trials; (g) identification of genes involved in conferring drug sensitivity and resistance; and (h) prediction of patients most likely to benefit from the drug and use in general pharmacogenomic studies. As a result of further technological improvements and decreasing costs, the use of microarrays will become an essential and potentially routine tool for cancer and biomedical research..
Bailey, S.M.
Lewis, A.D.
Patterson, L.H.
Fisher, G.R.
Knox, R.J.
Workman, P.
(2001). Involvement of NADPH: cytochrome P450 reductase in the activation of indoloquinone EO9 to free radical and DNA damaging species. Biochemical pharmacology,
Vol.62
(4),
pp. 461-8.
Anderson, H.
Price, P.
Blomley, M.
Leach, M.O.
Workman, P.
Canc Res Campaign PK PD Technolog,
(2001). Measuring changes in human tumour vasculature in response to therapy using functional imaging techniques. Brit j cancer,
Vol.85
(8),
pp. 1085-1093.
show abstract
Antiangiogenic and antivascular agents provide new approaches to treating tumours. These may avoid many of the problems experienced with current approaches such as inherent and acquired resistance to treatment. Tumours do not grow beyond 1-2 mm(3) in size without the development of new vessels (Folkman, 1971). Such neo-vascularization (angiogenesis) allows tumour cells to increase their nutrient supply, survive and proliferate despite the new vessels often having structural and functional differences compared to normal tissue vasculature. Treatments targeted at tumour vasculature have produced impressive results in animal models (Lindsay et al, 1996; Watson et al, 1996; O'Reilly, 1997; Horsman et al, 1998). These therapies are now entering clinical trials, However, the successful introduction of these therapies into clinical practice will require the development of reliable ways to assess angiogenesis and its modification or inhibition in vivo. Here we discuss some of the emerging imaging techniques that may be useful. (C) 2001 Cancer Research Campaign..
Hardcastle, A.
Clinton, S.
Goddard, P.
Brunton, L.
Valenti, M.
Workman, P.
Aherne, G.W.
(2000). Localisation of hypoxic cells using a polyclonal antiserum which recognises protein adducts of the 2-nitroimidazole probe SR4554. British journal of cancer,
Vol.83,
pp. 32-1.
Smith, V.
Workman, P.
Kelland, L.R.
(2000). Characterisation of a human colon cell line possessing acquired resistance to flavopiridol. British journal of cancer,
Vol.83,
pp. 62-1.
Raynaud, F.I.
Nutley, B.P.
Goddard, P.M.
Fischer, P.
McClue, S.
Lane, D.
Workman, P.
(2000). Cassette dosing is a valid approach to evaluate the pharmacokinetics of trisubstituted purines such as olomoucine, roscovitine and bohemine. British journal of cancer,
Vol.83,
pp. 68-1.
Workman, P.
Garrett, M.D.
Kelland, L.R.
Nutley, B.P.
Eccles, S.A.
Jarman, M.
Hardcastle, I.
Bannister, A.
Kouzarides, T.
Aherne, G.W.
(2000). Contemporary mechanism-based screening, combinatorial chemistry and drug development against new cancer targets in an academic research institute environment. Clinical cancer research,
Vol.6,
pp. 4476S-1.
Aherne, G.W.
Hardcastle, A.
Turlais, F.
Newbatt, Y.
Boxall, C.
Clarke, E.
Jarman, M.
Dick, A.
Bannister, A.
Kouzarides, T.
Workman, P.
(2000). Experience of high-throughput screening (HTS) in an academic drug discovery organisation. Clinical cancer research,
Vol.6,
pp. 4497S-1.
Raynaud, F.I.
Nutley, B.P.
Goddard, P.M.
Kelland, L.R.
Valenti, M.
Brunton, L.
Eady, D.
Bell, G.
Marriage, H.
Fisher, P.
McClue, S.
Lane, D.
Workman, P.
(2000). Antitumour activity and oral bioavailability of the cyclin dependent kinase (CDK) inhibitor roscovitine. Clinical cancer research,
Vol.6,
pp. 4529S-1.
Price, T.
Walton, M.I.
Titley, J.
Cunningham, D.
Workman, P.
(2000). Studies on the mechanism of action of the cyclin dependent kinase inhibitors 9-nitro and 10-bromopaullone in human colon tumour cell lines. Clinical cancer research,
Vol.6,
pp. 4530S-1.
Maloney, A.
Clarke, P.A.
Walton, M.I.
Prodromou, C.
Pearl, L.
Sharp, S.
Kelland, L.
Workman, P.
(2000). Gene expression profiling of HT29 colon carcinoma cells after treatment with analogues of the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17AAG) and the structurally dissimilar compound radicicol. Clinical cancer research,
Vol.6,
pp. 4532S-1.
Price, T.
Walton, M.
Cunningham, D.
Workman, P.
(2000). Activity of paullones in colon cell lines does not correlate to cell cycle protein expression. Annals of oncology,
Vol.11,
pp. 13-1.
Smith, N.F.
Raynaud, F.I.
Workman, P.
(2000). Development of an HPLC-APCI-MS/MS method for the simultaneous determination of 17-allylamino-17-demethoxygeldanamycin (17-AAG) analogues in mouse plasma. Clinical cancer research,
Vol.6,
pp. 4545S-2.
Smith, V.
Workman, P.
Kelland, L.
(2000). Drug resistance to the farnesyl transferase inhibitor R115777. Clinical cancer research,
Vol.6,
pp. 4546S-2.
Poele, R.T.
Maillard, K.
Wooster, R.
Workman, P.
(2000). The use of expression profiling to identify genes regulated by the PI3K inhibitor LY294002. Clinical cancer research,
Vol.6,
pp. 4548S-1.
Troy, H.
McSheehy, P.M.
Judson, I.R.
Workman, P.
Leach, M.O.
Griffiths, J.R.
(2000). Early detection of the response of human HT29 adenocarcinomas to the thioredoxin inhibitor, PX12, using P-31-magnetic resonance spectroscopy (MRS) in vivo. Clinical cancer research,
Vol.6,
pp. 4563S-1.
Sharp, S.Y.
Kelland, L.R.
Valenti, M.R.
Brunton, L.A.
Hobbs, S.
Workman, P.
(2000). Establishment of an isogenic human colon tumor model for NQO1 gene expression: application to investigate the role of DT-diaphorase in bioreductive drug activation in vitro and in vivo. Mol pharmacol,
Vol.58
(5),
pp. 1146-1155.
show abstract
Many tumors overexpress the NQO1 gene, which encodes DT-diaphorase (NADPH:quinone oxidoreductase; EC 1.6.99.2). This obligate two-electron reductase deactivates toxins and activates bioreductive anticancer drugs. We describe the establishment of an isogenic human tumor cell model for DT-diaphorase expression. An expression vector was used in which the human elongation factor 1alpha promoter produces a bicistronic message containing the genes for human NQO1 and puromycin resistance. This was transfected into the human colon BE tumor line, which has a disabling point mutation in NQO1. Two clones, BE2 and BE5, were selected that were shown by immunoblotting and enzyme activity to stably express high levels of DT-diaphorase. Drug response was determined using 96-h exposures compared with the BE vector control. Functional validation of the isogenic model was provided by the much greater sensitivity of the NQO1-transfected cells to the known DT-diaphorase substrates and bioreductive agents streptonigrin (113- to 132-fold) and indoloquinone EO9 (17- to 25-fold) and the inhibition of this potentiation by the DT-diaphorase inhibitor dicoumarol. A lower degree of potentiation was seen with the clinically used agent mitomycin C (6- to 7-fold) and the EO9 analogs, EO7 and EO2, that are poorer substrates for DT-diaphorase (5- to 8-fold and 2- to 3-fold potentiation, respectively), and there was no potentiation or protection with menadione and tirapazamine. Exposure time-dependent potentiation was seen with the diaziquone analogs methyl-diaziquone and RH1 [2, 5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], the latter being an agent in preclinical development. In contrast to the in vitro potentiation, there was no difference in the response to mitomycin C when BE2 and BE vector control were treated as tumor xenografts in vivo. This isogenic model should be valuable for mechanistic studies and bioreductive drug development..
Clarke, P.A.
Hostein, I.
Banerji, U.
Stefano, F.D.
Maloney, A.
Walton, M.
Judson, I.
Workman, P.
(2000). Gene expression profiling of human colon cancer cells following inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, an inhibitor of the hsp90 molecular chaperone. Oncogene,
Vol.19
(36),
pp. 4125-4133.
show abstract
A number of molecular therapeutic agents, derived from exploiting our knowledge of the oncogenic pathways that are frequently deregulated in cancer, are now entering clinical trials. One of these is the novel agent 17-allylamino-17-demethoxygeldanamycin that acts to inhibit the hsp90 molecular chaperone. Treatment of four human colon cancer cell lines with iso-effective concentrations of this agent resulted in depletion of c-raf-1 and akt and inhibition of signal transduction. We have used gene expression array analysis to identify genes responsive to treatment with this drug. The expression of hsp90 client protein genes was not affected, but hsc hsp70, hsp90beta, keratin 8, keratin 18 and caveolin-1 were deregulated following treatment. These observations were consistent with inhibition of signal transduction and suggested a possible mechanism of resistance or recovery from 17-allylamino-17-demethoxygeldanamycin treatment. The results shed light on the molecular mode of action of the hsp90 inhibitors, and suggest possible molecular markers of drug action for use in hypothesis testing clinical trials. Oncogene (2000) 19, 4125 - 4133.
Workman, P.
(2000). Biotechnology and the discovery of new anticancer drugs Part 1: Locating new targets. Helix,
Vol.1,
pp. 31-33.
Workman, P.
(2000). Biotechnology and the discovery of new anticancer drugs Part 2: From genes to drugs. Helix,
Vol.2,
pp. 32-33.
Workman, P.
(2000). Overcoming cross-talk related signalling in tumours with therapeutic agents. Pezcoller foundation year 7,
Vol.Number 15,
pp. 16-17.
Workman, P.
(2000). Towards genomic cancer pharmacology: Innovative drugs for the new millenium. Current opinion in oncologic, endocrine & metabolic investigational drugs,
Vol.2,
pp. 21-25.
Workman, P.
(2000). Towards intelligent drug design in the post-genome era?. Anticancer drug des,
Vol.1386,
pp. 525-531.
Workman, P.
(2000). At the cutting edge of discovery (interview by D Bonn). Lancet oncol,
Vol.2,
pp. 113-118.
Plumb, J.A.
Sludden, J.
Workman, P.
(1999). Increased activity of DT-diaphorase in ras transformed mouse fibroblasts may be due to DNA hypomethylation. British journal of cancer,
Vol.80,
pp. 81-1.
Goddard, P.M.
Raynaud, F.
Nutley, B.
Fisher, P.
Lane, D.
Workman, P.
(1999). Pharmacokinetics of olomoucine, a cyclin dependent kinase inhibitor following IV administration to mice. British journal of cancer,
Vol.80,
pp. 92-1.
Pestell, K.E.
Walton, M.I.
Titley, J.C.
Kelland, L.R.
Lane, D.
Workman, P.
(1999). The role of p53 in determining chemosensitivity of two novel aminopurine based cyclin dependent kinase inhibitors. Clinical cancer research,
Vol.5,
pp. 3755S-1.
Maloney, A.
Walton, M.I.
Sharp, S.
Kelland, L.R.
Jarman, M.
Prodromou, C.
Pearl, L.
Workman, P.
(1999). Structure-activity relationships of the Hsp90 inhibitor 17-allylamino 17-demethoxy geldanamycin analogues (17AAG). Clinical cancer research,
Vol.5,
pp. 3781S-1.
Kelland, L.R.
Sharp, S.Y.
Valenti, M.R.
Brunton, L.A.
Workman, P.
(1999). An isogenic human colon model for NQO1 and its application in determining the role of DT-diaphorase in the antitumour activity of a range of quinone-based agents. Clinical cancer research,
Vol.5,
pp. 3818S-2.
Raynaud, F.I.
Nutley, B.P.
Goddard, P.
Fisher, P.
Marriage, H.
Lane, D.
Workman, P.
(1999). Pharmacokinetics of the cyclin dependent kinase inhibitors Olomoucine, CYC201 and CYC202 in Balb C- mice after iv administration. Clinical cancer research,
Vol.5,
pp. 3838S-1.
Orr, R.M.
Valenti, M.R.
Brunton, L.A.
Carnochan, P.
Brooks, R.C.
Wancewicz, E.
Monia, B.P.
Workman, P.
(1999). Mouse strain differences affect the antitumor activity of ISIS 2503, an antisense phosphorothioate oligodeoxynucleotide targeted to Ha-ras, against the MDA-MB-231 breast carcinoma. Clinical cancer research,
Vol.5,
pp. 3849S-1.
Padhani, A.
Hayes, C.
Judson, I.
Workman, P.
Langecker, P.
Leach, M.
Husband, J.
(1999). Dynamic contrast enhanced MR imaging in the evaluation of antiangiogenesis therapy. Clinical cancer research,
Vol.5,
pp. 3828S-1.
Gelmon, K.A.
Eisenhauer, E.A.
Harris, A.L.
Ratain, M.J.
Workman, P.
(1999). Anticancer agents targeting signaling molecules and cancer cell environment: Challenges for drug development?. J natl cancer i,
Vol.91
(15),
pp. 1281-1287.
Maloney, A.
Walton, M.I.
Kelland, L.R.
Workman, P.
(1999). DNA damage is not involved in the potentiation of the cytotoxicity of 17-allyamino 17-demethoxy geldanamycin (17AAG) by DT-diaphorase (DTD). Brit j cancer,
Vol.80,
pp. 46-46.
Garrett, M.D.
Workman, P.
(1999). Discovering novel chemotherapeutic drugs for the third millennium. Eur j cancer,
Vol.35
(14),
pp. 2010-2030.
show abstract
There is enormous potential for the discovery of innovative cancer drugs with improved efficacy and selectivity for the third millennium. In this review we show how novel mechanism-based agents are being discovered by focusing on the molecular targets and pathways that are causally involved in cancer formation, maintenance and progression. We also show how new technologies, from genomics through high through-put bioscience, combinatorial chemistry, rational drug design and molecular pharmacodynamic and imaging techniques, are accelerating the pace of cancer drug discovery. The process of contemporary small molecule drug discovery is described and progress and current issues are reviewed. New and potential targets and pathways for therapeutic intervention are illustrated. The first examples of a new generation of molecular therapeutics are now entering hypothesis-testing clinical trials and showing activity. The early years of the new millennium will see a range of exciting new agents moving from bench to bedside and beginning to impact on the management and cure of cancer..
Kelland, L.R.
Sharp, S.Y.
Rogers, P.M.
Myers, T.G.
Workman, P.
(1999). DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90. J natl cancer inst,
Vol.91
(22),
pp. 1940-1949.
show abstract
To our knowledge, 17-allylamino,17-demethoxygeldanamycin (17AAG) is the first inhibitor of heat shock protein 90 (Hsp90) to enter a phase I clinical trial in cancer. Inhibition of Hsp90, a chaperone protein (a protein that helps other proteins avoid misfolding pathways that produce inactive or aggregated states), leads to depletion of important oncogenic proteins, including Raf-1 and mutant p53 (also known as TP53). Given its ansamycin benzoquinone structure, we questioned whether the antitumor activity of 17AAG was affected by expression of the NQO1 gene, which encodes the quinone-metabolizing enzyme DT-diaphorase..
Workman, P.
(1999). 94 United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) (P Workman Member of Committee) United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) Strategy Group Workshop. Br j cancer,
Vol.79,
pp. 2-3.
Workman, P.
(1999). The impact of biotechnology on the discovery of new anticancer drugs. Helix,
.
Workman, P.
(1999). Where are the drugs of the future?. Cancer topics,
Vol.11,
pp. 15-19.
Bailey, S.M.
Lewis, A.D.
Knox, R.J.
Patterson, L.H.
Fisher, G.R.
Workman, P.
(1998). Reduction of the indoloquinone anticancer drug EO9 by purified DT-diaphorase: A detailed kinetic study and analysis of metabolites. Biochemical pharmacology,
Vol.56
(5),
pp. 613-9.
Workman, P.
Kelland, L.
(1998). Preclinical assessment: Should we modify our preclinical evaluation for agents aimed at new molecular targets?. Annals of oncology,
Vol.9,
pp. 5-1.
Workman, P.
Raynaud, F.
Judson, I.
Kelson, A.
Tracy, M.
Lewis, A.
Obagye, E.
(1998). Oxygen-sensitive reductive metabolism of the fluorinated 2-nitroimidazole SR 4554 (CRC 94/17): Support for clinical development as a non-invasive hypoxia detection probe. Annals of oncology,
Vol.9,
pp. 29-1.
Aboagye, E.O.
Kelson, A.B.
Tracy, M.
Workman, P.
(1998). Preclinical development and current status of the fluorinated 2-nitroimidazole hypoxia probe N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR 4554, CRC 94/17): a non-invasive diagnostic probe for the measurement of tumor hypoxia by magnetic resonance spectroscopy and imaging, and by positron emission tomography. Anti-cancer drug design,
Vol.13
(6),
pp. 703-28.
Stratford, I.J.
Workman, P.
(1998). Bioreductive drugs into the next millennium. Anti-cancer drug des,
Vol.13
(6),
pp. 519-528.
Workman, P.
(1998). Cancer research in the clinic: Translating modern research into clinical reality. Id weekly highlights,
Vol.3,
pp. 17-20.
Workman, P.
Twentyman, P.
Balkwill, F.
Balmain, A.
Chaplin, D.
Double, J.
Embleton, J.
Newell, D.
Raymond, R.
Stables, J.
Stephens, T.
(1998). United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia (second edition). B j cancer,
Vol.77,
pp. 1-10.
Aboagye, E.O.
Maxwell, R.J.
Horsman, M.R.
Lewis, A.D.
Workman, P.
Tracy, M.
(1998). The relationship between tumour oxygenation determined by oxygen electrode measurements and magnetic resonance spectroscopy of the fluorinated 2-nitroimidazole SR-4554. Br j cancer,
Vol.77,
pp. 65-70.
full text
Brunton, V.G.
Steele, G.
Lewis, A.D.
Workman, P.
(1998). Geldanamycin-induced cytotoxicity in human colon-cancer cell lines: Evidence against the involvement of c-Src or DT-diaphorase. Cancer chemother pharmacol,
Vol.41,
pp. 417-422.
Bailey, S.M.
Wyatt, M.D.
Friedlos, F.
Hartley, J.A.
Knox, R.J.
Lewis, A.D.
Workman, P.
(1997). Involvement of DT-diaphorase (EC 1 6 99 2) in the DNA cross-linking and sequence selectivity of the bioreductive anti-tumour agent EO9. British journal of cancer,
Vol.76
(12),
pp. 1596-8.
Cross, M.J.
Hodgkin, M.N.
Plumb, J.A.
Brunton, V.G.
Stewart, A.
MacAully, G.
Hill, R.
Kerr, D.J.
Workman, P.
Wakelam, M.J.
(1997). Inhibition of phospholipid signalling and proliferation of Swiss 3T3 cells by the wortmannin analogue demethoxyviridin. Biochimica et biophysica acta-molecular basis of disease,
Vol.1362
(1),
pp. 29-10.
Aboagye, E.O.
Lewis, A.D.
Tracy, M.
Workman, P.
(1997). Bioreductive metabolism of the novel fluorinated 2-nitroimidazole hypoxia probe N-(2-hydroxy-3,3,3-trifluoroproply)-2-(2-nitroimidazolyl) acetamide (SR-4554). Biochem pharmacol,
Vol.54,
pp. 1217-1224.
Aboagye, E.O.
Maxwell, R.J.
Kelson, A.B.
Tracy, M.
Lewis, A.D.
Graham, M.A.
Horsman, M.R.
Griffiths, J.R.
Workman, P.
(1997). Preclinical evaluation of the fluorinated 2-nitroimidazole N-(2- hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) as a probe for the measurement of tumor hypoxia. Cancer res,
Vol.57,
pp. 3314-3318.
Cross, M.J.
Hodgkin, M.N.
Plumb, J.A.
Brunton, V.G.
Stewart, A.
McAully, G.
Hill, R.
Kerr, D.J.
Workman, P.
Wakelam, M.J.
(1997). Involvement of DT-diaphorase (EC 1 6 99 2) in the DNA cross- linking and sequence selectivity of the bioreductive anti-tumour agent EO9. Biochim biophys acta,
Vol.1362,
pp. 29-38.
Graham, M.A.
Senan, S.
Robin Jnr, H.
Eckhardt, N.
Lendrem, D.
Hincks, J.
greenslade, D.
Rampling, R.
Kaye, S.B.
von Roemeling, R.
Workman, P.
(1997). Pharmacokinetics of the hypoxic cell cytotoxic agent tirapazamine and its major bioreductive metabolites in mice and humans: Retrospective analysis of a pharmacokinetically guided dose-escalation. Cancer chemother pharmacol,
Vol.40,
pp. 1-10.
Senan, S.
Rampling, R.
Graham, M.A.
Wilson, P.
Robin Jr, H.
Eckhardt, N.
Lawson, N.
McDonald, A.
von Roeming, R.
Workman, P.
Kaye, S.B.
(1997). Phase I and pharmacokinetic study of single agent tirapazamine (SR 4233) administered 3-weekly. Clin cancer res,
Vol.3,
pp. 31-38.
Workman, P.
(1997). The changing delivery of healthcare: New molecular targets for cancer chemotherapy. Id weekly highlights,
Vol.45,
pp. 24-27.
Workman, P.
(1997). Towards intelligent anticancer drug screening in the post-genome era?. Anticancer drug des,
Vol.12,
pp. 525-531.
Aboagye, E.O.
Lewis, A.D.
Graham, M.A.
Tracy, M.
Kelson, A.B.
Ryan, K.J.
Workman, P.
(1996). The pharmacokinetics, bioavailability and biodistribution in mice of a rationally designed 2-nitroimidazole hypoxia probe SR-4554. Anticancer drug des,
Vol.11
(3),
pp. 231-242.
show abstract
N-(2-Hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) is a fluorinated 2-nitroimidazole which has been rationally designed as a non-invasive probe for tumor hypoxia. The key selection criteria for this molecule were low central nervous system penetration and toxicity, high metabolic stability other than nitroreduction, good tumor uptake and high sensitivity for detection by magnetic resonance spectroscopy. As part of the pre-clinical development strategy, pharmacokinetic, bioavailability and biodistribution studies were performed in mice. Pharmacokinetic studies in mice demonstrated that SR-4554 was rapidly absorbed into plasma following i.p. administration and eliminated with a half-life of 42 min, similar to other 2-nitroimidazoles. By comparing the areas under the concentration-time curve (AUC), the tumor exposure towards SR-4554 was on average 84% of the value obtained for the plasma exposure. SR-4554 penetrated tumor tissue extremely well but, in contrast to misonidazole and certain other fluorinated analogues, its distribution into brain tissue was poor (AUCbrain/AUCplasma = 0.07), suggesting potentially lower toxicity in spite of its higher lipophilicity (P = 0.43 versus 0.63, respectively). The bioavailability of SR-4554 from i.p. and p.o. routes was 100 and 96% respectively. In non-tumor-bearing mice, SR-4554 was excreted mainly as unchanged drug. The percentage of the injected i.p. dose of SR-4554 excreted unchanged in the urine over 24 h was 68 +/- 8%. Neither SR-4554 nor its metabolites were detected in mouse feces. We propose that these favorable pharmacokinetic properties of SR-4554 are due to the hydrophilic character and hydrogen-bonding capability of the amide and hydroxyl functions in the compound..
Brunton, V.G.
Kelland, L.R.
Lear, M.J.
Montgomery, G.J.
Robertson, J.H.
Robins, D.J.
Queen, J.
Workman, P.
(1996). Synthesis and biological evaluation of a series of tyrphostins containing nitrothiophene moieties as possible epidermal growth factor receptor tyrosine kinase inhibitors. Anti-cancer drug des,
Vol.11
(4),
pp. 265-295.
show abstract
A series of 36 nitrothiophene tyrphostins were synthesized, 32 of which were novel structures. Their ability to inhibit the epidermal growth factor (EGF) receptor tyrosine kinase was assessed in a cell-free assay. Compounds containing a dinitrile, 2-aminoethene-1,1-dinitrile or a thioamide group were good inhibitors of the receptor tyrosine kinase. Although anti-proliferative and cytotoxic activity was seen, no evidence of inhibition of EGF receptor autophosphorylation in intact cells was observed. The compounds showed no preferential inhibition of EGF-dependent proliferation of fibroblasts transfected with the EGF receptor. Furthermore, in a panel of squamous cell carcinoma cell lines with varying levels of EGF receptor expression, there was no selective cell kill of lines with the highest EGF receptor expression. The 2-nitro-5-substituted-thiophenes and the 2-nitro-3-substituted-thiophenes showed reduction potentials falling within the range likely to be reduced by cellular reducing agents, while the 2-nitro-4-substituted-thiophenes and 4-nitro-2-substituted-thiophenes did not. Compounds from the 2-nitro-5-substituted-thiophene series were shown to induce DNA damage, while no evidence of DNA damage was demonstrated with compounds from the 2-nitro-4-substituted-thiophene series. The 2-nitro-5-substituted-thiophene compound 4 showed significant tumour-type selectivity in the US National Cancer Institute human tumour cell line panel. The leukaemia cell lines were particularly sensitive to the compound, as were the majority of the colon cancer, melanoma and breast cancer cell lines, while the central nervous system-derived lines and the non-small cell lung cancer lines were particularly resistant. Further work is required to determine the precise mechanisms involved in these effects..
Workman, P.
Fitzsimmons, S.A.
Grever, M.
Paull, K.
Camalier, R.
Lewis, A.D.
(1996). Reductase enzyme expression across the National Cancer Institute tumor cell line panel: Correlation with sensitivity to mitomycin C and EO9 - Response. Journal of the national cancer institute,
Vol.88
(16),
pp. 1161-3.
Aboagye, E.O.
Lewis, A.D.
Graham, M.A.
Tracy, M.
Kelson, A.B.
Ryan, K.
Workman, P.
(1996). Pharmacokinetics, bioavailability and biodistribution in mice of a rationally designed fluorinated 2-nitromidazole probe SR-4554 for the detection of hypoxia by magnetic resonance spectroscopy. Anticancer drug des,
Vol.11,
pp. 231-242.
Brunton, V.G.
Lear, M.J.
McKeown, P.
Robins, D.J.
Workman, P.
(1996). Synthesis and antiproliferative activity of tyrphostins containing quinoline moieties. Anticancer drug des,
Vol.11,
pp. 463-483.
Fitzsimmons, S.A.
Workman, P.
Grever, M.
Paull, K.
Camelier, R.
Lewis, A.D.
(1996). Reductase expression across the National Cancer Institute tumor cell line panel: Correlation with sensitivity to mitomycin C and EO9. J natl cancer inst,
Vol.88,
pp. 259-269.
Henderson, N.D.
Plumb, J.A.
Robins, D.J.
Workman, P.
(1996). Synthesis and anti-cancer activity of 2,6-disubstituted N-methylpiperidine derivatives and their N-oxides. Anticancer drug des,
Vol.11,
pp. 421-438.
McLeod, H.L.
Brunton, V.G.
Eckardt, N.
Lear, M.J.
Robins, D.J.
Workman, P.
Graham, M.A.
(1996). In vivo pharmacology and anti-tumour evaluation of the tyrphostin tyrosine kinase inhibitor RG13022. Br j cancer,
Vol.74,
pp. 1714-1716.
CHEN, S.
KNOX, R.
LEWIS, A.D.
FRIEDLOS, F.
WORKMAN, P.
DENG, P.S.
FUNG, M.
EBENSTEIN, D.
WU, K.B.
TSAI, T.M.
(1995). CATALYTIC PROPERTIES OF NAD(P)H-QUINONE ACCEPTOR OXIDOREDUCTASE - STUDY INVOLVING MOUSE, RAT, HUMAN, AND MOUSE-RAT CHIMERIC ENZYMES. Molecular pharmacology,
Vol.47
(5),
pp. 934-6.
Aboagye, E.O.
Graham, M.A.
Lewis, A.D.
Workman, P.
Kelson, A.B.
Tracy, M.
(1995). Development and validation of a solid-phase extraction and high-performance liquid chromatographic assay for a novel fluorinated 2-nitroimidazole hypoxia probe (SR-4554) in Balb/c mouse plasma. J chromatogr b biomed appl,
Vol.672,
pp. 125-132.
Aboagye, E.O.
Lewis, A.D.
Johnson, A.
Workman, P.
Tracy, M.
Huxham, I.M.
(1995). The novel fluorinated hypoxia probe SR-4554: Reductive metabolism and semi-quantitative localisation in human ovarian cancer multicellular spheroids as measured by electron loss spectroscopic analysis. Br j cancer,
Vol.72,
pp. 312-318.
Lohmeyer, M.
Workman, P.
(1995). Growth arrest vs direct cytotoxicity and the importance of molecular structure for the in vitro antitumour activity of ether lipids. Br j cancer,
Vol.72,
pp. 277-286.
full text
Robin, H.J.
Senan, S.
Workman, P.
Graham, M.A.
(1995). Development and validation of a sensitive solid-phase-extraction and high-performance liquid chromatographic assay for the bioreductive agent tirapazamine and its major metabolites in mouse and human plasma for pharmacokinetically-guided dose escalation. Cancer chemother pharmacol,
Vol.36,
pp. 266-270.
Houlihan, W.J.
Lohmeyer, M.
Workman, P.
Cheon, H.
(1995). Phospholipid antitumor agents. Med res rev,
Vol.15,
pp. 157-223.
Workman, P.
(1995). To pop or not to pop: p53 as a critical modulator of tumour responsiveness to therapy in vivo?. Hum exp toxicol,
Vol.14,
pp. 222-225.
WORKMAN, P.
DINCALCI, M.
BURSCH, W.
HARRAP, K.R.
HAWKINS, R.E.
NEIDLE, S.
POWIS, G.
(1994). EUROPEAN SCHOOL OF ONCOLOGY TASK-FORCE REPORT - NEW APPROACHES IN CANCER PHARMACOLOGY - DRUG DESIGN AND DEVELOPMENT 2. European journal of cancer,
Vol.30A
(8),
pp. 1148-13.
CROSS, M.J.
STEWART, A.
PLUMB, J.
MCAULEY, G.
HILL, R.A.
ISAACS, N.
WORKMAN, P.
KERR, D.
WAKELAM, M.J.
(1994). INHIBITION OF PHOSPHOLIPASES AND CELL-PROLIFERATION BY DEMETHOXYVIRIDIN. Journal of cellular biochemistry,
,
pp. 56-1.
Plumb, J.A.
Workman, P.
(1994). Unusually marked hypoxic sensitization to indoloquinone EO9 and mitomycin C in a human colon-tumour cell line that lacks DT-diaphorase activity. Int j cancer,
Vol.56
(1),
pp. 134-139.
show abstract
Studies with purified DT-diaphorase have shown that the enzyme is capable of catalyzing a two-electron reduction of the novel indoloquinone EO9 to a DNA-damaging alkylating species. The aim of this study was to determine to what extent DT-diaphorase may be involved in the metabolic activation of EO9 and mitomycin C in both aerobic and hypoxic conditions. Two human colon-carcinoma cell lines were used; HT29 has high levels of DT-diaphorase whilst BE lacks this activity because of a point mutation in the NQOI gene. In aerobic conditions the 2 cell lines show similar sensitivities to a number of cytotoxic drugs including cisplatin, doxorubicin and etoposide. They are equally sensitive to the benzotriazine di-N-oxide SR 4233 but HT29 is more sensitive than BE to mitomycin C and EO9. Sensitivity to SR 4233 is increased by about 100-fold for both cell lines in hypoxic conditions. DT-diaphorase-deficient BE cells show markedly increased sensitivity to mitomycin C and particularly EO9 in hypoxic conditions, whereas DT-diaphorase-rich HT29 cells show little hypoxic sensitization to these agents unless exposed in the presence of dicoumarol. These results suggest that DT-diaphorase can reduce EO9 and mitomycin C to potent cytotoxic species in aerobic conditions, and this activity predominates over the one-electron-reducing enzymes even in hypoxic conditions. In the absence of DT-diaphorase activity, EO9 and mitomycin C are reduced in hypoxic conditions, presumably by one-electron-reducing enzymes, to a similar or greater extent than is achieved with DT-diaphorase..
Brunton, V.G.
Lear, M.J.
Robins, D.J.
Williamson, S.
Workman, P.
(1994). Synthesis and antiproliferative activity of tyrphostins containing heteroaromatic moieties. Anticancer drug des,
Vol.9
(4),
pp. 291-309.
show abstract
A series of benzylidenemalononitrile derivatives previously synthesized by condensing aromatic aldehydes with malononitrile derivatives are known as tyrphostins. In this study, 32 tyrphostins were synthesized, 19 of which are novel compounds. Both hydroxylated derivatives and compounds containing heteroaromatic moieties were prepared. We have confirmed and extended the observation that the tyrphostins displayed an enhancement in their ability to inhibit the epidermal growth factor (EGF) receptor tyrosine kinase domain as the number of hydroxyl groups on the aromatic portion was increased. IC50 values of 1-5 microM were readily achieved. Some inhibitory activity was seen with the heteroaromatic structures, with two compounds exhibiting IC50 values of 56 and 77 microM. However, these derivatives were poor inhibitors of the EGF receptor tyrosine kinase activity as compared to the hydroxylated derivatives. The ability of the 32 tyrphostins synthesized in the present study to inhibit proliferation of a human breast adenocarcinoma cell line (MCF-7) was determined using [3H]thymidine incorporation as a measure of DNA synthesis. Some of the compounds containing pyridine, imidazole or thiophene portions displayed antiproliferative activity comparable to that of tyrphostins prepared from 3,4,5-trihydroxybenzaldehyde. The lack of inhibitory effect of these heteroaromatic compounds on the EGF receptor tyrosine kinase activity suggests that their antiproliferative activity is not related to inhibition of EGF receptor function. As the growth of the MCF-7 cell line is governed by other factors, such as the insulin-like growth factors (IGFs) and oestradiol, it is also still to be established whether the antiproliferative activity of the hydroxylated tyrphostins is directly related to inhibition of the EGF receptor tyrosine kinase activity..
Brunton, V.G.
Carlin, S.
Workman, P.
(1994). Alterations in EGF-dependent proliferative and phosphorylation events in squamous cell carcinoma cell lines by a tyrosine kinase inhibitor. Anticancer drug des,
Vol.9
(4),
pp. 311-329.
show abstract
The epidermal growth factor (EGF) receptor is overexpressed in squamous cell carcinoma and the EGF receptor has been proposed as a potential target for new therapeutic agents in this tumour type. We have utilized a tyrphostintype inhibitor of the EGF receptor tyrosine kinase domain (RG50864) to study EGF-dependent proliferation and phosphorylation in two human squamous cell carcinoma cell lines. There were selected on the basis that whereas both cell lines have a large number of EGF receptors, one is growth inhibited by EGF (A431) while the proliferation of the other cell line (B2A4) is stimulated by EGF. EGF induced receptor autophosphorylation in each of the two cell lines; however, the level of phosphorylation was greater in the A431 cells than in the B2A4 cells. The pattern of proteins phosphorylated in response to EGF was different in the two squamous cell lines. RG50864 antagonized the EGF-dependent proliferation of B2A4 cells, but was unable to reverse the inhibitory effect of EGF on A431 cell growth. RG50864 partially inhibited EGF receptor autophosphorylation in both cell lines and completely inhibited the EGF-dependent phosphorylation of other cellular proteins, one of which co-migrated with MAP2kinase in both cell lines. Moreover, different dose-response relationships for the inhibition of phosphorylation of various proteins were observed in A431 versus B2A4 cells. As a substrate competitive inhibitor of the EGF receptor tyrosine kinase, the primary mode of action of RG50864 may be to prevent the association and/or phosphorylation of multiple specific substrates of the receptor in a fashion which may be cell line dependent. The precise relationship of these phosphorylation events to tyrphostin sensitivity remains to be established..
Fitzsimmons, S.A.
Lewis, A.D.
Riley, R.J.
Workman, P.
(1994). Reduction of 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, WIN 59075, SR 4233) to a DNA-damaging species: a direct role for NADPH:cytochrome P450 oxidoreductase. Carcinogenesis,
Vol.15
(8),
pp. 1503-1510.
show abstract
3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, WIN 59075, SR 4233, NSC 130181) has entered phase 1 clinical trials as a bioreductive hypoxic cell cytotoxin because of its novel structure and impressive selective cytotoxicity towards hypoxic cells. Understanding the enzymology and underlying mechanism of oxidative and reductive DNA damage may allow more optimal development and use of this agent and contribute to the rational design of new bioreductive drugs. Here we provide unambiguous evidence that WIN 59075 undergoes one-electron reduction by purified rat liver NADPH:cytochrome P450 oxidoreductase to generate single- and double-strand breaks in plasmid DNA. The DNA damage caused may be important for the therapeutic toxicity of the drug. Enzyme kinetic parameters for this oxidoreductase reaction are in the range 1.01-1.61 mM for Km and 4416-5099 nmol/min/mg for Vmax. The relative levels of expression and cellular localization of target tumour NADPH:cytochrome P450 oxidoreductase may contribute to the therapeutic selectivity of WIN 59075..
Plumb, J.A.
Gerritsen, M.
Workman, P.
(1994). DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9. Br j cancer,
Vol.70
(6),
pp. 1136-1143.
show abstract
full text
Aerobic sensitivity to indoloquinone EO9 has been shown to correlate with cellular levels of the two-electron reducing enzyme DT-diaphorase. However, little is known about the relative roles of one- and two-electron reducing enzymes in the hypoxic cytotoxicity of EO9. We have characterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to EO9. Eight cell lines were then selected for a comparison of aerobic and hypoxic sensitivities. Activities of DT-diaphorase showed a wide range (> 10,000-fold), while activities of the one-electron reducing cytochrome b5 and cytochrome P450 reductases were generally lower and showed only a 15- and 25-fold range respectively. The aerobic cytotoxicity of EO9 was clearly related to the cellular levels of DT-diaphorase (r = 0.87), with higher levels giving increased sensitivity, but not to the levels of one-electron reducing enzymes. In contrast, there was no relationship between sensitivity to BCNU, cisplatin or the bioreductive agent SR 4233 (tirapazamine) and activities of any of these reducing enzymes. Under hypoxic conditions sensitivity to EO9 was markedly increased in cell lines with low levels of DT-diaphorase activity, while cell lines with high levels show only a small increase in sensitivity. This is reflected by a clear correlation (r = 0.98) between cellular DT-diaphorase activity and the ratio of aerobic to hypoxic sensitivity to EO9. However, we have now for the first time demonstrated an inverse correlation (r = 0.93) between the cellular activity of DT-diaphorase and hypoxic sensitivity to EO9, that is sensitivity decreases with increasing DT-diaphorase activity. Moreover, this correlation was lost when cells were exposed to drug in the presence of dicoumarol, supporting an involvement of DT-diaphorase in this relationship. These observations question the previously straightforward role for DT-diaphorase in the metabolic activation of EO9. Whereas DT-diaphorase is associated with increased toxicity in air, it appears to reduce the cytotoxicity of EO9 in hypoxic conditions. This suggests either that the one-electron reduction product of EO9 metabolism, the semiquinone, is more toxic than the two-electron reduction product, the hydroquinone, or that the hydroquinone is not cytotoxic and aerobic toxicity is due to the transient appearance of the semiquinone upon back oxidation of the hydroquinone..
Lohmeyer, M.
McNaughton, L.
Hunt, S.P.
Workman, P.
(1994). Stimulation of intracellular free calcium increases by platelet-activating factor in HT29 colon carcinoma cells Spectrofluorimetric and preliminary spatio-temporal analysis using confocal laser scanning fluorescence imaging microscopy. Biochem pharmacol,
Vol.47
(6),
pp. 975-985.
show abstract
We examined the ability of platelet-activating factor (PAF) and its lyso derivative (lyso-PAF) to elicit increases in intracellular free calcium concentration ([Ca2+]i) in HT29 human colon carcinoma cells. Using spectrofluorimetric analysis with indo-1 as the [Ca2+]i reporter molecule, we found that 1-10 microM concentrations of both lipids stimulated substantial, reversible, monophasic [Ca2+]i elevations. Evidence was obtained that the two lipids may act via specific receptors to release Ca2+ from internal stores. Homologous desensitization was observed in both cases and PAF and lyso-PAF were also able to desensitize cells reciprocally (heterologous desensitization). The potent PAF receptor antagonist WEB 2086 (3-[4-(chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2, 4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4-morpholinyl)-1-propano ne) successfully blocked PAF-induced [Ca2+]i elevations, but did not affect rises in response to lyso-PAF, suggesting that lyso-PAF may act through a different cellular receptor or mechanism. Higher concentrations (> 10 microM) of PAF resulted in non-reversible [Ca2+]i elevations which were caused by Ca2+ influx following membrane lysis. However, the WEB 2086 insensitivity of these effects and the resultant cellular toxicity clearly showed that such events were mechanistically distinct from the reversible [Ca2+]i elevations apparently operating via WEB 2086-sensitive receptors. Preliminary spatio-temporal observations, using confocal microscopy and fluo-3 as the [Ca2+]i reporter molecule, suggested that PAF can also induce [Ca2+]i elevations in the absence of cell lysis in monolayer HT29 cells. Visual impressions were obtained of cellular and subcellular heterogeneity and of [Ca2+]i oscillations in responding cells. However, these need to be interpreted with caution because of the intrinsic limitations of the methodology, particularly using non-ratiometric dyes. The significance of a receptor-mediated, reversible elevation of [Ca2+]i by sub-toxic concentrations of PAF in HT29 colon cancer cells remains to be elucidated, but it is tempting to speculate that PAF might function as a locally acting signalling mediator in these and possibly other tumour cells..
Plumb, J.A.
Gerritsen, M.
Milroy, R.
Thomson, P.
Workman, P.
(1994). Relative importance of DT-diaphorase and hypoxia in the bioactivation of EO9 by human lung tumor cell lines. Int j radiat oncol biol phys,
Vol.29
(2),
pp. 295-299.
show abstract
PURPOSE: Although a number of bioreductive agents are substrates for purified DT-diaphorase the role of this enzyme in either activation or detoxification of these agents in the whole cell is unclear. The aim of this study was to determine the role of DT-diaphorase in the metabolic activation of EO9 under both aerobic and hypoxic conditions. METHODS AND MATERIALS: A panel of lung cancer cell lines was used and drug sensitivity was determined by clonogenic or tetrazolium-dye-based assays. Activities of DT-diaphorase, cytochrome P450 and cytochrome b5 reductase were determined spectrophotometrically by following the reduction of cytochrome c. RESULTS: Small-cell lung cancer cell lines showed a 600-fold range in DT-diaphorase activities but levels were much higher in three of the four non-small-cell lines. Activities of cytochromes P450 and b5 reductase were much lower than those of DT-diaphorase and showed much less variation between cell lines. There was no relationship between the activities of any of the enzymes and aerobic sensitivity to SR 4233, BCNU and cis-platin. Under aerobic conditions there was a clear correlation between DT-diaphorase activity and sensitivity to EO9. The small-cell lines were much more resistant to EO9 than the DT-diaphorase rich non-small-cell lines. A doxorubicin resistant variant of one of the small-cell lines (H69LX10) did not show cross resistance to EO9 but did show a small degree (3-fold) of cross resistance to SR 4233. Under hypoxic conditions, cell lines with high levels of DT-diaphorase showed only a small increase in sensitivity to EO9 (1.5-7 fold); cell lines with low levels of activity showed a 10-37-fold increase in sensitivity. CONCLUSION: These results suggest that under hypoxic conditions, EO9 is metabolized by 1-electron reducing enzymes to a toxic species. This reduction product is oxygen sensitive but a similar degree of activation is obtained under aerobic conditions in cell lines with high levels of 2-electron reducing DT-diaphorase..
Brunton, V.G.
Workman, P.
(1994). Protein tyrosine kinase inhibitors as potential cancer drugs. Cancer topics,
Vol.10,
p. 14.
Reilly, J.J.
Workman, P.
(1994). Is body composition an important variable in the pharmacokinetics of anticancer drugs? A review and suggestions for further research. Cancer chemother pharmacol,
Vol.34,
pp. 3-13.
Workman, P.
(1994). Enzyme-directed bioreductive drug development revisited: A commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly DT-diaphorase. Oncol res,
Vol.6,
pp. 461-475.
Workman, P.
Graham, M.A.
(1994). Pharmacokinetics and cancer chemotherapy. Eur j cancer,
Vol.30A,
pp. 706-710.
SCHWARTSMANN, G.
WORKMAN, P.
(1993). ANTICANCER DRUG SCREENING AND DISCOVERY IN THE 1990S - A EUROPEAN PERSPECTIVE. European journal of cancer,
Vol.29A
(1),
pp. 3-12.
DEVRIES, E.G.
GIETEMA, J.A.
WORKMAN, P.
SCOTT, J.E.
CRAWSHAW, A.
DOBBS, H.J.
DENNIS, I.
MULDER, N.H.
SLEIJFER, D.T.
WILLEMSE, P.H.
(1993). A PHASE-II AND PHARMACOKINETIC STUDY WITH ORAL PIRITREXIM FOR METASTATIC BREAST-CANCER. British journal of cancer,
Vol.68
(3),
pp. 641-4.
full text
Brunton, V.G.
Workman, P.
(1993). In vitro antitumour activity of the novel imidazoisoquinoline SDZ 62-434. Br j cancer,
Vol.67
(5),
pp. 989-995.
show abstract
The novel imidazoisoquinoline SDZ 62-434, originally identified as a platelet-activating factor (PAF) antagonist, has antiproliferative activity in a range of cell lines from human solid and haematological malignancies. Using an MTT cytotoxicity assay, IC50 values of 5 microM - 111 microM were observed following a 24 h exposure. Similar results were obtained using a clonogenic assay. The HT29 colon adenocarcinoma was particularly sensitive while the MCF-7 breast carcinoma was the most resistant in our panel. Only a 2-3 fold cross-resistance was seen in the doxorubicin and cisplatin resistant variants of the A2780 ovarian carcinoma; the drug did not modulate sensitivity to doxorubicin in either parent or resistant lines. No cross-resistance to SDZ 62-434 was seen in a doxorubicin-resistant MCF-7 variant. Cytotoxicity was not due to non-specific membrane lysis. The potent PAF antagonist WEB 2086 did not modulate SDZ 62-434 cytotoxicity, indicating no role for PAF receptors. Precursor incorporation studies in A2780 cells showed that DNA synthesis was inhibited more effectively than protein synthesis while RNA synthesis was unaffected. SDZ 62-434 inhibited both bombesin and platelet-derived growth factor-induced DNA synthesis in quiescent Swiss 3T3 cells. This suggests a possible role for SDZ 62-434 as an inhibitor of signal transduction in cancer cells..
Walton, M.I.
Workman, P.
(1993). Pharmacokinetics and bioreductive metabolism of the novel benzotriazine di-N-oxide hypoxic cell cytotoxin tirapazamine (WIN 59075; SR 4233; NSC 130181) in mice. J pharmacol exp ther,
Vol.265
(2),
pp. 938-947.
show abstract
The novel benzotriazine di-N-oxide SR 4233 (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide) shows high selective cytotoxicity toward hypoxic tumor cells. We investigated its pharmacokinetics and bioreductive metabolism in mouse plasma, brain, liver and tumor in vivo and also tumor metabolism in vitro. Plasma elimination T1/2 increased slightly with dose, and metabolite kinetics were dose-dependent. Peak concentration and area under the curve0-infinity increased linearly with dose from 0.1 to 0.3 mmol kg-1 i.v. After 0.2 mmol kg-1 i.v., elimination was biphasic (T1/2 alpha < 2 min; T1/2 beta, 26.5 min). Peak plasma concentration and area under the curve0-infinity were 26 and 13.6 micrograms ml-1 hr, respectively. Peak plasma concentration for the two-electron reduction product SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) was 7 to 9 micrograms ml-1 and for the four-electron reduction product SR 4330 (3-amino-1,2,4-benzotriazine) peak plasma concentration was 0.5 to 1.0 micrograms ml-1. Identical results were obtained after i.p. administration. Oral dosing gave lower peak plasma drug concentrations (2-3 micrograms ml-1) but reasonable bioavailability (75%). SR 4233 underwent extensive bioreduction in KHT tumors. Tumor/plasma ratios (percentages) for SR 4233 were 32% compared to 174 (SR 4317) and 196% (SR 4330), respectively. Similar SR 4233 tissue/plasma percentages were obtained in RIF-1 and 16C tumors, but EMT6 tumors were markedly lower at 7%. Reduction also occurred with tumor homogenates in vitro (KHT = EMT6 > RIF-1). Conversion to SR 4317 and SR 4330 was more extensive in liver, with tissue/plasma percentages between 50 to 220 and 500 to 1800%, respectively. The brain showed a similar pattern to tumors. Urinary recoveries (0-8 hr) were low at 4.5% for SR 4233 and 0.4% for the reduced metabolites. A further 30% occurred as a glucuronide. Concentrations of SR 4233 required for effective in vitro cytotoxicity are achieved in vivo, and extensive bioreductive metabolism occurs in tumor and normal tissues..
WORKMAN, P.
BELPOMME, D.
ROBERT, J.
(1993). NEW DRUG DESIGN AND DEVELOPMENT IN CANCER - PRESENT STATUS AND FUTURE PERSPECTIVES. Cancer chemoth pharm,
Vol.32
(2),
pp. 83-84.
Coley, H.M.
Amos, W.B.
Twentyman, P.R.
Workman, P.
(1993). Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines. Br j cancer,
Vol.67
(6),
pp. 1316-1323.
show abstract
full text
This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention..
Workman, P.
Stratford, I.J.
(1993). The experimental development of bioreductive drugs and their role in cancer therapy. Cancer metastasis rev,
Vol.12
(2),
pp. 73-82.
show abstract
Bioreductive drugs undergo metabolic reduction to generate cytotoxic metabolites. This process is facilitated by bioreductive enzymes and the lower oxygen conditions present in solid tumours compared to normal tissues. Because of this specificity, bioreductive drugs have enormous potential to contribute to modern cancer therapy. Examples undergoing clinical trials include N-oxides such as tirapazamine, aziridinylnitroimidazoles RSU 1069/RBU 6145 and quinones such as indoloquinone EO9. Other novel structures are also under study. Here we review the experimental development of bioreductive drugs and their role in cancer therapy..
Lohmeyer, M.
Workman, P.
(1993). The role of intracellular free calcium mobilization in the mechanism of action of antitumour ether lipids SRI 62-834 and ET18-OMe. Biochem pharmacol,
Vol.45
(1),
pp. 77-86.
show abstract
Membrane-active antitumour ether lipids such as ET18-OMe (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) and SRI 62-834 ((+-)-2-(Hydroxy[tetrahydro-2-(octadecyloxy) methylfuran-2-yl] methoxyl phosphinyloxy)-N,N,N-trimethylethaniminium hydroxide) are selectively cytotoxic to tumour cells in vitro. Their precise mechanisms of action are unclear, but they are known to have effects on cell membranes and cell signalling. A previous report suggested that ether lipids cause a biphasic sustained rise in intracellular free calcium [Lazenby et al., Cancer Res 50: 3327-3330, 1990]. We show here that the second phase is an experimental artefact due to cell membrane permeabilization by ether lipids in serum-free buffers. In serum-free medium, the membrane toxicity of antitumour ether lipids was increased 50-60 fold, when compared to medium containing 10% serum. Membrane disruption was neither dependent on extracellular calcium, nor modulated by preloading cells with the calcium chelators bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]methyl]-6- methoxy-8-[bis(carboxymethyl)amino]quinoline. This indicates that the mechanism of membrane damage by ether lipids does not involve changes in calcium homeostasis. Using indo-1 and fura-2 as calcium probes, we established that lower concentrations of antitumour ether lipids do elicit a genuine monophasic and transient rise in intracellular free calcium, predominantly mobilized from internal stores. This acute calcium agonist activity of ether lipids is distinct from the inhibitory effects on cell signalling reported previously after more prolonged exposure. It appears that the calcium elevation induced by antitumour ether lipids is unlikely to be instrumental in their selective and potent antitumour activity..
Riley, R.J.
Hemingway, S.A.
Graham, M.A.
Workman, P.
(1993). Initial characterization of the major mouse cytochrome P450 enzymes involved in the reductive metabolism of the hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, SR 4233, WIN 59075). Biochem pharmacol,
Vol.45
(5),
pp. 1065-1077.
show abstract
The benzotriazine di-N-oxide SR 4233 (tirapazamine, WIN 59075) is currently in phase I clinical trials as the lead compound in a series of novel and highly selective antitumour hypoxic cytotoxins. Reductive bioactivation is thought to proceed via a one-electron reduced, oxidizing nitroxide radical and also forms the inactive single N-oxide SR 4317 via radical disproportionation or a second one-electron reduction. In mouse liver microsomes reductive metabolism is catalysed predominantly by cytochrome P450 (70%) and cytochrome P450 reductase (30%). The aim of the present study was to examine which cytochrome P450 isozymes may be involved. Reduction of SR 4233 to SR 4317 was monitored by HPLC analysis. Metabolism by microsomes from both control and dexamethasone-induced BALB/c male mice was 70% inhibited by carbon monoxide. The cytochrome P450 inhibitor SKF 525A, following aerobic preincubation, also inhibited SR 4233 reduction by 58%. Reduction was induced 2-3-fold by dexamethasone and was not accountable by increases in cytochrome P450 reductase or DT-diaphorase. The induction data and the greater degree of inhibition of SR 4233 reduction by metyrapone compared to alpha-naphthoflavone suggested a possible involvement of Cyp2b, Cyp2c and Cyp3a cytochrome P450 subfamilies. Both Cyp3a (7.4-fold) and Cyp2b (1.8-fold) type enzymes were shown by western immunoblot analysis to be induced by dexamethasone, the latter correlating more closely with increased SR 4233 reductase activity and also with the 2-fold induction of benzphetamine N-demethylase, a Cyp2b-type enzyme. No inhibition of SR 4233 reduction was seen with erythromycin or cyclosporin A which act as substrates/inhibitors for Cyp3a-type enzymes, but inhibition was seen with p-nitrophenol and tolbutamide which are substrates for Cyp2el- and Cyp2c-type enzymes, respectively (11% and 25% inhibition in induced microsomes). SR 4233 itself inhibited benzphetamine N-demethylase, which is catalysed by Cyp2b-type enzymes but not erythromycin N-demethylase which is catalysed by Cyp3a-type isoforms. Immunoinhibition studies with epitope specific monoclonal antibodies were consistent with the major involvement of phenobarbitone- and steroid-inducible products of the Cyp2b and Cyp2c subfamilies. These forms contributed at least 53% and 26%, respectively, of the cytochrome P450-associated SR 4233 reductase activity in the induced microsomes. The findings support our earlier conclusion that cytochrome P450 is the major SR 4233 reductase in mouse liver and provides leads as to the possible involvement of specific isoforms in human tumours and normal tissues..
Dive, C.
Workman, P.
Watson, J.V.
(1993). Can flow cytoenzymology be applied to measure membrane-bound enzyme kinetics? Assessment by analysis of gamma-glutamyl transpeptidase activity. Biochem pharmacol,
Vol.46
(4),
pp. 643-650.
show abstract
We describe an improved technique which allows the analysis of enzyme reaction kinetics for gamma-glutamyl transpeptidase (gamma-GT) by flow cytometry. This is technically difficult because of the location of the enzyme on the external surface of the cell membrane leading to the rapid escape of the product. The reaction is determined by monitoring the conversion of gamma-glutamyl aminomethylcoumarin to aminomethylcoumarin. Reaction kinetics are described for BL8 hepatocyte and JB1 hepatoma cells lines, together with inhibition kinetics for the active site-directed glutamine analogue L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid. We show that it is possible to follow the reaction dynamics in a heterogeneous mixture of BL8 and JB1 cells allowing discrimination of the two cell types based on gamma-GT activity. Improvements for further optimizing the assay of this important enzyme are suggested..
Coley, H.M.
Twentyman, P.R.
Workman, P.
(1993). The efflux of anthracyclines in multidrug-resistant cell lines. Biochem pharmacol,
Vol.46
(8),
pp. 1317-1326.
show abstract
In order to address the association of enhanced drug efflux with the multidrug-resistant (MDR) phenotype, we have studied the cellular pharmacokinetics of anthracyclines in the P-glycoprotein (Pgp)-positive MDR cell lines H69/LX4 (human small cell lung cancer) and EMT6/AR1.0 (mouse mammary tumour). Both doxorubicin (DOX) and daunorubicin (DNR) were accumulated to a lesser extent and effluxed at a higher rate by MDR cells than by their drug-sensitive counterparts. In contrast, the 9-alkyl substituted compound, aclacinomycin A (ACL), was accumulated and effluxed from parent and MDR cells at an identical rate. In experiments designed to examine energy-dependent efflux, DOX and DNR were shown to be efficiently effluxed against the concentration gradient in the presence of glucose. However, in the same experiments the analogues ACL and Ro 31-3294 (9-alkyl and morpholinyl substituted), which have previously been shown to retain activity against MDR cell lines, were accumulated and effluxed at identical rates in parent and MDR EMT6 cells. Hence, 9-alkyl and morpholinyl substituted compounds appear to behave less favourably as substrates for energy-driven drug efflux by Pgp-positive MDR cells than do DOX or DNR. Resistance modifiers verapamil and cyclosporin A appeared to abolish energy-dependent efflux for DOX and DNR in both the EMT6 and H69 MDR lines whereas they had no effect on the cellular efflux of ACL. The altered cellular pharmacology in MDR cell lines may provide a rational basis for the use of modified anthracycline analogues (e.g. 9-alkyl and morpholinyl (substituted) and resistance of modifying agent in the treatment of tumours expressing a Pgp-mediated phenotype..
Workman, P.
(1993). Pharmacokinetics and cancer: successes, failures and future prospects. Cancer surv,
Vol.17,
pp. 1-26.
show abstract
Cancer pharmacokinetics has made an enormous contribution to the development and optimization of cancer therapy. While consolidating its position and subjecting itself to prospective clinical trials in areas of established pharmacology, it needs also to keep constantly on the move in order to respond to the challenging demands of more futuristic approaches. Antibodies, cytokines, nucleic acid drugs and therapeutic genes will be metabolized and excreted and will in addition display special difficulties in terms of tissue and cell uptake. Pharmacokinetic considerations must accompany all new experimental therapies for scientific reasons as well as to fulfil regulatory requirements (Peck et al, 1992). The very latest approaches will understandably be seen by many practising clinicians as science fiction--for example obtaining a genetic fingerprint of a patient's tumour in order to guide corrective therapy, whether it be based on a chemical drug or a replacement gene. But it is from such speculative attempts that the real breakthroughs of the future may follow and pharmacokinetic/pharmacodynamic studies are essential to these efforts. In terms of the role of pharmacokinetics and pharmacodynamics in day to day management of individual patients, we do need to be more pragmatic. We must address tough questions such as: Can the assays be conducted on a routine basis? Do the measurements affect treatment outcome? Are they cost effective? Will they actually be used by clinical and nursing staff in a busy hospital environment? There is ample evidence in the following pages that pharmacokinetics will continue to prove vital to support both routine patient management and the exciting new approaches to cancer therapy..
Kaye, S.B.
Workman, P.
Graham, M.A.
Cassidy, J.
Jodrell, D.
(1993). Pharmacokinetics and early clinical studies of selected new drugs. Cancer surv,
Vol.17,
pp. 371-396.
show abstract
The five examples given here illustrate new cytotoxic agents at different stages of evaluation. In all cases, considerable effort has gone into detailed pharmacokinetic studies conducted before and during the clinical phase I studies. Has this effort contributed significantly to the development of these agents? At present, it has to be said that the contribution made in the case of these particular agents has been modest. For the anthrapyrazoles, the availability of the pharmacokinetic data did not permit a pharmacokinetically guided dose escalation to be performed because of non-linear kinetics, and a similar comment can be made for rhizoxin, since the human plasma AUC values at the MTD were much lower than in the mouse. For the camptothecin analogues, a detailed knowledge of the kinetics of the closed and open forms of the various agents did not influence the way in which the studies were conducted, nor did pharmacokinetic information appreciably do so for EO9, although some comfort was gained by clinical investigators when the short half-life seen in preclinical species was also observed in humans. For suramin, therapeutic drug monitoring is clearly essential, although toxicity remains a problem. Of course, a proper understanding of the pharmacokinetics and metabolism of these agents greatly improves the interpretation of the clinical observations made and is often critical in planning the next stages of development. This is more clearly seen with agents that have unusual forms of toxicity, such as flavone acetic acid, for which the achievement of notional target concentrations is a key element in clinical trials (Kerr et al, 1987; Maughan et al, 1992). Moreover, as reviewed elsewhere (Graham and Workman, 1992; see also Graham and Kaye, this volume), there are several other instances where pharmacokinetically guided dose escalation has greatly improved the conduct of a phase I study. Good examples of this are iododoxorubicin (Gianni et al, 1990), mitotic inhibitor CI-980 (Brodfuehrer et al, 1992) and DNA intercalator CI-958 (Whitfield et al, 1992). Not surprisingly then, pharmacokinetics can help guide early clinical studies of some compounds but not others and whether they will be of value can only be determined by carrying out the pharmacokinetic measurements. The real value of the pharmacokinetic studies for the five compounds reviewed may not yet have been seen. Interpatient variations in drug handling can play a major part in determining levels of anti-tumour activity as well as toxicity.(ABSTRACT TRUNCATED AT 400 WORDS).
Brunton, V.G.
Workman, P.
(1993). Cell-signaling targets for antitumour drug development. Cancer chemother pharmacol,
Vol.32
(1),
pp. 1-19.
Dennis, I.F.
Ramsay, J.R.
Workman, P.
Bleehen, N.M.
(1993). Pharmacokinetics of BW12C and mitomycin C, given in combination in a phase 1 study in patients with advanced gastrointestinal cancer. Cancer chemother pharmacol,
Vol.32
(1),
pp. 67-72.
show abstract
The effect of combining the oxygen-transport-modifying drug BW12C with mitomycin C was investigated in a phase 1 study of 26 patients with advanced gastrointestinal cancer. The dose of BW12C was increased from 20 mg/kg to 60 mg/kg. Dose-limiting toxicity of vomiting was experienced at doses greater than 50 mg/kg. This corresponded to whole blood levels > or = 700 micrograms/ml and to > 50% haemoglobin modification. Whole blood concentrations of BW12C and modification of the haemoglobin oxygen saturation curve were linearly dependent on dose. BW12C whole blood pharmacokinetics were best described by a one-compartment model and were clearly dose-dependent. The half-life increased from 2.1 h at a dose of 20 mg/kg to 7.2 h at a dose of 60 mg/kg. The AUC increased in a similar non-linear fashion with increasing dose. Mitomycin C was given at a fixed dose of 20 mg/m2 at the end of the BW12C infusion. Mitomycin C plasma pharmacokinetics fitted a two-compartment model, giving a mean beta half-life of 50 +/- 7 min and AUC of 1.1 +/- 0.08 micrograms/ml h, and were unaffected by the combined treatment. There was no evidence of increased mitomycin C toxicity..
HENDRIKS, H.R.
PIZAO, P.E.
BERGER, D.P.
KOOISTRA, K.L.
BIBBY, M.C.
BOVEN, E.
DREEFVANDERMEULEN, H.C.
HENRAR, R.E.
FIEBIG, H.H.
DOUBLE, J.A.
HORNSTRA, H.W.
PINEDO, H.M.
WORKMAN, P.
SCHWARTSMANN, G.
(1993). E09 - A NOVEL BIOREDUCTIVE ALKYLATING INDOLOQUINONE WITH PREFERENTIAL SOLID TUMOR-ACTIVITY AND LACK OF BONE-MARROW TOXICITY IN PRECLINICAL MODELS. European journal of cancer,
Vol.29A
(6),
pp. 897-10.
Reilly, J.J.
Workman, P.
(1993). Normalisation of drug dosage: Some theoretical and practical considerations. Cancer chemother pharmacol,
Vol.32,
pp. 411-418.
Workman, P.
(1993). New strategies for targeted cancer chemotherapy. Helix,
Vol.1,
p. 50.
Workman, P.
(1993). Relevance of pharmacology in clinicl practice The moderator thinks that. Ann oncol,
Vol.4,
pp. 466-469.
Workman, P.
Harris, A.L.
(1993). Translating advances in molecular oncology into improved therapy: New targets for drug discovery. Cancer topics,
Vol.9,
p. 97.
MAUGHAN, T.S.
WARD, R.
DENNIS, I.
HONESS, D.J.
WORKMAN, P.
BLEEHEN, N.M.
(1992). TUMOR CONCENTRATIONS OF FLAVONE ACETIC-ACID (FAA) IN HUMAN-MELANOMA - COMPARISON WITH MOUSE DATA. British journal of cancer,
Vol.66
(3),
pp. 579-4.
full text
WALTON, M.I.
BIBBY, M.C.
DOUBLE, J.A.
PLUMB, J.A.
WORKMAN, P.
(1992). DT-DIAPHORASE ACTIVITY CORRELATES WITH SENSITIVITY TO THE INDOLOQUINONE-EO9 IN MOUSE AND HUMAN COLON CARCINOMAS. European journal of cancer,
Vol.28A
(10),
pp. 1597-4.
HUXHAM, I.M.
GAZE, M.N.
WORKMAN, P.
MAIRS, R.J.
(1992). THE USE OF PARALLEL EEL SPECTRAL IMAGING AND ELEMENTAL MAPPING IN THE RAPID ASSESSMENT OF ANTICANCER DRUG LOCALIZATION. Journal of microscopy,
Vol.166,
pp. 367-14.
Graham, M.A.
Workman, P.
(1992). The impact of pharmacokinetically guided dose escalation strategies in phase I clinical trials: critical evaluation and recommendations for future studies. Ann oncol,
Vol.3
(5),
pp. 339-347.
show abstract
Phase I studies requiring multiple dose escalation steps have led to the development of pharmacokinetically guided dose escalation (PGDE) strategies to expedite the conduct of early clinical trials. This article critically reviews PGDE strategies for a number of new anticancer agents including amphethinile, brequinar sodium, iodo-doxorubicin, the anthrapyrazoles (DuP 941, DuP 942 and DuP 937), rhizoxin, and aphidicolin glycinate. The benefits and problems associated with PGDE are examined. Recommendations are made for the optimal deployment of pharmacological information in future phase I studies..
WORKMAN, P.
(1992). SIGNAL TRANSDUCTION INHIBITORS AS NOVEL ANTICANCER DRUGS - WHERE ARE WE. Ann oncol,
Vol.3
(7),
pp. 527-531.
Workman, P.
Maxwell, R.J.
Griffiths, J.R.
(1992). Non-invasive MRS in new anticancer drug development. Nmr biomed,
Vol.5
(5),
pp. 270-272.
show abstract
In the rational development of anticancer drugs it is important to employ all the available pharmacological information. Early clinical trials provide an opportunity for hypothesis testing. MRS techniques have the potential to provide valuable data on the preclinical and clinical pharmacokinetics and pharmacodynamics of drugs non-invasively. Here we illustrate advantages and pitfalls of MRS using studies of two fluorine-containing cancer drugs: a beta,beta-difluoro analogue of the alkylating agent chlorambucil and a fluorinated derivative of the nitroimidazole misonidazole, Ro 07-0741. Limitations include signal quenching via protein binding and inadequate sensitivity for more potent drugs like beta,beta-difluorochlorambucil; but fluoromisonidazole was shown to accumulate in tumours and shows promise as a chemical probe for tumour hypoxia, detectable by 19F MRS..
Workman, P.
(1992). Emerging from a crisis in anticancer drug discovery? Screening versus design: confessions of an optimistic fence-sitter. Ann oncol,
Vol.3
(9),
pp. 699-705.
Workman, P.
(1992). Introduction: new anticancer drug design and discovery based on advances in molecular oncology. Semin cancer biol,
Vol.3
(6),
pp. 329-333.
Workman, P.
Brunton, V.G.
Robins, D.J.
(1992). Tyrosine kinase inhibitors. Semin cancer biol,
Vol.3
(6),
pp. 369-381.
show abstract
Tyrosine kinases are associated with the cytoplasmic domains of growth factor receptors as well as oncoproteins and many have the potential to cause transformation if mutated or hyperexpressed. Tyrosine kinases therefore represent an excellent target for the development of cancer drugs. A large number of inhibitors have now been identified and many show promising cytostatic activity, particularly using in vitro models. Some in vivo activity has been reported. Progress with various structural classes is reviewed. It is not clear whether specific or broad spectrum tyrosine kinase inhibitors should be developed as potential anticancer drugs. It does seem likely, however, that tyrosine inhibitors will enter clinical trial in cancer patients..
Coley, H.M.
Twentyman, P.R.
Workman, P.
(1992). Further examination of 9-alkyl- and sugar-modified anthracyclines in the circumvention of multidrug resistance. Anticancer drug des,
Vol.7
(6),
pp. 471-481.
show abstract
Anthracyclines possessing either a 9-alkyl modification in the A-ring of the tetracyclic aglycone and/or specific changes to the amino sugar moiety retain effective cytotoxic activity against multidrug resistant (MDR) cell lines. To obtain a better understanding of the structural features responsible for this potentially valuable behaviour, we used the MTT tetrazolium dye reduction assay to calculate resistance factors (RF = the ratio of ID50 for the drug-resistant line to that for the parental line) for the EMT6/P mouse mammary tumour and its MDR variant EMT6/AR1.0, and the H69/P human small cell lung cancer line and its MDR counterpart H69/LX4. Both MDR lines exhibit marked resistance to doxorubicin, MDR 1 gene amplification, hyperexpression of the membrane P-glycoprotein and reduced drug accumulation. RF values for doxorubicin were 34 and 131 in the EMT6 and H69 cell line pairs, respectively. The 9-alkyl-substituted anthracyclines were confirmed as having RF values 9- to 15-fold lower than those for doxorubicin. The 9-ethyl analogues Ro 31-1966 (RF for EMT6 2.2, RF for H69 4.7) and Ro 31-1749 (RF for EMT6 3.9, RF for H69 9.5) were superior to the previously studied 9-methyl analogue Ro 31-1215 (RF for EMT6 8.1 RF for H69 12.4). A clear trend for RF values to decrease with increasing 9-alkyl chain length was also noted in the structurally more complex aclacinomycin series. For example, 13-methyl-aclacinomycin (RF for EMT6 1.0, RF for H69 2.2) featuring a 9-isopropyl moiety was superior to the 9-alkyl-containing aclacinomycin A (RF for EMT6 4.7, RF for H69 5.8), and this was in turn more effective than the 9-methyl analogue sulfurmycin A (RF for EMT6 6.4, RF for H69 14.2). The trisaccharide moiety was not an essential feature for activity against MDR lines in the aclacinomycins, as shown by the low RF value with aklavine (RF for EMT6 2.1, RF for H69 2.5). However, a small change in one of the sugar moieties of aclacinomycin A, as in marcellomycin, resulted in a considerable increase in RF values (RF for EMT6 18.5, RF for H69 25.3). The complex anthracyclines AD 32 (RF for EMT6 6.5, RF for H69 11.7) and particularly tetrahydropyranyl-doxorubicin (RF for EMT6 1.4, RF for H69 3.2) were effective against MDR lines.(ABSTRACT TRUNCATED AT 400 WORDS).
Workman, P.
Kaye, S.B.
Schwartsmann, G.
(1992). Laboratory and phase I studies of new cancer drugs. Curr opin oncol,
Vol.4
(6),
pp. 1065-1072.
show abstract
New drug discovery continues to follow the time-honored paths of screening and novel target identification combined with analogue development and serendipity. The agents selected here reflect these various approaches. They include drugs already showing significant antitumor activity, eg, anthrapyrazoles, temozolomide, camptothecin analogues, and taxotere, and updated information is provided on their development. Other drugs just entering or currently in phase I trials include rhizoxin, which seems capable of overcoming multidrug resistance; the novel bioreductive agent EO9 [corrected]; and bryostatin, a highly potent protein kinase C agonist. Sequence specificity could well prove to be an important factor in the development of DNA-interactive agents, and information is provided on the progress with distamycin mustard and the new cyclopropylpyrroloindole analogues carzelesin and adozelesin..
Riley, R.J.
Workman, P.
(1992). DT-diaphorase and cancer chemotherapy. Biochem pharmacol,
Vol.43
(8),
pp. 1657-1669.
Lohmeyer, M.
Workman, P.
(1992). Lack of enantio-selectivity in the in vitro antitumour cytotoxicity and membrane-damaging activity of ether lipid SRI 62-834: further evidence for a non-receptor-mediated mechanism of action. Biochem pharmacol,
Vol.44
(4),
pp. 819-823.
show abstract
SRI 62-834 ([tetrahydro-2-(octadecycloxy)methylfuran- 2-yl]methoxylphosphocholine; CRC 86-05; NSC 614383) is a cyclic antitumour ether lipid (AEL) with a novel, but ill-defined, mechanism of action. AELs are believed to act on membranes and cell signals, but the precise mechanisms of selectivity are unclear. Receptor-mediated mechanisms can often be identified by the differential activity of the individual stereoisomers of a drug. We have therefore compared the R- and S-enantiomers of SRI 62-834 for: (1) cytotoxicity against the human HT29 colon carcinoma cell line using a tetrazolium dye reduction assay and (2) membrane-damaging effects monitored by 51Cr radiolabel release. The tetrazolium assay revealed near-identical mean ID50 values around of 2-3 microM for the R- and S-isomers as well as for the racemic mixture. Moreover, pre- and co-incubation of the cells with the potent platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine;PAF) receptor antagonist WEB 2086BS (3-[4-(chorophenyl)-9-methyl-6H-thieno[3,2- f][1,2,4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4- morpholinyl)-1-propanone) had no effect on the cytotoxicity of either isomer or the racemate. Short-term membrane damage was not evident at low micromolar concentrations and between 139 and 163 microM either lipid was required to release 50% of the incorporated 51Cr label. Again, there was no difference in potency between the enantiomers and the racemate. Coincubation with WEB 2086BS also failed to modulate the membrane-lytic potency of the AELs. These results indicate that the site(s) of cytotoxic action of SRI 62-834 is (are) not stereospecific and also appear to rule out the involvement of a conventional PAF receptor in the mechanism of action of SRI 62-834..
Walton, M.I.
Wolf, C.R.
Workman, P.
(1992). The role of cytochrome P450 and cytochrome P450 reductase in the reductive bioactivation of the novel benzotriazine di-N-oxide hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233, WIN 59075) by mouse liver. Biochem pharmacol,
Vol.44
(2),
pp. 251-259.
show abstract
SR 4233 or WIN 59075 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel and highly selective hypoxic cell cytotoxin requiring reductive bioactivation for its impressive antitumour effects. Expression of appropriate reductases will contribute to therapeutic selectivity. Here we provide more detailed information on the role of cytochrome P450 and cytochrome P450 reductase in SR 4233 reduction by mouse liver microsomes. Reduction of SR 4233 to the mono-N-oxide SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) is NADPH, enzyme and hypoxia dependent. An inhibitory antibody to cytochrome P450 reductase decreased the microsomal SR 4233 reduction rate by around 20%. Moreover, studies with purified rat cytochrome P450 reductase showed unequivocally that this enzyme was able to catalyse SR 4233 reduction at a rate of 20-30% of that for microsomes with equivalent P450 reductase activity. Exposure to the specific cytochrome P450 inhibitor carbon monoxide (CO) inhibited microsomal reduction by around 70% and CO plus reductase antibody blocked essentially all activity. Additional confirmation of cytochrome P450 involvement was provided by the use of other P450 ligands: beta-diethylaminoethyl diphenylpropylacetate hydrochloride gave a slight stimulation while aminopyrine, n-octylamine and 2,4-dichloro-6-phenylphenoxyethylamine were inhibitory. Induction of SR 4233 reduction was seen with phenobarbitone, pregnenalone-16-alpha-carbonitrile and beta-napthoflavone, suggesting that cytochrome P450 subfamilies IIB, IIC and IIIA may be involved. Since cytochrome P450 and P450 reductase catalyse roughly 70 and 30%, of mouse liver microsomal SR 4233 reduction respectively, we propose that expression of these and other reductases in normal and tumour tissue is likely to be a major factor governing the toxicity and antitumour activity of the drug..
Riley, R.J.
Workman, P.
(1992). Enzymology of the reduction of the potent benzotriazine-di-N-oxide hypoxic cell cytotoxin SR 4233 (WIN 59075) by NAD(P)H: (quinone acceptor) oxidoreductase (EC 1 6 99 2) purified from Walker 256 rat tumour cells. Biochem pharmacol,
Vol.43
(2),
pp. 167-174.
show abstract
3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233; WIN 59075) is a highly selective hypoxic cell cytotoxin soon to enter phase I clinical trial. The compound is thought to exert its action through a toxic one-electron reduced free radical intermediate. Preliminary data have suggested that SR 4233 may be metabolized by DT-diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2)] to both two- and four-electron reduced products and that this route of biotransformation may represent a bioprotection pathway. In this study, a highly purified enzyme preparation was employed in order to investigate further the metabolism of SR 4233 by DT-diaphorase and to examine the mechanism of reduction in more detail. Spectrophotometric analysis showed that SR 4233 underwent reduction by DT-diaphorase with an apparent Km of 1.23 +/- 0.27 mM and Vmax of 8.55 +/- 1.67 nmol/min/microgram protein. This reaction was inhibited completely by dicoumarol (100 microM) and partially by an antiserum raised against the purified enzyme. Characterization of the products of SR 4233 reduction by reverse-phase HPLC confirmed that both two- (SR 4317) and four- (SR 4330) electron reduction products were generated, the latter being the predominant metabolite, particularly in prolonged incubations. Further experiments showed that the four-electron reduction product, but not the two-electron reduction product, was also a substrate for DT-diaphorase with an apparent Km of 1.14 mM and a Vmax of 57.12 nmol/min/micrograms protein. The results presented confirm that SR 4233 is indeed a substrate for DT-diaphorase and that a mixture of two-, four- and six-electron reduced products may be formed. The possible toxicological and pharmacodynamic significance of this metabolism is discussed..
WORKMAN, P.
DINCALCI, M.
BERDEL, W.
EGORIN, M.
HELENE, C.
HICKMAN, J.
JARMAN, M.
SCHWARTSMANN, G.
SIKORA, K.
(1992). NEW APPROACHES IN CANCER PHARMACOLOGY - DRUG DESIGN AND DEVELOPMENT. Eur j cancer,
Vol.28A
(6-7),
pp. 1190-1200.
Workman, P.
(1992). Bioreductive mechanisms. Int j radiat oncol biol phys,
Vol.22
(4),
pp. 631-637.
show abstract
The design, development, and application of bioreductive antitumor agents in a rational way requires a detailed understanding of the mechanisms involved in their action. In addition to measuring and manipulating tumor hypoxia, we need to elucidate the particulars of the activation versus bioprotection pathways and the nature and properties of the participating enzymes. These areas are reviewed with particular reference to the development of novel quinone, nitro and N-oxide bioreductives..
Walton, M.I.
Sugget, N.
Workman, P.
(1992). The role of human and rodent DT-diaphorase in the reductive metabolism of hypoxic cell cytotoxins. Int j radiat oncol biol phys,
Vol.22
(4),
pp. 643-647.
show abstract
DT-diaphorase is a unique two electron (2e) donating reductase catalyzing either bioactivation or bioprotection reactions. Using human and rodent DT-diaphorase preparations (cell extracts and purified enzyme) we have characterized the reductive metabolism of the hypoxic cell cytotoxins EO9, mitomycin C (MMC), CB 1954, and SR 4233 in vitro. Drug metabolism was assayed spectrophotometrically or by HPLC, with dicoumarol as a selective inhibitor. DNA damage was measured using an agarose gel mobility technique with plasmid pBR322 DNA. The developmental indoloquinone, EO9, was metabolized by both rat Walker and human HT29 tumor DT-diaphorases. Reduction proceeded 5-fold more efficiently with the rat than the human tumor enzyme and resulted in single-strand breaks in plasmid DNA. The structurally related MMC was metabolized much more slowly than EO9 by the rat Walker tumor enzyme and there was no detectable reaction with the human HT29 tumor DT-diaphorase. No DNA damage was seen with MMC for either enzyme. The dinitrophenylaziridine CB 1954 was reduced by both human and rat enzymes forming, preferentially, the highly toxic 4-hydroxylamine as a 4e reduction product. Rates were 3-fold lower than for the human tumor enzyme. SR 4233 was also reduced by the rat tumor enzyme predominantly via 4e reduction to the benzotriazine SR 4330, in a novel reaction mechanism. This appears to be a bioprotection pathway that bypasses the toxic 1e radical formed by other reductases. Such information may be valuable in the selection of hypoxic cell cytoxins to treat human tumors high or low in DT-diaphorase and should facilitate 'enzyme-directed' analogue development..
Bailey, S.M.
Suggett, N.
Walton, M.I.
Workman, P.
(1992). Structure-activity relationships for DT-diaphorase reduction of hypoxic cell directed agents: indoloquinones and diaziridinyl benzoquinones. Int j radiat oncol biol phys,
Vol.22
(4),
pp. 649-653.
show abstract
The flavoenzyme DT-diaphorase has the potential either to bioactivate or to detoxify different bioreductive cytotoxins. Elucidation of structural features governing the ability to act as a substrate for DT-diaphorase should facilitate rational optimization or elimination of this reductive pathway for a particular class of bioreductive drug. We have examined structure-activity relationships governing both the cytotoxicity and the DT-diaphorase mediated reduction of two groups of bioreductive alkylating agents: (1) Indoloquinones related to EO9 [3-hydroxy-methyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta - en-alpha-ol]; and (2) derivatives of diaziridinyl benzoquinone or diaziquone [2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone]. The rat U.K. 256 Walker tumor cell line and the human HT29 colon carcinoma line were studied because of their high DT-diaphorase content. Enzyme activity was measured spectrophotometrically by dicoumarol inhibitable cytochrome c reduction in the presence of drug, and aerobic cytotoxicity was assessed by the MTT assay. EO9 acted as a good substrate for both enzyme preparations and was highly potent in each cell line, especially in Walker tumor cells (ID50 0.039 nM). AZQ was also reduced efficiently and gave an ID50 of 6 nM in the Walker tumor line. Slight modifications in structure resulted in large variations in both DT-diaphorase metabolism and toxicity for both types of agent. There was a clear tendency for the most efficiently reduced analogues to exhibit greater cytotoxic potency. Inclusion of an aziridine moiety in the structure appears to be desirable, but not essential, for both rapid reduction and cytotoxicity. There was no evidence of active site-directed enzyme inhibition..
Workman, P.
Binger, M.
Kooistra, K.L.
(1992). Pharmacokinetics, distribution, and metabolism of the novel bioreductive alkylating indoloquinone EO9 in rodents. Int j radiat oncol biol phys,
Vol.22
(4),
pp. 713-716.
show abstract
The indoloquinone EO9 is a novel and potent bioreductive agent related in structure to mitomycin C but differing in many aspects of its antitumor activity, toxicity, and enzymatic activation. Because it is about to undergo clinical trial, we have investigated the pharmacokinetics of EO9 in mice and rats. At the highest tolerated dose in male C3H/He mice (12 mg/kg iv) the initial plasma concentration (Co) was 1.8 micrograms/ml. The drug was cleared rapidly with a t1/2 of 1.9 min. The volume of distribution (Vd) was large (7.5 ml g-1) and the plasma clearance (Clp) correspondingly high (2.6 ml g-1 min-1). The AUCo-infinity was 4.8 micrograms ml-1 min. Equally rapid elimination was noted at the lower dose of 6 mg/kg iv. For comparison, the t1/2 for the same dose of mitomycin C was much longer at 16 min and the peak plasma level 4-fold higher. In male Sprague-Dawley rats receiving 3 mg kg-1 the Co was 1.5 micrograms/ml and the t1/2 was again short at 3.0 min. Vd was 2.2 ml g-1, Clp was 0.5 ml g-1 min-1, and AUCo-infinity was 6.2 micrograms ml-1 min. No parent drug was detected in urine, but extensive biotransformation was confirmed by the detection of around 20% of the dose as metabolites, including the aziridine ring-opened hydrolysis product EO5A. No drug or metabolite was detected in tumor or tissues. The results show that cytotoxic drug levels can be achieved for a short period in rodent plasma. The extremely fast excretion is consistent with the rapid rates of bioreductive metabolism in vitro. These data should be useful in the forthcoming clinical trials of EO9, where a pharmacokinetically guided dose escalation may be used, and also in the design and development of second generation analogues..
Wasserman, T.H.
Siemann, D.
Workman, P.
(1992). Editor's Note Seventh International Conference on Chemical Modifiers of Cancer Treatment. Int j radiat oncol biol phys,
,
p. 22.
Workman, P.
(1992). Infusional anthracyclines: Is slower better? If so, why?. Ann oncol,
Vol.3,
pp. 591-594.
DIVE, C.
WATSON, J.V.
WORKMAN, P.
(1991). MULTIPARAMETRIC FLOW-CYTOMETRY OF THE MODULATION OF TUMOR-CELL MEMBRANE-PERMEABILITY BY DEVELOPMENTAL ANTITUMOR ETHER LIPID SRI 62-834 IN EMT6 MOUSE MAMMARY-TUMOR AND HL-60 HUMAN PROMYELOCYTIC LEUKEMIA-CELLS. Cancer research,
Vol.51
(3),
pp. 799-8.
Coley, H.M.
Workman, P.
Twentyman, P.R.
(1991). Retention of activity by selected anthracyclines in a multidrug resistant human large cell lung carcinoma line without P-glycoprotein hyperexpression. Br j cancer,
Vol.63
(3),
pp. 351-357.
show abstract
full text
A subline (COR-L23/R) of the human large cell lung line [corrected] COR-L23, derived by in vivo exposure to doxorubicin, exhibits an unusual multidrug resistant (MDR) phenotype. This subline shows cross-resistance to daunorubicin, vincristine, colchicine and etoposide but does not express P-glycoprotein. Interestingly, COR-L23/R [corrected] shows little or no resistance to a range of structurally-modified analogues of doxorubicin comprising 9-alkyl and/or sugar modified anthracyclines. We have previously identified these same compounds as effective agents against P-glycoprotein-positive MDR cell lines. In contrast to typical MDR cell lines, COR-L23/R [corrected] shows only minimal chemosensitisation by verapamil and no collateral sensitivity to verapamil. Compared to the parental cell line, COR-L23/R [corrected] displays reduced accumulation of doxorubicin and daunorubicin. Accumulation defects were apparent only after 0.5-1 h of incubation of cells with these agents. The rate of daunorubicin efflux was shown to be enhanced by COR-L23/R [corrected] and this efflux was demonstrated to be energy-dependent. The use of anthracyclines which retain activity in MDR cells thus appears to be a valid approach for the circumvention of MDR, not only in cells which express P-glycoprotein, but also where defective drug accumulation is due to other mechanisms possibly involving an alternative multidrug transporter..
Walton, M.I.
Smith, P.J.
Workman, P.
(1991). The role of NAD(P)H: quinone reductase (EC 1 6 99 2, DT-diaphorase) in the reductive bioactivation of the novel indoloquinone antitumor agent EO9. Cancer commun,
Vol.3
(7),
pp. 199-206.
show abstract
EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(H-indole-4, 7-indione)-propenol] is a novel indoloquinone structurally related to mitomycin C, a quinone anticancer drug that requires reductive bioactivation. NAD(P)H: (quinone-acceptor) oxidoreductase (quinone reductase, DT-diaphorase, EC 1.6.99.2) is an obligate 2-electron donating enzyme that can reduce a variety of quinones resulting either in bioactivation or bioprotection. Using quinone reductase (QR) preparations from rat Walker 256 mammary tumor cells and human HT29 colon carcinoma cells, we have characterized the role of this enzyme in EO9 reductive metabolism. QR activity was assayed under optimal conditions by following cytochrome c reduction at 550 nm in the presence of enzyme, quinone substrate, NADH, and bovine albumin, and confirmed by loss of EO9 absorbance at 550 nm. Both the rat and human tumor cell enzymes catalyzed reduction of the benchmark quinone menadione with a similar Km of 1.4-3.1 microM, although the Vmax was 7 to 8-fold lower for the human preparation. EO9 was readily reduced by the rat Walker QR. The mean Km was about 5-fold higher than for menadione at around 15 microM and the Vmax was 6-fold lower at around 2.5 mumol of cytochrome c reduced mg-1 of protein. EO9 was also metabolized by QR from HT29 human colon carcinoma cells but rather less efficiently than by the rat tumor enzyme. For example, the rate was 6-fold lower than that for the Walker tumor enzyme at 100 microM substrate concentration after correcting for the 7- to 8-fold difference in specific activity for the two preparations.(ABSTRACT TRUNCATED AT 250 WORDS).
Workman, P.
(1991). Antitumor ether lipids: endocytosis as a determinant of cellular sensitivity. Cancer cells,
Vol.3
(8),
pp. 315-317.
Workman, P.
Donaldson, J.
Lohmeyer, M.
(1991). Platelet-activating factor (PAF) antagonist WEB 2086 does not modulate the cytotoxicity of PAF or antitumour alkyl lysophospholipids ET-18-O-methyl and SRI 62-834 in HL-60 promyelocytic leukaemia cells. Biochem pharmacol,
Vol.41
(2),
pp. 319-322.
Binger, M.
Workman, P.
(1991). Pharmacokinetic contribution to the improved therapeutic selectivity of a novel bromoethylamino prodrug (RB 6145) of the mixed-function hypoxic cell sensitizer/cytotoxin alpha-(1-aziridinomethyl)-2-nitro-1H-imidazole-1-ethanol (RSU 1069). Cancer chemother pharmacol,
Vol.29
(1),
pp. 37-47.
show abstract
RB 6145 is a novel hypoxic cell sensitizer and cytotoxin containing both an essential bioreductive nitro group and a bromoethylamino substituent designed to form an alkylating aziridine moiety under physiological conditions. In mice, RB 6145 is 2.5 times less toxic but only slightly less active than the aziridine analogue RSU 1069, giving rise to an improved therapeutic index. However, the mechanism for the enhanced selectivity is not clear. Reasoning that this may lie in a more beneficial pharmacokinetic profile, we investigated the plasma pharmacokinetics, tissue distribution and metabolism of RB 6145 in mice using a specially developed reversed-phase HPLC technique. An i.p. dose of 190 mg kg-1 (0.5 mmol kg-1) RB 6145 produced peak plasma concentrations of about 50 micrograms ml-1 of the pharmacologically active target molecule RSU 1069 as compared with levels of around twice this value that were obtained using an equimolar i.p. dose of RSU 1069 itself. The plasma AUC0-infinity value for administered RSU 1069 was ca. 47 micrograms ml-1 h and that for the analogue RSU 1069 was ca. 84 micrograms ml-1 h. No prodrug was detectable. Another major RB 6145 metabolite in plasma was the corresponding oxazolidinone, apparently formed on interaction of the drug with hydrogen carbonate. The oxazolidinone initially occurred at higher concentrations than did RSU 1069, with the levels becoming very similar from 30 min onwards. Post-peak plasma concentrations of both RB 6145 metabolites declined exponentially, displaying an elimination t1/2 of ca. 25 min, very similar to the 30-min value observed for injected RSU 1069. The plasma AUC0-infinity value for the metabolite RSU 1069 was about 1.3 and 1.6 times higher following i.p. injection of 95 mg kg-1 (0.25 mmol kg-1) of the prodrug as compared with administration via the oral and i.v. routes, respectively. After i.v. injection, peak levels of the oxazolidinone metabolite were twice those observed following both i.p. and oral dosing and possibly contributed to the acute toxicity. After an i.p. dose of 190 mg kg-1 RB 6145, concentrations of RSU 1069 and the oxazolidinone metabolites rose to 40% and 33%, respectively, of the ambient plasma level in i.d. KHT tumours. The peak level of metabolite RSU 1069 was ca. 6 micrograms g-1 as compared with 10 micrograms g-1 following an equimolar dose of RSU 1069 itself; the tumour AUC0-infinity value for the metabolite RSU 1069 was some 35% lower.(ABSTRACT TRUNCATED AT 400 WORDS).
Workman, P.
Newman, H.F.
Bleehen, N.M.
Ward, R.
Smithen, C.E.
(1991). Lack of stereoselectivity in the pharmacokinetics and metabolism of the radiosensitizer Ro 03-8799 in man. Cancer chemother pharmacol,
Vol.28
(2),
pp. 118-122.
show abstract
During a clinical toxicity study it was possible to obtain urine samples from six patients receiving either the R-(-)- or S-(+)-stereoenantiomeric forms of the developmental 2-nitroimidazole radiosensitizer Ro 03-8799 (pimonidazole). Paired plasma samples were also obtained from four patients. The pharmacokinetic data were compared with those for the racemic mixture in the same individuals. The results revealed no major differences in the plasma pharmacokinetics, urinary clearance or N-oxidation of the individual enantiomers as compared with the racemic mixture. A similar lack of stereoselectivity with respect to the acute dose-limiting CNS toxicity syndrome suggests that this may not involve a specific CNS receptor interaction..
Birchmeier, W.
Cavalli, F.
Garrattini, S.
Heldin, C.-.
Mach, J.
Monier, R.
Nusse, R.
Ponder, B.A.
Rietmuller, G.
Rajewski, M.F.
Tubiana, M.
Hansen, H.H.
Aapro, M.S.
Double, J.A.
Gunzburg, W.H.
Helene, C.
Jahde, E.
Leake, R.E.
Mantovani, A.
Pluckthun, A.
Schwartsmann, G.
Stevens, M.F.
Workman, P.
(EORTC Scientific Advisory Board),
(1991). Towards improved cancer diagnosis and treatment founded on current developments in the basic sciences: Options for intensified European efforts. Eur j cancer,
Vol.27,
p. 936.
Hartman, N.G.
Patterson, L.H.
Workman, P.
Suarato, A.
Angelucci, F.
(1990). Doxorubicin-3'-NH-oestrone-17-oxime-ethyl-carbonyl, a doxorubicin-oestrone conjugate that does not redox cycle in rat liver microsomes. Biochem pharmacol,
Vol.40
(5),
pp. 1164-1167.
show abstract
In summary doxorubicin-3'-NH-oestrone-17-oximethyl-carbonyl (Dox-Oes) is a covalent adduct of the anthracycline antitumor agent doxorubicin and oestrogen. Dox-Oes does not generate free radicals in rat liver microsomes as detected by electron spin resonance spectroscopy or redox cycle as shown by lack of superoxide anion formation and NADPH oxidation. Furthermore Dox-Oes actually inhibits free radical formation by doxorubicin used in equimolar amounts. The lack of free radical formation by doxorubicin when covalently linked to oestrone supports the development of Dox-Oes as a non-cardiotoxic derivative whilst potentially improving its targeting to oestrogen positive breast tumour cells..
Walton, M.I.
Workman, P.
(1990). Enzymology of the reductive bioactivation of SR 4233 A novel benzotriazine di-N-oxide hypoxic cell cytotoxin. Biochem pharmacol,
Vol.39
(11),
pp. 1735-1742.
show abstract
SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel benzotriazine di-N-oxide which shows unusually high selective toxicity towards hypoxic cells, probably as a result of reductive bioactivation. Using an HPLC assay for the parent drug and its 2- and 4-electron reduction products (SR 4317 and SR 4330, respectively), we have examined the enzymology of SR 4233 reductive metabolism in vitro using a variety of different enzyme preparations. SR 4233 was converted extremely rapidly to SR 4317 under N2 by mouse liver microsomes, and showed a marked preference for NADPH over NADH as a reduced cofactor. The reaction was inhibited completely in air and boiled preparations. It was also inhibited by 78-86% in carbon monoxide (CO), implicating cytochrome P-450 as the major microsomal SR 4233 reductase. The kinetics of reductive metabolism of SR 4233 to SR 4317 by mouse liver microsomes conformed to Michaelis-Menten kinetics, with a Km of 1.4 mM and a Vmax of 950 nmol/min/mg protein. SR 4233 reduction was also catalysed by mouse liver cytosol under N2. However, rates were markedly slower than for microsomes and showed an equal dependency on NADH and NADPH. The cytosolic enzymes aldehyde oxidase and xanthine oxidase both catalysed SR 4233 reduction to SR 4317 under N2. Purified buttermilk xanthine oxidase also catalysed this reaction. In contrast to other enzyme preparations, DT-diaphorase from Walker 256 tumour cells reduced SR 4233 predominantly to SR 4330, and this reaction occurred under aerobic conditions. These data illustrate that SR 4233 is reduced rapidly by a wide variety of reductases. We propose that the therapeutic selectivity of SR 4233 will be controlled by the relative expression of reductases in tumour versus normal tissues, and in particular by the differential participation of putative activating versus detoxifying enzymes..
WORKMAN, P.
WALTON, M.I.
(1990). ENZYME-DIRECTED BIOREDUCTIVE DRUG DEVELOPMENT - IS DT-DIAPHORASE A 2-EDGED-SWORD. International journal of radiation biology,
Vol.58
(5),
pp. 907-2.
COLEY, H.M.
TWENTYMAN, P.R.
WORKMAN, P.
(1990). 9-ALKYL, MORPHOLINYL ANTHRACYCLINES IN THE CIRCUMVENTION OF MULTIDRUG RESISTANCE. European journal of cancer,
Vol.26
(6),
pp. 665-3.
WORKMAN, P.
WALTON, M.I.
BINGER, M.
BAILEY, S.M.
SUGGETT, N.R.
(1990). ENZYME-DIRECTED BIOREDUCTIVE DRUGS - METABOLISM OF DEVELOPMENTAL INDOLOQUINONE E09 AND RELATED AGENTS BY DT-DIAPHORASE. British journal of cancer,
Vol.62
(3),
pp. 495-1.
WALTON, M.I.
SMITH, P.J.
WORKMAN, P.
(1990). DEMONSTRATION OF LOW AND BIOLOGICALLY INSIGNIFICANT BIOREDUCTION RATES FOR MITOMYCIN-C (MMC) BY DT-DIAPHORASE. British journal of cancer,
Vol.62
(3),
pp. 495-1.
WORKMAN, P.
DIVE, C.
LOHMEYER, M.
DONALDSON, J.
(1990). INVITRO ANTITUMOR-ACTIVITY OF MEMBRANE-ACTIVE ANALOGS OF PLATELET ACTIVATING FACTOR (PAF). British journal of cancer,
Vol.62
(3),
pp. 495-1.
WALTON, M.I.
WORKMAN, P.
(1990). COMPARATIVE ENZYMOLOGY OF THE BIOREDUCTIVE METABOLISM OF THE AZIRIDINYL DINITROBENZAMIDE CB-1954 - MICROSOMAL-ENZYMES VERSUS DT-DIAPHORASE. British journal of cancer,
Vol.62
(3),
pp. 508-1.
WALTON, M.I.
WORKMAN, P.
(1990). NOVEL, POTENTIAL BIOPROTECTION PATHWAY FOR THE DEVELOPMENTAL HYPOXIC CELL CYTOTOXIN SR-4233 - CATALYSIS BY DT-DIAPHORASE. British journal of cancer,
Vol.62
(3),
pp. 508-1.
COLEY, H.M.
WORKMAN, P.
TWENTYMAN, P.R.
AMOS, W.B.
(1990). CONFOCAL MICROSCOPY OF ANTHRACYCLINE SUBCELLULAR-LOCALIZATION IN CLASSIC AND ATYPICAL MULTIDRUG RESISTANCE (MDR). British journal of cancer,
Vol.62
(3),
pp. 512-1.
WORKMAN, P.
WALTON, M.I.
BIBBY, M.C.
DOUBLE, J.A.
(1990). INVIVO RESPONSE OF MOUSE ADENOCARCINOMA OF THE COLON (MAC) TUMORS TO INDOLOQUINONE E09 - CORRELATION WITH BIOREDUCTIVE ENZYME CONTENT. British journal of cancer,
Vol.62
(3),
pp. 515-2.
LAMONT, A.
WORKMAN, P.
DENNIS, I.
BLEEHEN, N.M.
(1990). PHASE-I CLINICAL AND PHARMACOKINETICS STUDY OF PHYLLANTHOSIDE (NSC-328426, CRC85/13) USING A SINGLE DOSE 30 MINUTE INFUSION SCHEDULE. British journal of cancer,
Vol.62
(3),
pp. 520-1.
WORKMAN, P.
(1990). THE CELL-MEMBRANE AND CELL SIGNALS - NEW TARGETS FOR NOVEL ANTICANCER DRUGS. Annals of oncology,
Vol.1
(4),
pp. A100-12.
HOBAN, P.R.
WALTON, M.I.
ROBSON, C.N.
GODDEN, J.
STRATFORD, I.J.
WORKMAN, P.
HARRIS, A.L.
HICKSON, I.D.
(1990). DECREASED NADPH - CYTOCHROME-P-450 REDUCTASE-ACTIVITY AND IMPAIRED DRUG ACTIVATION IN A MAMMALIAN-CELL LINE RESISTANT TO MITOMYCIN-C UNDER AEROBIC BUT NOT HYPOXIC CONDITIONS. Cancer research,
Vol.50
(15),
pp. 4692-6.
MAUGHAN, T.S.
NEWMAN, H.F.
BLEEHEN, N.M.
WARD, R.
WORKMAN, P.
(1990). ABNORMAL CLINICAL PHARMACOKINETICS OF THE DEVELOPMENTAL RADIOSENSITIZERS PIMONIDAZOLE (RO03-8799) AND ETANIDAZOLE (SR2508). International journal of radiation oncology biology physics,
Vol.18
(5),
pp. 1151-6.
POWIS, G.
HICKMAN, J.
WORKMAN, P.
TRITTON, T.
ABITA, J.
BERDEL, W.
GESCHER, A.
MOSES, H.
NICOLSON, G.
(1990). THE CELL-MEMBRANE AND CELL SIGNALS AS TARGETS IN CANCER-CHEMOTHERAPY AACR, EORTC, AND BACR SPECIAL CONFERENCE IN CANCER-RESEARCH. Cancer res,
Vol.50
(7),
pp. 2203-2211.
BINGER, M.
WORKMAN, P.
(1990). GRADIENT HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR THE DETERMINATION OF THE NOVEL INDOLOQUINONE ANTITUMOR AGENT-E09 IN BIOLOGICAL SPECIMENS. Journal of chromatography-biomedical applications,
Vol.532
(2),
pp. 321-16.
Workman, P.
(1990). The cell membrane and cell signals: new targets for novel anticancer drugs. Ann oncol,
Vol.1
(2),
pp. 100-111.
show abstract
In the concluding Discussion session, emphasis focussed on the potential for interfering selectively with cell membranes and cell signalling in tumour as against normal tissues. There could be no doubt that tremendous advances are being made in our understanding of the molecular changes associated with malignancy and that the information available for the rational design of inhibitors of particular signalling pathways is increasingly sophisticated. There was a consensus that we need more information on the qualitative and quantitative differences in the structure and function of membranes and the signalling machinery in various normal tissues as compared to their cancerous counterparts. Ideally we will develop drug against, for example, specific forms of, let us say, protein kinase C or tyrosine kinase which are found to be predominantly active in neoplastic cells. This may well prove possible, at least in some instances, in which case a safe therapeutic margin will be assured. But differences may in other situations turn out to be in the level of expression rather than purely qualitative in nature, and the scale of the disparate expression may not always be great. Even in such situations, adequate therapeutic selectivity may still be achieved. This may derive from a "damping down" of signalling in the hyperactive tumour. Although there are legitimate concerns regarding the possible toxic effects of administering signal-wrecking molecules in man, we should not be pessimistic as there are clear precedents elsewhere in medicine for drugs acting on membrane signals proving to be safe and effective against expectation informed by hindsight. There may also be concerns about new forms of drug resistance. But this will be so for any new agent or novel target. And with mechanism of action clearly to the fore we should be able to predict resistance pathways in advance and devise appropriate circumvention strategies or targeted second line therapies. There was a palpable buzz at the meeting that this is a valid, different and above all rational approach. Not only that, but the new therapeutic molecules which we discover will themselves prove to be valuable tools with which to probe further into the mechanisms of malignancy and signal transduction. We had expected to see a bewildering amount of new information from the basic sciences of molecularbiology and cell physiology, and we got it. But it was also impressive to witness the number of new compounds coming through which look like real drugs or at least exciting lead compounds. The membrane-active ether lipids are in clinical trial. Bryostatin 1 will shortly join them.(ABSTRACT TRUNCATED AT 400 WORDS).
Dive, C.
Watson, J.V.
Workman, P.
(1990). Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes. Cytometry,
Vol.11
(2),
pp. 244-252.
show abstract
We describe an improved twin-probe multiparameter flow cytometric technique to examine cell membrane permeability. Ability to retain preloaded intracellular bis-carboxyethyl carboxy fluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) is measured, simultaneously with forward and right-angle scatter. This has significant advantages over an earlier method using fluorescein together with ethidium. In addition to the two expected cell populations which were stained green positive, red negative (by convention membrane "intact" and "viable," Region 1) and green negative, red positive ("membrane-damaged" and "non-viable," Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 2). This was true for each of 9 cell types in vitro. For EMT6 mouse mammary tumour cells held under sub-optimal conditions or treated with membrane-active drugs, progression from Region 1 to Region 2 was observed, followed by further progression from Region 2 to Region 3. Cells eventually accumulated in Region 3. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and in turn to extracellular propidium..
Workman, P.
(1990). 31 Joint Steering Committee of the EORTC and CRC (Member of EORTC New Drug Development and Co-ordinating Committee and CRC Phase I/II Clinical Trials Subcommittee: P Workman) General guidelines for the preclinical toxicology of new cytotoxic anticancer agents in Europe. Eur j cancer,
Vol.26,
p. 411.
Workman, P.
(1990). Philosophies of new anticancer drug development. J drug dev,
Vol.2,
p. 193.
Walton, M.I.
Bleehen, N.M.
Workman, P.
(1989). Stimulation by localized tumor hyperthermia of reductive bioactivation of 2-nitroimidazole benznidazole in mice. Cancer res,
Vol.49
(9),
pp. 2351-2355.
show abstract
We have investigated the effects of localized tumor hyperthermia (LTH; 43.5 degrees C x 30 min) on the reductive bioactivation of the 2-nitroimidazole benznidazole in C3H mouse normal tissues and KHT tumors. Mice were allocated to one of three treatment groups: (a) unrestrained controls, (b) sham tumor treatment, and (c) LTH. Concentrations of benznidazole and its amine metabolite were determined by high-performance liquid chromatography. Conscious mice were given LTH or sham treatment 2.5 h after 2.5 mmol/kg benznidazole i.p. This gave steady-state plasma benznidazole concentrations of 120-170 micrograms/ml at 2-5 h in all three groups. Plasma amine concentrations were very low at 0.1-1 micrograms/ml in all cases. Liver benznidazole concentrations were similar to plasma but amine concentrations were 30-40-fold greater at 20-40 micrograms/g in all three groups, implicating the liver as a major site of reductive metabolism. Benznidazole concentrations in tumors from unrestrained mice were comparable to those in plasma and liver, with tumor/plasma ratios of 85-113%. Tumor amine concentrations were intermediate at about 2-3 micrograms/g, indicating reductive bioactivation had occurred. Sham treatment decreased tumor benznidazole concentrations by 25-50%, particularly at later times, and amine concentrations were correspondingly increased. This may be a result of sham tumor treatment at 37 degrees C, a temperature 3-4 degrees C higher than in unrestrained controls. More importantly, LTH further decreased tumor benznidazole concentrations over sham treatment, e.g., by 59% from 114 to 47 micrograms/g (P less than 0.01) immediately after heating. Amine concentrations were correspondingly elevated, e.g., by 40% from 5.1 to 8.4 micrograms/g (P less than 0.01). These results clearly show that LTH can selectively enhance the reductive bioactivation of benznidazole in KHT tumors in mice, and support a particular role for the use of bioreductive agents with heat..
WORKMAN, P.
WARD, R.
MAUGHAN, T.S.
NEWMAN, H.F.
BLEEHEN, N.M.
(1989). PHARMACOKINETIC ASPECTS OF PLASMA DRUG CONCENTRATION MONITORING FOR PREDICTION OF ETANIDAZOLE (SR 2508) TOXICITY. British journal of cancer,
Vol.60
(3),
pp. 464-1.
DIVE, C.
THOMPSON, M.G.
HICKMAN, J.A.
WATSON, J.V.
WORKMAN, P.
(1989). SPECIFIC BIOCHEMICAL OR LESS-SPECIFIC BIOPHYSICAL ACTION OF ETHER LIPID SRI 62-834 ON TUMOR-CELL MEMBRANES INVITRO. British journal of cancer,
Vol.60
(3),
pp. 472-1.
WALTON, M.I.
HOBAN, P.R.
ROBSON, C.N.
WORKMAN, P.
HARRIS, A.L.
HICKSON, I.D.
(1989). MITOMYCIN-C RESISTANCE - ASSOCIATION WITH DECREASED NADPH CYTOCHROME-P-450 REDUCTASE-ACTIVITY IN CHINESE-HAMSTER OVARY (CHO) CELLS-INVITRO. British journal of cancer,
Vol.60
(3),
pp. 474-1.
WALTON, M.I.
WORKMAN, P.
(1989). BIODISTRIBUTION AND MOLECULAR ENZYMOLOGY OF SR-4233 REDUCTIVE METABOLISM IN MICE. British journal of cancer,
Vol.60
(3),
pp. 475-1.
COLEY, H.M.
TWENTYMAN, P.R.
WORKMAN, P.
REEVE, J.G.
(1989). ANTHRACYCLINE CROSS-RESISTANCE AND DRUG ACCUMULATION IN A MULTIDRUG RESISTANT HUMAN-LUNG CANCER CELL-LINE WHICH DOES NOT EXPRESS P-170 GLYCOPROTEIN. British journal of cancer,
Vol.60
(3),
pp. 496-1.
MAXWELL, R.J.
WORKMAN, P.
GRIFFITHS, J.R.
(1989). DEMONSTRATION OF TUMOR-SELECTIVE RETENTION OF FLUORINATED NITROIMIDAZOLE PROBES BY F-19 MAGNETIC-RESONANCE SPECTROSCOPY INVIVO. International journal of radiation oncology biology physics,
Vol.16
(4),
pp. 925-5.
BLEEHEN, N.M.
NEWMAN, H.F.
MAUGHAN, T.S.
WORKMAN, P.
(1989). A MULTIPLE DOSE STUDY OF THE COMBINED RADIOSENSITIZERS RO-03-8799 (PIMONIDAZOLE) AND SR-2508 (ETANIDAZOLE). International journal of radiation oncology biology physics,
Vol.16
(4),
pp. 1093-4.
WORKMAN, P.
(1989). SPETH,PAUL,A J - OBITUARY. Cancer chemotherapy and pharmacology,
Vol.24
(4),
pp. 268-1.
Workman, P.
(1989). Accelerated elimination of pimonidazole following microsomal enzyme induction in mice: a possible approach to reduced neurotoxicity of the pimonidazole-etanidazole combination. Int j radiat oncol biol phys,
Vol.16
(4),
pp. 1011-1014.
show abstract
The pimonidazole-etanidazole combination shows promise for improved radiosensitization, although pimonidazole may increase the dose-limiting peripheral neurotoxicity of etanidazole in man. Induction of liver microsomal drug metabolizing enzymes by phenobarbitone resulted in reduced exposure to pimonidazole in plasma, brain, and tumor in mice. Peak tumor concentrations were lowered, but to a lesser extent. Phenobarbitone induction caused no change in the urinary ratio of pimonidazole to its N-oxide metabolite, and in fact, markedly reduced the circulating metabolite concentrations in plasma. There was no effect of phenobarbitone on plasma or tissue pharmacokinetics of etanidazole, which is eliminated by renal clearance. The results suggest that hepatic microsomal enzyme induction may be a possible approach to reducing the toxicity of the pimonidazole-etanidazole combination, and may also provide valuable information on the enzymology of pimonidazole metabolism..
Workman, P.
(1989). Micropharmacokinetics of chemical modifiers. Int j radiat oncol biol phys,
Vol.16
(4),
pp. 897-906.
show abstract
Classical pharmacokinetic analysis of plasma, urine and tissue specimens continues to be of major value to the rational development of chemical modifiers of cancer treatment. However, in addition, increasingly sophisticated analytical techniques are becoming available which allow the pathways of microdistribution and micrometabolism of drugs to be traced down to the cellular and molecular level. New developments described here include flow cytometry, magnetic resonance spectroscopy, and molecular enzymology. These are predicted to have a major impact on the optimization of chemical modification..
Walton, M.I.
Wolf, C.R.
Workman, P.
(1989). Molecular enzymology of the reductive bioactivation of hypoxic cell cytotoxins. Int j radiat oncol biol phys,
Vol.16
(4),
pp. 983-986.
show abstract
The hypoxic cell cytotoxins SR 4233, benznidazole (Benzo), and CB 1954 were readily reduced by anaerobic mouse liver microsomes in vitro to their respective amino or single N-oxide derivatives. The reactions were inhibited in air and required reduced cofactors, particularly NADPH. The rates of reductive bioactivation were markedly different for each drug, with SR 4233 much greater than CB 1954 greater than Benzo. Using purified cytochrome P-450 reductase (P-450 reductase) and an inhibitory antibody to this enzyme, we demonstrated that P-450 reductase was involved in the reductive bioactivation of all 3 compounds. It had a minor role in SR 4233 reduction, but a more important involvement in CB 1954 metabolism to its 4-amino metabolite. Using carbon monoxide, a specific inhibitor of cytochrome P-450 (P-450), we demonstrated that P-450 was involved in both SR 4233 and Benzo reduction. P-450 had a major role both in SR 4233 conversion to SR 4317 and in the latter steps of Benzo amine formation. Purified xanthine oxidase was shown to reduce SR 4233 and Benzo in vitro, but cytosolic aldehyde oxidase activity was only detectable with Benzo as substrate. Characterizing the relative participation of the various reductases in tumor versus normal tissues may allow a more rational selection and application of hypoxic cell cytotoxins in cancer therapy..
Walton, M.I.
Bleehen, N.M.
Workman, P.
(1989). Effects of localised tumour hyperthermia on pimonidazole (Ro 03-8799) pharmacokinetics in mice. Br j cancer,
Vol.59
(5),
pp. 667-673.
show abstract
full text
We have investigated the effects of localised tumour hyperthermia (LTH; 43.5 degrees C x 30 min) on the acute toxicity and pharmacokinetics of the hypoxic cell sensitizer pimonidazole (Ro 03-8799) in mice. There were three treatment groups: unrestrained controls, sham-treated and LTH treated mice. LTH had minimal effects on the acute toxicity (LD50/7d) of pimonidazole with no significant difference between the three treatment groups. Pharmacokinetic studies were carried out at the maximum tolerated dose (MTD; approximately 60% LD50) of 437 micrograms g-1 i.v. in plasma, brain and tumour. Sham tumour treatment consistently increased plasma drug concentrations compared to unrestrained controls but had minimal effects on the elimination t1/2. The AUC0-infinitive was increased by 35% and the plasma clearance decreased by 26%. By contrast, LTH had minimal effects on these parameters compared to sham treatment. Brain pimonidazole concentrations were increased in restrained mice (sham and LTH treatments) compared to unrestrained controls, but average brain/plasma ratios were similar in all three groups at between 400 and 500%. Sham tumour treatment markedly reduced peak tumour pimonidazole concentrations compared to unrestrained controls giving a 29% lower AUC0-180min. Average tumour/plasma ratios were reduced from 236 to 129%. The most important finding was that LTH further reduced pimonidazole tumour concentrations, giving a 31% lower AUC0-180 min compared to sham treated tumours. Tumour/plasma ratios for pimonidazole were reduced by 41%. Plasma exposure to the pimonidazole N-oxide metabolite, Ro 31-0313, was unaltered by LTH. The markedly reduced drug concentrations in heated tumours may be a result of hyperthermia-stimulated bioreductive drug activation..
Workman, P.
Ward, R.
Maughan, T.S.
Newman, H.F.
Bleehen, N.M.
(1989). Estimation of plasma area under the curve for etanidazole (SR 2508) in toxicity prediction and dose adjustment. Int j radiat oncol biol phys,
Vol.17
(1),
pp. 177-181.
show abstract
The hydrophilic 2-nitroimidazole radiosensitizer etanidazole is currently undergoing clinical evaluation. Although considerably less neurotoxic than misonidazole because of its rapid renal clearance and partial exclusion from the nervous system, total dose is limited by peripheral neuropathy. Monitoring plasma etanidazole concentration in patients to determine the area under the curve (AUC0-infinity) has been proposed as a method of predicting patients at risk, and of providing a quantitative basis for dose reduction in such patients. Successful application of this policy requires accurate assessment of AUC0-infinity. We have analyzed plasma data for 18 patients receiving 2 g/m2 etanidazole to determine the errors introduced in the estimation of AUC0-infinity caused by omitting selected time points from the analysis. A 'baseline' AUC0-infinity value was calculated by integration of the rate equation for the 2-compartment model using data points at 0, 15, and 30 min and 1, 2, 4, 8, 12, and 24 hr after the end of infusion. The mean +/- SD area for AUC0-infinity was 502 +/- 152 micrograms ml-1 h (2.35 +/- 0.71 mM.h). Omitting the zero or the 24 hr time point, the average errors were quite small (2.5% in both cases), but errors of up to 16.4 and 7.3%, respectively, were seen for individual patients. Leaving out both the 8 hr and 12 hr points at the same time gave a similar low average error of 2.9%, with a highest error of 7.3%. Omitting all data points after 4 hr, the mean error was 24.7% and 15 of 18 patients had errors in excess of 10%. In addition, failure to correct for infusion time results in an underestimation of AUC0-infinity averaging 4.5% (range 1.9-8.7%). The choice of sampling times for toxicological monitoring will depend upon the accuracy with which the AUC0-infinity must be known. Including all data points between 0 and 24 hr will minimize errors. Considering the general similarity in the errors introduced by omitting the 8 hr and 12 hr points together compared to those seen with exclusion of the single 24 hr point, the choice between these truncated sampling options would be expected to lie in the relative inconvenience caused to patients and medical staff for the particular dose schedule used. The short sampling schedule (0-4 hr) should not be used..
Workman, P.
Walton, M.I.
Powis, G.
Schlager, J.J.
(1989). DT-diaphorase: questionable role in mitomycin C resistance, but a target for novel bioreductive drugs?. Br j cancer,
Vol.60
(5),
pp. 800-802.
full text
Coley, H.M.
Twentyman, P.R.
Workman, P.
(1989). Improved cellular accumulation is characteristic of anthracyclines which retain high activity in multidrug resistant cell lines, alone or in combination with verapamil or cyclosporin A. Biochem pharmacol,
Vol.38
(24),
pp. 4467-4475.
show abstract
We have examined the cellular accumulation of anthracycline compounds, alone or in conjunction with resistance modifiers, in an attempt to identify mechanisms by which multidrug resistance (MDR) can be circumvented. This was facilitated by using the EMT6 mouse mammary tumour cell line EMT6/P and its MDR subline EMT6/AR1.0 with 30-fold resistance to Adriamycin (ADM), and the human small cell lung cancer line H69/P together with its MDR subline H69/LX4 with 100-fold resistance to ADM. Both MDR lines hyperexpress membrane P-170 glycoprotein. The accumulation of ADM was compared to that seen for the anthracycline analogues aclacinomycin A (ACL), Ro 31-1215 and 4'-deoxy-4'-iodo-Adriamycin (iodo-ADM). These analogues were selected because of their high activity against MDR sublines, including H69/LX4 and EMT6/AR1.0. Both MDR cell lines exhibited a deficiency in ADM accumulation compared to the parent lines. Smaller differentials were seen using Ro 31-1215 or iodo-ADM. Both resistant sublines were able to accumulate ACL in identical amounts to their respective parental sublines. Improved drug accumulation is likely to contribute to the improved activity of the analogues against MDR cell lines. However, the relative accumulation defects in the resistant lines did not correlate exactly with the degree of resistance to a particular compound. Cyclosporin A (5 micrograms/ml) or verapamil (3.3 micrograms/ml) caused a preferential increase in uptake in both MDR sublines, with a small or negligible effect for the parental line. A smaller effect was observed with iodo-ADM and Ro 31-1215, and levels of ACL were unchanged in the MDR lines in the presence of either resistance modifier. These results indicate two mechanisms for circumventing drug resistance due to reduced drug accumulation. Structurally modified derivatives can partially or completely eliminate uptake differentials between parent and drug resistant cell lines. Any residual uptake can be eliminated using resistance modifiers. The two mechanisms may both operate via inhibition or circumvention of P-170 mediated efflux. The situation is complex, however, and this study indicates the possible involvement of additional resistance mechanisms..
Coley, H.M.
Twentyman, P.R.
Workman, P.
(1989). Identification of anthracyclines and related agents that retain preferential activity over adriamycin in multidrug-resistant cell lines, and further resistance modification by verapamil and cyclosporin A. Cancer chemother pharmacol,
Vol.24
(5),
pp. 284-290.
show abstract
A range of anthracyclines and related compounds were evaluated for activity against murine and human cell lines exhibiting multidrug resistance (MDR). Cell lines used were the NCI-H69 human small-cell lung cancer line and the EMT6 murine mammary tumour line, together with their multidrug-resistant counterparts produced by in vitro exposure to Adriamycin (ADM). Chemosensitivity testing was carried out using the tetrazolium (MTT) dye assay. Results were expressed as the ratio of the ID50 for the resistant line to that obtained in the parent, i.e. the resistance factor (RF). Compounds exhibiting much lower RF values than ADM in both resistant cell lines were identified as those anthracyclines with 9-alkyl substituents and those with certain changes to the amino sugar residue at position 3' and 4', together with the anthracenedione mitoxantrone (MIT). In a further attempt to overcome resistance, we used four of these compounds, Ro 31-1215, 4'-deoxy-4'-iodo-ADM (iodo-ADM), aclacinomycin A (ACL) and MIT (all yielding low RF values), in combination with the resistance modifiers verapamil (VRP) and cyclosporin A (CYA). Additional enhancement of chemosensitivity was achieved in the ADM-resistant sublines, as shown by the further decrease in RF values. At the concentrations used, the largest effects were generally seen with CYA, and the combination of this modifier with ACL and MIT was particularly effective. For the H69/LX4 resistant line, the latter combinations gave RF values approaching unity. These findings point to the use of analogues with the 9-alkyl substituent and/or specific changes to the sugar residue in combination with resistance modifiers as a therapeutic strategy for circumvention of the MDR phenotype..
Dive, C.
Workman, P.
Watson, J.V.
(1989). Inhibition of cellular esterases by the antitumour imidazotetrazines mitozolomide and temozolomide: demonstration by flow cytometry and conventional spectrofluorimetry. Cancer chemother pharmacol,
Vol.25
(3),
pp. 149-155.
show abstract
Using flow cytometry and conventional spectrofluorimetry we have previously shown that chloroethylnitrosoureas (CNUs) can exhibit marked inhibition of cellular enzymes catalysing hydrolysis of fluorescein diacetate (FDA). More potent inhibition was seen for the carbamoylating CNUs, whereas alkylating agents were largely inactive. We now report results obtained with the developmental imidazotetrazines mitozolomide and temozolomide in comparison with BCNU, the novel alkylating agents clomesome and cyclodisone, and the active mitozolomide metabonate MCTIC. Inhibition of EMT6 mouse mammary-tumour esterases was seen for mitozolomide and temozolomide, and activity against purified porcine carboxylesterase was demonstrated. Flow cytometric analysis showed that inhibition occurred across the entire EMT6 cell population, with no evidence of a subpopulation resistant to enzyme inhibition. Inhibitory potency for the imidazotetrazines was much weaker than for BCNU. With EMT6 cells, I50 values from flow cytometry were 9.7 x 10(-3) M and 1.5 x 10(-3) M for mitozolomide and temozolomide compared with 3.7 x 10(-4) M for BCNU. These were higher than the ID50 values for in vitro antitumour activity (MTT assay), 8.5 x 10(-6) M in the case of mitozolomide and 1.2 x 10(-5) M for BCNU, but similar to that of 5.6 x 10(-4) M for the less toxic temozolomide. MCTIC and cyclodisone showed very low activity, but significant inhibition was seen for clomesome. The results are consistent with the view that the imidazotetrazines do not exhibit major carbamoylating ability, although significant effects are seen at cytotoxic concentrations of temozolomide. In addition, the potential for the generation of carbamoylating species at the enzyme active site cannot be ruled out..
Walton, M.I.
Bleehen, N.M.
Workman, P.
(1989). Effects of elevated temperature on misonidazole O-demethylation by mouse liver microsomes: kinetic and stability studies of a model mixed-function oxidase reaction. Cancer chemother pharmacol,
Vol.24
(3),
pp. 172-176.
show abstract
We investigated the effects of a range of temperatures (33 degrees-44 degrees C) on the stability and kinetics of C3H mouse liver microsomal misonidazole (MISO) O-demethylase in vitro. Microsomal O-demethylase activity was stable for 60 min at 37 degrees C and for 30 min at 41 degrees C but was steadily inactivated with longer incubation times. Inactivation at 44 degrees and 47 degrees C was exponential, with half-lives of 41 and 11 min, respectively. MISO O-demethylation followed Michaelis-Menten kinetics from 33 degrees to 44 degrees C. The apparent Vmax for desmethylmisonidazole (Ro 05-9963) formation was decreased by 32% (from 2.14 to 1.47 nmol min-1 mg-1 protein) with a 4 degrees decrease from 37 degrees to 33 degrees C. An increase of 4 degrees from 37 degrees to 41 degrees C enhanced the Vmax by 47%, but there was only an additional 9% increase for a further 3 degrees rise to 44 degrees C. Apparent Km values were unaltered at about 1.6 mM. These results show that elevated temperatures in the clinically relevant hyperthermia range (41 degrees-44 degrees C) can enhance a model mixed-function oxidase reaction in vitro. Such effects may be important for the metabolism, activity and toxicity of anticancer drugs combined with hyperthermia in vivo..
Workman, P.
(1989). New drugs and novel agents. Curr opin oncol,
Vol.1,
pp. 213-221.
WORKMAN, P.
WALTON, M.I.
KOOISTRA, K.L.
(1988). MOLECULAR ENZYMOLOGY OF BIOREDUCTIVE METABOLISM. British journal of cancer,
Vol.58
(4),
pp. 548-1.
WORKMAN, P.
KOOISTRA, K.L.
LEE, F.Y.
DONALDSON, J.
MARGISON, G.P.
(1988). INVIVO-INDUCED RESISTANCE TO CHLOROETHYLATING AGENTS CORRELATES WITH INCREASED O-6-ALKYLGUANINE DNA ALKYLTRANSFERASE EXPRESSION IN A MURINE SOLID TUMOR. British journal of cancer,
Vol.58
(2),
pp. 233-1.
COLEY, H.M.
TWENTYMAN, P.R.
WORKMAN, P.
(1988). RESISTANCE MODIFIERS VERAPAMIL AND CYCLOSPORIN-A - DIFFERENTIAL-EFFECTS ON THE CHEMOSENSITIVITY AND CELLULAR PHARMACOLOGY OF ANTHRACYCLINES IN MULTIDRUG RESISTANT CELLS. British journal of cancer,
Vol.58
(2),
pp. 234-1.
DIVE, C.
WORKMAN, P.
WATSON, J.V.
(1988). IMPROVED FLUORESCEIN-BASED SUBSTRATES FOR FLOW CYTOMETRIC MEASUREMENT OF CELLULAR ESTERASES CAN BE USED IN COMBINATION WITH PROPIDIUM IODIDE (PI) IN A MULTIPARAMETRIC ASSAY OF CELL VIABILITY AND MEMBRANE-PERMEABILITY. British journal of cancer,
Vol.58
(2),
pp. 236-1.
KOOISTRA, K.L.
WORKMAN, P.
(1988). ANALYSIS AND PRECLINICAL PHARMACOLOGY OF THE NOVEL BIOREDUCTIVE ALKYLATING INDOLOQUINONE E09. British journal of cancer,
Vol.58
(2),
pp. 237-1.
HONESS, D.J.
WASSERMAN, T.H.
WORKMAN, P.
WARD, R.
BLEEHEN, N.M.
(1988). ADDITIVITY OF RADIOSENSITIZATION BY PIMONIDAZOLE (RO-03-8799) AND ETHANIDAZOLE (SR-2508) IN A MOUSE MODEL SYSTEM. British journal of radiology,
Vol.61
(729),
pp. 867-1.
NEWMAN, H.F.
WORKMAN, P.
BLEEHEN, N.M.
(1988). CLINICAL ASPECTS OF HYPOXIC CELL RADIOSENSITIZATION USING ETHANIDAZOLE AND PIMONIDAZOLE. British journal of radiology,
Vol.61
(729),
pp. 867-1.
WORKMAN, P.
MAXWELL, R.J.
WALTON, M.I.
GRIFFITHS, J.R.
(1988). POTENTIAL F-19 MAGNETIC-RESONANCE SPECTROSCOPY PROBES FOR NON-INVASIVE IDENTIFICATION OF HYPOXIC TUMOR-CELLS - SELECTIVE RETENTION IN MOUSE-TUMORS INVIVO. Proceedings of the american association for cancer research,
Vol.29,
pp. 229-1.
WORKMAN, P.
OPPITZ, M.
DONALDSON, J.
COE, P.
(1988). INHIBITION OF THE ADVERSE BETA-OXIDATION PATHWAY RESULTS IN REDUCED TOXICITY WITH NOVEL BETA-SUBSTITUTED AND GAMMA-SUBSTITUTED ANALOGS OF CHLORAMBUCIL. Proceedings of the american association for cancer research,
Vol.29,
pp. 335-1.
Dive, C.
Cox, H.
Watson, J.V.
Workman, P.
(1988). Polar fluorescein derivatives as improved substrate probes for flow cytoenzymological assay of cellular esterases. Mol cell probes,
Vol.2
(2),
pp. 131-145.
show abstract
Fluorescein esters are employed in assays of cell viability, membrane permeability and esterase activity. The ester most widely used, fluorescein diacetate (FDA), has the disadvantage of rapid cellular efflux of its hydrolysis product fluorescein. This is particularly problematic for flow cytoenzymology (FCE), where fluorescence is measured in individual cells allowing identification of subpopulations differing in esterase activity and/or membrane characteristics. We present a comparison of FDA with two potentially improved substrate probes for FCE, carboxyfluorescein diacetate (CFDA) and bis(carboxyethyl)-carboxyfluorescein-tetra acetoxy methyl ester (BCECF-AM). Substrates were characterized in terms of reaction and product efflux kinetics in EMT6 mouse mammary tumour cells, together with inhibition kinetics for the carbamoylating agent BCNU. Intact viable cells were analysed by FCE and spectrofluorimetry, and the latter was also used for cell sonicates and purified esterase. CFDA and BCECF-AM enter cells and are hydrolysed more slowly than FDA. CFDA and FDA hydrolyses obey Michaelis-Menten kinetics with Km values of around 19 and 2 microM, respectively, whereas BCECF-AM hydrolysis deviates from this classical behaviour. BCNU (5 X 10(-4) M) inhibits FDA and BCECF-AM hydrolyses by approximately 50%, compared to 30% for CFDA. CFDA may be partly hydrolysed by membrane-bound esterases. Efflux half-lives were 16 min, 94 min and greater than 2 h for products of FDA, CFDA and BCECF-AM, respectively. We conclude that BCECF-AM is the optimal substrate probe for FCE. This study emphasizes the need to optimize various parameters when selecting a substrate for flow cytoenzymological assay or when loading other reporter fluorochromes into cells via lipophilic esters..
Workman, P.
Balmain, A.
Hickman, J.A.
McNally, N.J.
Rohas, A.M.
Mitchison, N.A.
Pierrepoint, C.G.
Raymond, R.
Rowlatt, C.
Stephens, T.C.
(1988). UKCCCR guidelines for the welfare of animals in experimental neoplasia. Lab anim,
Vol.22
(3),
pp. 195-201.
Newman, H.F.
Bleehen, N.M.
Ward, R.
Workman, P.
(1988). Hypoxic cell radiosensitizers in the treatment of high grade gliomas: a new direction using combined Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int j radiat oncol biol phys,
Vol.15
(3),
pp. 677-684.
show abstract
The hypoxic cell radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole) have been evaluated for their simultaneous penetration into human brain tumors and surrounding normal tissue. Thirteen patients received a dose of 1 g of each agent, infused over a 10 minute period during neurosurgery. Samples of glioma (20), brain (10) and cerebrospinal fluid (1) were obtained at a mean time (+/- SD) of 31 +/- 18 min from the end of infusion. A 24 hr plasma time course was measured in six patients. Nitroimidazole concentrations were determined by HPLC. For a mean dose of 0.55 g/m2 of each agent, the mean tumor concentrations (+/- SD) were 17.0 +/- 12.0 micrograms/g for Ro 03-8799 and 13.5 +/- 10.9 micrograms/g for SR 2508. The tumor/plasma ratios were 279 +/- 230% and 47 +/- 34% respectively. For adjacent 'normal' brain tissue, the radiosensitizer concentrations were 29.9 +/- 13.1 micrograms/g for Ro 03-8799, and 4.0 +/- 1.7 micrograms/g for SR 2508, and the brain/plasma ratios were 430 +/- 29% and 14 +/- 8% respectively. There was a significant trend towards increasing accumulation of both agents with time, in both tumor and normal brain. Concentrations in cerebrospinal fluid were very low. Plasma pharmacokinetics for Ro 03-8799 were similar to previous experience, but for SR 2508 the terminal half-life was greater in this series by a factor of 1.3. The results confirm that Ro 03-8799 is distributed widely in the central nervous system, and demonstrate that SR 2508 can achieve high tumor concentrations when the blood-brain barrier is compromised. The concentrations achieved with the combination are indicative of a significant advantage over metronidazole, misonidazole, or either agent alone, and normalized to the therapeutic dose of 0.75 g/m2 plus 2.0 g/m2 SR 2508 are consistent with those giving additive sensitization in an in vivo mouse tumor model..
Walton, M.I.
Workman, P.
(1988). High-performance liquid chromatographic assay for the benzotriazine di-N-oxide (SR 4233) and its reduced metabolites in biological materials. J chromatogr,
Vol.430
(2),
pp. 429-437.
HONESS, D.J.
WASSERMAN, T.H.
WORKMAN, P.
WARD, R.
BLEEHEN, N.M.
(1988). ADDITIVITY OF RADIOSENSITIZATION BY THE COMBINATION OF SR-2508 (ETANIDAZOLE) AND RO-03-8799 (PIMONIDAZOLE) IN A MURINE TUMOR SYSTEM. International journal of radiation oncology biology physics,
Vol.15
(3),
pp. 671-5.
Newman, H.F.
Ward, R.
Workman, P.
Bleehen, N.M.
(1988). The multi-dose clinical tolerance and pharmacokinetics of the combined radiosensitizers, Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int j radiat oncol biol phys,
Vol.15
(5),
pp. 1073-1083.
show abstract
The hypoxic cell radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole) have differing physico-chemical properties and clinical toxicities. The former is basic, lipophilic and produces an acute but transient central nervous system syndrome; the latter is neutral, hydrophilic and causes cumulative peripheral neuropathy. We therefore investigated the possibility of combining these agents to achieve additive radiosensitization with no enhancement of toxicity, as demonstrated in a rodent tumor model. Following a single dose study which showed a lack of interaction with respect to both toxicity and pharmacokinetics, twenty-one patients have now completed simultaneous drug administration on an escalating, multiple dose schedule. There has been no adverse acute interaction up to 0.75 g/m2 Ro 03-8799 with 2 g/m2 SR 2508 for 15 doses. At this dose-level, however, all patients experienced peripheral neuropathy. There was no adverse pharmacokinetic interaction, or perturbation of plasma pharmacokinetics between initial and final infusions. Tumor concentrations were determined in 48 biopsy samples 0-60 min after administration. Mean values normalized to a dose of 0.75 g/m2 Ro 03-8799 plus 2 g/m2 SR 2508 were 33 micrograms/g Ro 03-8799 and 74 micrograms/g SR 2508. These would be expected to produce a single-dose sensitizer enhancement ratio of around 1.5. The combination is predicted to be around 6.8 times more active than misonidazole, and superior to any single agent tested to date. The current schedules are reaching the limits of clinical tolerance, and an attempt is now being made to define the optimal regimen for use in a randomized clinical trial of the combination..
Mbidde, E.K.
Selby, P.J.
Perren, T.J.
Dearnaley, D.P.
Whitton, A.
Ashley, S.
Workman, P.
Bloom, H.J.
McElwain, T.J.
(1988). High dose BCNU chemotherapy with autologous bone marrow transplantation and full dose radiotherapy for grade IV astrocytoma. Br j cancer,
Vol.58
(6),
pp. 779-782.
show abstract
full text
In a series of 22 patients, high dose BCNU (800-1,000mg m-2) with autologous bone marrow transplantation was given as the first post-surgical treatment for grade IV astrocytoma and followed by full dose radiotherapy. When compared to historical experience and matched to control patients in national studies, there appeared to be a small prolongation of survival but no increase in the proportion of long survivors. Acute myelosuppression was mild but toxicity to lung and liver was substantial and limited further dose escalation. Late bone marrow failure was seen in 4 patients. Pharmacokinetic studies were performed and suggested that the late marrow failure was due to persistence of BCNU at the time of marrow return. Despite the suggestion of a prolongation of survival this approach is not routinely recommended and a randomised trial is probably not justified..
Dive, C.
Workman, P.
Watson, J.V.
(1988). Inhibition of intracellular esterases by antitumour chloroethylnitrosoureas Measurement by flow cytometry and correlation with molecular carbamoylation activity. Biochem pharmacol,
Vol.37
(20),
pp. 3987-3993.
show abstract
Antitumour chloroethylnitrosoureas (Cnus) decompose in physiological conditions yielding alkylating species and organic isocyanates. While antitumour activity is mainly attributed to the alkylation of DNA, carbamoylation of intracellular proteins by isocyanates may also have pharmacological and toxicological relevance. We previously reported a novel dynamic flow cytoenzymological assay for esterase inhibition in intact murine cells by BCNU and related isocyanates, and proposed that this might form the basis of an assay for intracellular carbamoylation. We have now examined a wide range of Cnus, isocyanates, and alkylating agents for their ability to inhibit cellular esterases. BCNU, CCNU, their derived isocyanates, and the 4-OH metabolites of CCNU exhibited potent inhibitory activity (I50 values 5.5 x 10(-5)-7.3 x 10(-4) M). Chlorozotocin and GANU were relatively inactive (I50 much greater than 10(-2) M). ACNU, TCNU and the 2-OH metabolites of CCNU exhibited intermediate activity (I50 values 1.1 x 10(-3)-2.3 x 10(-2) M). Compounds not able to form isocyanates were essentially inactive. Poor membrane permeability was also implicated in the weak activity of chlorozotocin and GANU. There was overall a good correlation between esterase inhibition and chemical carbamoylating activity, but some particular differences were identified. We concluded that flow cytoenzymological assay appears to have the potential to provide useful measurement of intracellular protein carbamoylation by existing Cnus and novel derivatives, and also offers the advantage of cell subpopulation identification for in vivo evaluation of these agents..
Freedman, L.S.
Workman, P.
(1988). When can the infusion period be safely ignored in the estimation of pharmacokinetic parameters of drugs in humans?. Cancer chemother pharmacol,
Vol.22
(2),
pp. 95-103.
show abstract
We describe a simple and rapid method to determine the amount by which the area under the curve (AUC) is underestimated when a drug is given by i.v. infusion, but the infusion period is ignored and the post-infusion data are analysed as if they derive from a bolus injection. Charts are provided that allow the investigator to determine the approximate underestimation for drugs following the one- and two-compartment models, and hence to decide whether it is acceptable to ignore the infusion period in a given case (e.g., underestimation less than 5% or 10%). Equations are also provided that allow the exact underestimation to be calculated, together with the true value of the AUC..
Walton, M.I.
Workman, P.
(1988). Pharmacokinetics and metabolism of the mixed-function hypoxic cell sensitizer prototype RSU 1069 in mice. Cancer chemother pharmacol,
Vol.22
(4),
pp. 275-281.
show abstract
RSU 1069 is a leading compound in the class of mixed-function hypoxic cell sensitizers. Possessing an alkylating aziridine function as well as a nitro group, it represents an important prototype molecule for new sensitizer development. Using a novel HPLC assay for RSU 1069 and its metabolites with a cyanopropyl column, we studied the detailed pharmacokinetics and metabolism of this drug in mice. An i.v. dose of 100 mg kg-1 produced peak plasma concentrations of about 100 micrograms ml-1. Absorption was rapid after i.p. injection but peak plasma concentrations were some three- to fourfold lower, giving an i.p. bioavailability of 55%. The elimination t1/2 was route-dependent; e.g. after 50 mg kg-1 the t1/2 was 37.2 and 22.4 min for the i.v. and i.p. routes respectively (P less than 0.001). There was also an indication of dose-dependent kinetics, with a 37% increase in elimination t1/2 when the i.p. dose was doubled from 50 to 100 mg kg-1. Oral bioavailability was low. The volume of distribution was 0.65-1.31 ml g-1 at 50 mg kg-1, but tissue penetration was limited. Brain/plasma ratios ranged from 9.3% to 66.8%, while the mean steady-state tumour/plasma ratio was 28.4%, a value considerably less than the 80%-100% ratios occurring with the neutral 2-nitroimidazole misonidazole. About 18% and 8% of a dose were excreted as the parent drug and the ring-opened hydrolysis product (RSU 1137) in the 8 h urine, indicating the likelihood of extensive metabolism via aziridine-ring removal and nitroreduction. RSU 1137 was also detected in mouse plasma and tissues and, in contrast to the aziridine ring-intact parent compound, gave tumour/plasma ratios of 100%. These studies should provide a pharmacokinetic basis for the evaluation and development of improved mixed-function sensitizers..
Workman, P.
(1988). UKCCCR Ad Hoc Committee (Chairman: P Workman) UKCCCR guidelines for the welfare of animals in experimental neoplasia. Br j cancer,
Vol.58,
p. 109.
Bleehen, N.M.
Walton, M.I.
Workman, P.
(1988). The interactions of hyperthermia with hypoxic cell sensitizers. Recent results cancer res,
Vol.109,
pp. 136-148.
Dive, C.
Workman, P.
Watson, J.V.
(1987). Novel dynamic flow cytoenzymological determination of intracellular esterase inhibition by BCNU and related isocyanates. Biochem pharmacol,
Vol.36
(21),
pp. 3731-3738.
show abstract
We present a novel dynamic flow cytoenzymological demonstration of the potent inhibition by the antitumour chloroethylnitrosourea BCNU of the intracellular hydrolysis of fluorescein diacetate by esterases of viable, intact murine and human tumour cells in vitro. The BCNU metabonate chloroethyl isocyanate and the related compound n-butyl isocyanate were also potent inhibitors. I50 values were in the range 4.2 X 10(-5)-2.0 X 10(-4) M. Generally similar quantitative results were obtained for intact cells and sonicates by conventional spectrofluorimetry, and inhibition of purified porcine liver carboxyl esterase (EC 3.1.1.1) was demonstrated. Little or no inhibitory activity was seen with the alkylating agents methyl methane-sulphonate, melphalan and nitrogen mustard. The results are consistent with carbamoylation of the esterase molecules by isocyanates, and this may provide a basis for the flow cytometric determination of intracellular carbamoylation in discrete subpopulations of heterogeneous samples..
WALTON, M.I.
BLEEHEN, N.M.
WORKMAN, P.
(1987). HYPERTHERMIA-STIMULATED NITROREDUCTIVE BIOACTIVATION OF THE 2-NITROMIDAZOLE BENZNIDAZOLE INVITRO AND INVIVO. British journal of cancer,
Vol.56
(2),
pp. 193-1.
DIVE, C.
WORKMAN, P.
WATSON, J.
(1987). FURTHER EVIDENCE THAT FLOW CYTOENZYMOLOGICAL ASSAY OF CELLULAR ESTERASE INHIBITION MEASURES INTRACELLULAR CARBAMOYLATION BY CHLOROETHYLNITROSOUREA-DERIVED ISOCYANATES. British journal of cancer,
Vol.56
(2),
pp. 222-1.
WORKMAN, P.
MAXWELL, R.J.
GRIFFITHS, J.R.
(1987). NONINVASIVE PHARMACOKINETICS OF MISONIDAZOLE AND CHLORAMBUCIL ANALOGS BY F-19 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY. British journal of cancer,
Vol.56
(2),
pp. 237-1.
TWENTYMAN, P.R.
WORKMAN, P.
BLEEHEN, N.M.
(1987). ANTHRACYCLINES WHICH RETAIN POTENCY IN AN ADRIAMYCIN-RESISTANT HUMAN CELL-LINE. Investigational new drugs,
Vol.5
(1),
pp. 76-1.
HARTMAN, N.G.
WORKMAN, P.
SMITH, P.J.
BLEEHEN, N.M.
(1987). ADRIAMYCIN-DEXTRAN CONJUGATES IN MACROMOLECULAR DRUG-TARGETING - MOLECULAR PHARMACOLOGY AND CYTOTOXICITY. Investigational new drugs,
Vol.5
(1),
pp. 92-1.
WORKMAN, P.
LEE, F.Y.
ROBERTS, J.T.
BLEEHEN, N.M.
MBIDDE, E.
SELBY, P.
MCELWAIN, T.J.
(1987). COMPARATIVE PHARMACOKINETICS AND TOXICITY OF CHLOROETHYL-NITROSOUREAS IN MOUSE AND MAN - BCNU, CCNU AND MITOZOLOMIDE. Investigational new drugs,
Vol.5
(1),
pp. 108-1.
WORKMAN, P.
MBIDDE, E.
SELBY, P.
MCELWAIN, T.J.
BLOOM, H.J.
(1987). PHARMACOKINETICS OF HIGH-DOSE BCNU IN MAN. Investigational new drugs,
Vol.5
(1),
pp. 129-1.
DIVE, C.
WORKMAN, P.
WATSON, J.
(1987). MEASUREMENT OF INTRACELLULAR CARBAMOYLATING ACTIVITY OF CHLOROETHYLNITROSOUREAS IN INTACT HUMAN SMALL CELL-CARCINOMA (NCI-H69) AND MURINE MAMMARY-TUMOR (EMT6) CELLS USING FLOW CYTOENZYMOLOGY. British journal of cancer,
Vol.55
(3),
pp. 338-1.
MBIDDE, E.
SELBY, P.
WORKMAN, P.
WHITTON, A.
MCELWAIN, T.J.
BLOOM, H.J.
(1987). HIGH-DOSE BCNU PRIMARY CHEMOTHERAPY WITH FULL DOSE RADIOTHERAPY FOR ASTROCYTOMA GRADE-IV. British journal of cancer,
Vol.55
(3),
pp. 341-1.
Ward, R.
Workman, P.
(1987). Gradient high-performance liquid chromatographic method for simultaneous assay of the radiosensitizers etanidazole (SR 2508) and pimonidazole (Ro 03-8799) in biological materials. J chromatogr,
Vol.420
(1),
pp. 223-227.
WALTON, M.I.
BLEEHEN, N.M.
WORKMAN, P.
(1987). THE EFFECTS OF WHOLE-BODY HYPERTHERMIA ON THE PHARMACOKINETICS AND TOXICITY OF THE BASIC 2-NITROIMIDAZOLE RADIOSENSITIZER RO-03-8799 IN MICE. British journal of cancer,
Vol.55
(5),
pp. 469-8.
full text
Dive, C.
Workman, P.
Watson, J.V.
(1987). Improved methodology for intracellular enzyme reaction and inhibition kinetics by flow cytometry. Cytometry,
Vol.8
(6),
pp. 552-561.
show abstract
Flow cytoenzymology is the determination of enzyme activities or concentrations in single intact cells. Using the flow cytometer built and designed in our laboratory and recent modifications to hardware and software, we have developed an improved dynamic flow cytoenzymological procedure for the assay of cellular enzyme kinetics. The reaction mixture is sampled continuously, and the computer clock incorporates time as a parameter for kinetic determinations. Conditions for cellular esterase analysis were optimized and the rates of hydrolysis of two fluorogenic substrates, fluorescein diacetate (FDA) and 4-methylumbelliferone acetate (MUA), by esterases in EMT6 mouse mammary tumor cells were studied. Reaction kinetics were characterized, and Km values of 19 and 72 microM were obtained for the hydrolysis of FDA and MUA respectively. The kinetics of the cellular efflux of fluorescein were investigated, and a half-life of 7.5 min obtained. Enzyme inhibition kinetics were investigated using the competitive substrates p-nitrophenyl acetate and phenyl acetate, and the carbamoylating agents physostigmine and n-butyl isocyanate. The latter was particularly potent with an I50 of 4.8 X 10(-5) M for FDA hydrolysis compared with 6.5 X 10(-3) M for physostigmine. The I50 of 8.8 X 10(-5) M for n-butyl isocyanate inhibition of MUA hydrolysis was similar to that obtained with FDA as substrate. By monitoring FDA and MUA reactions separately and simultaneously, we showed them to act as competitive substrates. A comparison of flow cytoenzymological and conventional spectrofluorimetric analysis was also made, and differences identified in some cases..
WORKMAN, P.
(1987). TCNU - A RAY OF HOPE FOR DESIGNER NITROSOUREAS - COMMENT. Eur j cancer clin on,
Vol.23
(12),
pp. 1823-1828.
WALTON, M.I.
BLEEHEN, N.M.
WORKMAN, P.
(1987). HEAT-STIMULATED NITROREDUCTIVE BIOACTIVATION OF THE 2-NITROIMIDAZOLE BENZNIDAZOLE INVITRO. Biochemical pharmacology,
Vol.36
(16),
pp. 2627-6.
LEE, F.Y.
WORKMAN, P.
CHEESEMAN, K.H.
(1987). MISONIDAZOLE AND BENZNIDAZOLE INHIBIT HYDROXYLATION OF CCNU BY MOUSE-LIVER MICROSOMAL CYTOCHROME-P-450 INVITRO. Biochemical pharmacology,
Vol.36
(8),
pp. 1349-7.
WALTON, M.I.
WORKMAN, P.
(1987). NITROIMIDAZOLE BIOREDUCTIVE METABOLISM - QUANTITATION AND CHARACTERIZATION OF MOUSE-TISSUE BENZNIDAZOLE NITROREDUCTASES INVIVO AND INVITRO. Biochemical pharmacology,
Vol.36
(6),
pp. 887-10.
Workman, P.
Oppitz, M.
Donaldson, J.
Lee, F.Y.
(1987). High-performance liquid chromatography of chlorambucil analogues. J chromatogr,
Vol.422,
pp. 315-321.
Workman, P.
(1987). 21 EORTC Pharmacokinetics and Metabolism Group (Secretary: P Workman) Pharmacokinetically guided dose escalation in Phase I clinical trials: Commentary and proposed guidelines. Eur j cancer,
Vol.23,
p. 1083.
WORKMAN, P.
WALTON, M.I.
LEE, F.Y.
(1986). BENZNIDAZOLE - NITROREDUCTION AND INHIBITION OF CYTOCHROME-P-450 IN CHEMOSENSITIZATION OF TUMOR RESPONSE TO CYTOTOXIC DRUGS. Biochemical pharmacology,
Vol.35
(1),
pp. 117-3.
WORKMAN, P.
WHITE, R.A.
TALBOT, K.
(1986). CB-1954 REVISITED 1 DISPOSITION KINETICS AND METABOLISM. Cancer chemotherapy and pharmacology,
Vol.16
(1),
pp. 1-8.
WORKMAN, P.
MORGAN, J.E.
TALBOT, K.
WRIGHT, K.A.
DONALDSON, J.
TWENTYMAN, P.R.
(1986). CB-1954 REVISITED 2 TOXICITY AND ANTITUMOR-ACTIVITY. Cancer chemotherapy and pharmacology,
Vol.16
(1),
pp. 9-6.
WORKMAN, P.
LEE, F.Y.
ROBERTS, J.T.
WALTON, M.I.
CHEESEMAN, K.H.
BLEEHEN, N.M.
(1986). BENZNIDAZOLE INHIBITION OF CCNU HYDROXYLATION BY HEPATIC-MICROSOMAL CYTOCHROME-P-450. British journal of cancer,
Vol.54
(1),
pp. 191-2.
NEWMAN, H.F.
BLEEHEN, N.M.
WORKMAN, P.
(1986). A STUDY OF A COMBINATION OF 2 HYPOXIC CELL RADIOSENSITIZERS, RO 03-8799 AND SR 2508 - CLINICAL TOXICITY AND PHARMACOLOGY. British journal of cancer,
Vol.54
(1),
pp. 197-1.
LEE, F.Y.
COE, P.
WORKMAN, P.
(1986). PHARMACOKINETIC BASIS FOR THE COMPARATIVE ANTITUMOR-ACTIVITY AND TOXICITY OF CHLORAMBUCIL, PHENYLACETIC ACID MUSTARD AND BETA,BETA-DIFLUOROCHLORAMBUCIL (CB-7103) IN MICE. Cancer chemotherapy and pharmacology,
Vol.17
(1),
pp. 21-9.
LEE, F.Y.
WORKMAN, P.
(1986). ALTERED PHARMACOKINETICS IN THE MECHANISM OF CHEMOSENSITIZATION - EFFECTS OF NITROIMIDAZOLES AND OTHER CHEMICAL MODIFIERS ON THE PHARMACOKINETICS, ANTITUMOR-ACTIVITY AND ACUTE TOXICITY OF SELECTED NITROGEN MUSTARDS. Cancer chemotherapy and pharmacology,
Vol.17
(1),
pp. 30-8.
NEWMAN, H.F.
BLEEHEN, N.M.
WORKMAN, P.
(1986). A PHASE-I STUDY OF THE COMBINED HYPOXIC CELL RADIOSENSITIZERS, RO 03-8799 AND SR 2508 - A PRELIMINARY-REPORT OF SINGLE-DOSE TOXICITY, PHARMACOKINETICS AND TUMOR CONCENTRATIONS. British journal of radiology,
Vol.59
(700),
pp. 423-3.
WORKMAN, P.
(1986). CHEMOSENSITIZATION OF LOMUSTINE BY MISONIDAZOLE, BENZNIDAZOLE, AND RSU-1069. Cancer treatment reports,
Vol.70
(9),
pp. 1139-2.
LEE, F.Y.
WORKMAN, P.
(1986). INTERACTION OF NITROIMIDAZOLE SENSITIZERS WITH DRUG-METABOLIZING ENZYMES-SPECTRAL AND KINETIC-STUDIES. International journal of radiation oncology biology physics,
Vol.12
(8),
pp. 1383-5.
NEWMAN, H.F.
BLEEHEN, N.M.
WORKMAN, P.
(1986). A PHASE-I STUDY OF THE COMBINATION OF 2 HYPOXIC CELL RADIOSENSITIZERS, RO-03-8799 AND SR-2508 - TOXICITY AND PHARMACOKINETICS. International journal of radiation oncology biology physics,
Vol.12
(7),
pp. 1113-4.
ROBERTS, J.T.
BLEEHEN, N.M.
WALTON, M.I.
WORKMAN, P.
(1986). A CLINICAL PHASE-1 TOXICITY STUDY OF RO-03-8799 - PLASMA, URINE, TUMOR AND NORMAL BRAIN PHARMACOKINETICS. British journal of radiology,
Vol.59
(698),
pp. 107-10.
TWENTYMAN, P.R.
FOX, N.E.
WRIGHT, K.A.
WORKMAN, P.
BROADHURST, M.J.
MARTIN, J.A.
BLEEHEN, N.M.
(1986). THE INVITRO EFFECTS AND CROSS-RESISTANCE PATTERNS OF SOME NOVEL ANTHRACYCLINES. British journal of cancer,
Vol.53
(5),
pp. 585-10.
NEWMAN, H.F.
DUNPHY, E.P.
BLEEHEN, N.M.
DISCHE, S.
SAUNDERS, M.I.
DESROCHERS, C.
WORKMAN, P.
LENOXSMITH, I.
SMITHEN, C.E.
(1986). A COMPARATIVE-STUDY OF RO-03-8799 - RACEMIC-MIXTURE AND ENANTIOMERS. British journal of radiology,
Vol.59
(706),
pp. 997-3.
WALTON, M.I.
WORKMAN, P.
(1986). REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE SIMULTANEOUS DETERMINATION OF THE 2-NITROIMIDAZOLE BENZNIDAZOLE AND ITS AMINE METABOLITE IN BIOLOGICAL-MATERIALS. Journal of chromatography,
Vol.375
(1),
pp. 190-7.
WATSON, J.V.
WORKMAN, P.
(1986). ESTIMATING THE KINETIC-PARAMETERS FOR ENZYMATIC DRUG-METABOLISM IN THE WHOLE ANIMAL. Biochemical pharmacology,
Vol.35
(2),
pp. 145-5.
WORKMAN, P.
(1986). PHARMACOKINETICS IN EXPERIMENTAL AND CLINICAL CHEMOTHERAPY. Cancer chemoth pharm,
Vol.16
(2),
pp. 198-199.
WALTON, M.I.
BLEEHEN, N.M.
WORKMAN, P.
(1985). THE REVERSIBLE N-OXIDATION OF THE NITROIMIDAZOLE RADIOSENSITIZER RO-03-8799. Biochemical pharmacology,
Vol.34
(21),
pp. 3939-2.
WORKMAN, P.
(1985). THE ROLE OF PRECLINICAL AND CLINICAL PHARMACOKINETICS IN PHASE-I TRIALS. British journal of cancer,
Vol.52
(3),
pp. 412-2.
LEE, F.Y.
COE, P.
WORKMAN, P.
(1985). METABOLIC SWITCHING AND IMPROVED ANTITUMOUR SELECTIVITY WITH BETA,BETA-DIFLUOROCHLORAMBUCIL. British journal of cancer,
Vol.52
(3),
pp. 414-2.
TWENTYMAN, P.R.
FOX, N.E.
WRIGHT, K.A.
WORKMAN, P.
BROADHURST, M.J.
MARTIN, J.A.
(1985). NEW ANTHRACYCLINES - INVITRO EFFECTS AND CROSS-RESISTANCE PATTERNS IN CONTROL AND ADRIAMYCINRESISTANT CELL-LINES. British journal of cancer,
Vol.52
(3),
pp. 423-1.
TWENTYMAN, P.R.
WORKMAN, P.
WRIGHT, K.A.
BLEEHEN, N.M.
(1985). THE EFFECTS OF ALPHA-INTERFERONS AND GAMMA-INTERFERONS ON HUMAN-LUNG CANCER-CELLS GROWN-INVITRO OR AS XENOGRAFTS IN NUDE-MICE. British journal of cancer,
Vol.52
(3),
pp. 427-1.
LEE, F.Y.
WORKMAN, P.
(1985). MISONIDAZOLE PROTECTS AGAINST ORAL CCNU. British journal of cancer,
Vol.52
(3),
pp. 434-1.
WORKMAN, P.
LEE, F.Y.
DONALDSON, J.
(1985). PHARMACOKINETIC FACTORS AFFECTING COMPARATIVE TUMOR RESPONSE TO CCNU AND MITOZOLAMIDE. British journal of cancer,
Vol.52
(3),
pp. 438-2.
WALTON, M.I.
BLEEHEN, N.M.
WORKMAN, P.
(1985). THE EFFECTS OF MILD HYPERTHERMIA ON THE METABOLISM OF 3 NITROIMIDAZOLE RADIOSENSITIZERS INVIVO AND INVITRO. British journal of cancer,
Vol.52
(3),
pp. 456-2.
METCALFE, S.
NEWMAN, H.
WORKMAN, P.
(1985). INCREASE OF OLEIC-ACID IN ERYTHROCYTES ASSOCIATED WITH MALIGNANCIES. British medical journal,
Vol.291
(6497),
pp. 740-1.
LEE, F.Y.
WORKMAN, P.
ROBERTS, J.T.
BLEEHEN, N.M.
(1985). CLINICAL PHARMACOKINETICS OF ORAL CCNU (LOMUSTINE). Cancer chemotherapy and pharmacology,
Vol.14
(2),
pp. 125-7.
WALTON, M.I.
BLEEHEN, N.M.
WORKMAN, P.
(1985). WHOLE-BODY HYPERTHERMIA - EFFECTS ON THE PHARMACOKINETICS OF 2 NITROIMIDAZOLE RADIOSENSITIZERS SR 2508 AND RO 03-8799 IN MICE. British journal of cancer,
Vol.51
(4),
pp. 600-2.
BAILLIEJOHNSON, H.
TWENTYMAN, P.R.
FOX, N.E.
WALLS, G.A.
WORKMAN, P.
WATSON, J.V.
JOHNSON, N.
REEVE, J.G.
BLEEHEN, N.M.
(1985). ESTABLISHMENT AND CHARACTERIZATION OF CELL-LINES FROM PATIENTS WITH LUNG-CANCER (PREDOMINANTLY SMALL CELL-CARCINOMA). British journal of cancer,
Vol.52
(4),
pp. 495-10.
full text
TWENTYMAN, P.R.
WORKMAN, P.
WRIGHT, K.A.
BLEEHEN, N.M.
(1985). THE EFFECTS OF ALPHA-INTERFERONS AND GAMMA-INTERFERONS ON HUMAN-LUNG CANCER-CELLS GROWN-INVITRO OR AS XENOGRAFTS IN NUDE-MICE. British journal of cancer,
Vol.52
(1),
pp. 21-9.
full text
HONESS, D.J.
DONALDSON, J.
WORKMAN, P.
BLEEHEN, N.M.
(1985). THE EFFECT OF SYSTEMIC HYPERTHERMIA ON MELPHALAN PHARMACOKINETICS IN MICE. British journal of cancer,
Vol.51
(1),
pp. 77-8.
full text
LEE, F.Y.
WORKMAN, P.
(1985). MISONIDAZOLE PROTECTS MOUSE-TUMOR AND NORMAL-TISSUES FROM THE TOXICITY OF ORAL CCNU. British journal of cancer,
Vol.51
(1),
pp. 85-7.
Workman, P.
(1985). British Association for Cancer Research (Meeting Report). Cancer topics,
Vol.5,
p. 80.
LEE, F.Y.
WORKMAN, P.
(1984). NITROIMIDAZOLES AS MODIFIERS OF NITROSOUREA PHARMACOKINETICS. International journal of radiation oncology biology physics,
Vol.10
(9),
pp. 1627-4.
ROBERTS, J.T.
BLEEHEN, N.M.
LEE, F.Y.
WORKMAN, P.
WALTON, M.I.
(1984). A PHASE-I STUDY OF THE COMBINATION OF BENZNIDAZOLE AND CCNU IN MAN. International journal of radiation oncology biology physics,
Vol.10
(9),
pp. 1745-4.
ROBERTS, J.T.
BLEEHEN, N.M.
WORKMAN, P.
WALTON, M.I.
(1984). A PHASE-I STUDY OF THE HYPOXIC CELL RADIOSENSITIZER RO-03-8799. International journal of radiation oncology biology physics,
Vol.10
(9),
pp. 1755-4.
WORKMAN, P.
WALTON, M.I.
(1984). PHARMACOLOGY OF THE MIXED-FUNCTION RADIOSENSITIZERS AND CHEMOSENSITIZERS CB-1954 AND RSU-1069. International journal of radiation oncology biology physics,
Vol.10
(8),
pp. 1307-4.
WORKMAN, P.
LEE, F.Y.
(1984). EXPERIMENTAL ANTITUMOUR ACTIVITY AND MECHANISM OF ACTION OF THE NOVEL ANTICANCER AGENT CCRG-81010. British journal of cancer,
Vol.50
(2),
pp. 251-2.
WALTON, M.I.
WORKMAN, P.
(1984). NITROREDUCTION OF THE CHEMOSENSITIZER AND RADIO-SENSITIZER BENZNIDAZOLE INVIVO AND INVITRO. British journal of cancer,
Vol.50
(2),
pp. 266-1.
LEE, F.Y.
WORKMAN, P.
(1984). INHIBITION OF LIVER MICROSOMAL METABOLISM OF CCNU BY NITROIMIDAZOLES - CORRELATION WITH CHEMOSENSITIZATION ACTIVITY. British journal of cancer,
Vol.50
(2),
pp. 267-1.
REEVE, J.G.
WRIGHT, K.A.
WORKMAN, P.
(1984). PATTERNS OF CROSS SENSITIVITY IN THE RESPONSES OF CLONAL SUBPOPULATIONS ISOLATED FROM RIF-1 MOUSE SARCOMA TO SELECTED NITROSOUREAS AND NITROGEN MUSTARDS. British journal of cancer,
Vol.50
(2),
pp. 273-2.
ROBERTS, J.T.
BLEEHEN, N.M.
WORKMAN, P.
WALTON, M.I.
(1984). PLASMA AND TUMOR PHARMACOKINETICS OF BENZNIDAZOLE IN MAN. British journal of cancer,
Vol.49
(3),
pp. 386-1.
WORKMAN, P.
WHITE, R.A.
WALTON, M.I.
OWEN, L.N.
TWENTYMAN, P.R.
(1984). PRECLINICAL PHARMACOKINETICS OF BENZNIDAZOLE. British journal of cancer,
Vol.50
(3),
pp. 291-13.
REEVE, J.G.
WRIGHT, K.A.
WORKMAN, P.
(1984). PATTERNS OF CROSS-SENSITIVITY IN THE RESPONSES OF CLONAL SUBPOPULATIONS ISOLATED FROM THE RIF-1 MOUSE SARCOMA TO SELECTED NITROSOUREAS AND NITROGEN MUSTARDS. British journal of cancer,
Vol.50
(2),
pp. 153-6.
LEE, F.Y.
WORKMAN, P.
(1984). MISONIDAZOLE AND CCNU - FURTHER EVIDENCE FOR A PHARMACOKINETIC MECHANISM OF CHEMOSENSITIZATION AND THERAPEUTIC GAIN. British journal of cancer,
Vol.49
(5),
pp. 579-7.
full text
Workman, P.
(1984). British Association of Cancer Research (Meeting Report). Cancer topics,
Vol.5,
p. 20.
WORKMAN, P.
DONALDSON, J.
SMITH, N.C.
(1983). EFFECTS OF CIMETIDINE, ANTIPYRINE, AND PREGNENOLONE CARBONITRILE ON MISONIDAZOLE PHARMACOKINETICS. Cancer treatment reports,
Vol.67
(7-8),
pp. 723-3.
WORKMAN, P.
TWENTYMAN, P.R.
LEE, F.Y.
WALTON, M.I.
OWEN, L.N.
WHITE, R.A.
(1983). BENZNIDAZOLE - CHEMOSENSITIZATION AND PHARMACOKINETICS. British journal of cancer,
Vol.48
(1),
pp. 122-2.
TWENTYMAN, P.R.
WORKMAN, P.
WRIGHT, K.A.
(1983). SENSITIZATION OF EMT6 MULTICELLULAR TUMOR SPHEROIDS TO CCNU AND MELPHALAN BY HYPOXIC PRE-INCUBATION WITH NITROIMIDAZOLES. British journal of cancer,
Vol.48
(1),
pp. 141-1.
LEE, F.
WORKMAN, P.
(1983). EFFECTS OF SINGLE DOSE MISONIDAZOLE ON THE PHARMACOKINETICS OF CCNU IN MICE. British journal of cancer,
Vol.48
(1),
pp. 141-2.
WORKMAN, P.
TWENTYMAN, P.R.
LEE, F.Y.
WALTON, M.I.
(1983). RADIOSENSITIZERS AND CYTO-TOXIC DRUGS - PHARMACOKINETIC ASPECTS. International journal of radiation biology,
Vol.44
(1),
pp. 104-2.
TWENTYMAN, P.R.
WORKMAN, P.
(1983). CHEMOSENSITIZATION BY LIPOPHILIC NITROIMIDAZOLES. International journal of radiation biology,
Vol.44
(1),
pp. 105-1.
WORKMAN, P.
LEE, F.Y.
(1983). SYSTEMIC PHARMACOKINETICS AND CELLULAR PHARMACOLOGY OF CYTO-TOXIC DRUGS. British journal of radiology,
Vol.56
(665),
pp. 351-1.
BLEEHEN, N.M.
GRANT, R.M.
JONES, D.H.
ROBERTS, J.T.
WORKMAN, P.
GLEAVE, J.R.
HARDY, D.G.
HOLMES, A.E.
(1983). THE PENETRATION OF MISONIDAZOLE AND DESMETHYLMISONIDAZOLE INTO BRAIN-TUMORS AND OTHER CENTRAL NERVOUS-SYSTEM TISSUES IN MAN. British journal of radiology,
Vol.56
(671),
pp. 859-5.
JONES, D.H.
BLEEHEN, N.M.
WORKMAN, P.
SMITH, N.C.
(1983). THE ROLE OF MICROSOMAL-ENZYME INDUCERS IN THE REDUCTION OF MISONIDAZOLE NEUROTOXICITY. British journal of radiology,
Vol.56
(671),
pp. 865-6.
JONES, D.H.
BLEEHEN, N.M.
WORKMAN, P.
WALTON, M.I.
(1983). THE ROLE OF DEXAMETHASONE IN THE MODIFICATION OF MISONIDAZOLE PHARMACOKINETICS. British journal of cancer,
Vol.48
(4),
pp. 553-5.
full text
TWENTYMAN, P.R.
WORKMAN, P.
(1983). CHEMOSENSITIZATION BY LIPOPHILIC NITROIMIDAZOLES. British journal of cancer,
Vol.48
(1),
pp. 17-10.
full text
LEE, F.Y.
WORKMAN, P.
(1983). MODIFICATION OF CCNU PHARMACOKINETICS BY MISONIDAZOLE - A MAJOR MECHANISM OF CHEMOSENSITIZATION IN MICE. British journal of cancer,
Vol.47
(5),
pp. 659-11.
full text
WORKMAN, P.
TWENTYMAN, P.R.
LEE, F.Y.
WALTON, M.I.
(1983). DRUG-METABOLISM AND CHEMOSENSITIZATION - NITROIMIDAZOLES AS INHIBITORS OF DRUG-METABOLISM. Biochemical pharmacology,
Vol.32
(5),
pp. 857-8.
TWENTYMAN, P.R.
WORKMAN, P.
(1983). AN INVESTIGATION OF THE POSSIBILITY OF CHEMOSENSITIZATION BY CLINICALLY ACHIEVABLE CONCENTRATIONS OF MISONIDAZOLE. British journal of cancer,
Vol.47
(2),
pp. 187-8.
full text
Twentyman, P.R.
Workman, P.
(1983). Second UK Workshop on the enhancement of chemotherapy by nitroimidazoles (Meeting Report). Br j cancer,
Vol.47,
p. 553.
Workman, P.
(1983). New drugs against hypoxic cells (part 1). Cancer topics,
Vol.4,
p. 42.
Workman, P.
(1983). New drugs against hypoxic cells (part 2). Cancer topics,
Vol.4,
p. 54.
TWENTYMAN, P.R.
WORKMAN, P.
(1982). THE COMBINATION OF CYTO-TOXIC DRUGS WITH CLINICALLY RELEVANT DOSE REGIMES OF MISONIDAZOLE. British journal of cancer,
Vol.46
(3),
pp. 469-1.
WORKMAN, P.
TWENTYMAN, P.
(1982). MODIFIERS OF DRUG-METABOLISM - THEIR EFFECTS ON THE RESPONSE OF TUMOR AND NORMAL-TISSUES TO CYTO-TOXIC AGENTS. British journal of cancer,
Vol.46
(3),
pp. 497-1.
TWENTYMAN, P.R.
WORKMAN, P.
(1982). THE EFFECT OF RADIOSENSITIZER PRETREATMENT ON THE RESPONSE OF THE RIF-1 MOUSE SARCOMA TO CYTO-TOXIC DRUGS. International journal of radiation oncology biology physics,
Vol.8
(3-4),
pp. 611-3.
WORKMAN, P.
TWENTYMAN, P.R.
(1982). ENHANCEMENT BY ELECTRON-AFFINIC AGENTS OF THE THERAPEUTIC EFFECTS OF CYTO-TOXIC AGENTS AGAINST THE KHT TUMOR - STRUCTURE-ACTIVITY-RELATIONSHIPS. International journal of radiation oncology biology physics,
Vol.8
(3-4),
pp. 623-4.
WORKMAN, P.
TWENTYMAN, P.R.
(1982). DO NITROIMIDAZOLES INCREASE THE THERAPEUTIC INDEXES OF CYTO-TOXIC AGENTS INVIVO. British journal of cancer,
Vol.45
(4),
pp. 631-1.
WORKMAN, P.
WHITE, R.A.
(1982). EFFECTS OF THE XANTHINE-OXIDASE INHIBITOR ALLOPURINOL ON THE RENAL CLEARANCE OF NITROIMIDAZOLES. Biochemical pharmacology,
Vol.31
(19),
pp. 3041-6.
WORKMAN, P.
TWENTYMAN, P.R.
(1982). STRUCTURE ACTIVITY RELATIONSHIPS FOR THE ENHANCEMENT BY ELECTRON-AFFINIC DRUGS OF THE ANTI-TUMOR EFFECT OF CCNU. British journal of cancer,
Vol.46
(2),
pp. 249-11.
TWENTYMAN, P.
WORKMAN, P.
(1982). EFFECT OF MISONIDAZOLE OR METRONIDAZOLE PRETREATMENT ON THE RESPONSE OF THE RIF-1 MOUSE SARCOMA TO MELPHALAN, CYCLOPHOSPHAMIDE, CHLORAMBUCIL AND CCNU. British journal of cancer,
Vol.45
(3),
pp. 447-9.
full text
WARENIUS, H.M.
WORKMAN, P.
BLEEHEN, N.M.
(1982). RESPONSE OF A HIGH-GLUCURONIDASE HUMAN-TUMOR XENOGRAFT TO ANILINE MUSTARD. British journal of cancer,
Vol.45
(1),
pp. 27-8.
full text
Workman, P.
(1982). Hypoxic cells and cancer therapy. Cancer topics,
Vol.4,
p. 22.
White, R.A.
Workman, P.
Owen, L.N.
(1982). The pharmacokinetics in mice and dogs of nitroimidazole radiosensitizers and chemosensitizers more lipophilic than misonidazole. Int j radiat oncol biol phys,
Vol.8,
pp. 473-476.
TWENTYMAN, P.R.
WORKMAN, P.
(1981). MODIFICATION BY MISONIDAZOLE AND METRONIDAZOLE OF THE RESPONSE OF THE RIF-1 MOUSE SARCOMA TO CYTO-TOXIC DRUGS. British journal of cancer,
Vol.44
(2),
pp. 283-1.
WORKMAN, P.
TWENTYMAN, P.R.
(1981). STRUCTURE-ACTIVITY-RELATIONSHIPS FOR THE ENHANCEMENT OF THE ANTI-TUMOR EFFECT OF CCNU BY ELECTRON-AFFINIC AGENTS. British journal of cancer,
Vol.44
(2),
pp. 283-1.
WHITE, R.A.
WORKMAN, P.
(1981). DISPOSITION KINETICS AND METABOLISM OF CB-1954 IN MICE AND DOGS. British journal of cancer,
Vol.44
(2),
pp. 283-2.
WORKMAN, P.
BROWN, J.M.
(1981). STRUCTURE-PHARMACOKINETIC RELATIONSHIPS FOR MISONIDAZOLE ANALOGS IN MICE. Cancer chemotherapy and pharmacology,
Vol.6
(1),
pp. 39-11.
WORKMAN, P.
(1981). PHARMACOKINETIC ASPECTS OF INTERACTIONS BETWEEN NITROIMIDAZOLES AND OTHER DRUGS. British journal of radiology,
Vol.54
(640),
pp. 369-2.
Workman, P.
(1981). Pharmacology of hypoxic cell sensitizers. Camb med,
Vol.1,
p. 12.
WORKMAN, P.
(1980). DOSE-DEPENDENCE AND RELATED STUDIES ON THE PHARMACOKINETICS OF MISONIDAZOLE AND DESMETHYLMISONIDAZOLE IN MICE. Cancer chemotherapy and pharmacology,
Vol.5
(1),
pp. 27-11.
WORKMAN, P.
(1980). EFFECTS OF DEXAMETHASONE ON THE PHARMACOKINETICS AND TOXICITY OF MISONIDAZOLE IN MICE. British journal of cancer,
Vol.42
(1),
pp. 179-2.
WHITE, R.A.
WORKMAN, P.
(1980). DRUG-INTERACTIONS WITH MISONIDAZOLE IN THE DOG. British journal of cancer,
Vol.42
(1),
pp. 180-1.
WHITE, R.A.
WORKMAN, P.
(1980). PHENYTOIN SODIUM-INDUCED ALTERATIONS IN THE PHARMACOKINETICS OF MISONIDAZOLE IN THE DOG. Cancer treatment reports,
Vol.64
(2-3),
pp. 360-2.
WORKMAN, P.
(1980). THE NEUROTOXICITY OF MISONIDAZOLE - POTENTIAL MODIFYING ROLE OF DEXAMETHASONE. British journal of radiology,
Vol.53
(631),
pp. 736-1.
WORKMAN, P.
BLEEHEN, N.M.
WILTSHIRE, C.R.
(1980). PHENYTOIN SHORTENS THE HALF-LIFE OF THE HYPOXIC CELL RADIOSENSITIZER MISONIDAZOLE IN MAN - IMPLICATIONS FOR POSSIBLE REDUCED TOXICITY. British journal of cancer,
Vol.41
(2),
pp. 302-3.
full text
WHITE, R.A.
WORKMAN, P.
BROWN, J.M.
(1980). THE PHARMACOKINETICS AND TUMOR AND NEURAL TISSUE PENETRATING PROPERTIES OF SR-2508 AND SR-2555 IN THE DOG - HYDROPHILIC RADIOSENSITIZERS POTENTIALLY LESS TOXIC THAN MISONIDAZOLE. Radiation research,
Vol.84
(3),
pp. 542-20.
WORKMAN, P.
(1980). DRUG-INTERACTIONS WITH MISONIDAZOLE - EFFECTS OF DEXAMETHASONE AND ITS DERIVATIVES ON THE PHARMACOKINETICS AND TOXICITY OF MISONIDAZOLE IN MICE. Biochemical pharmacology,
Vol.29
(20),
pp. 2769-8.
WORKMAN, P.
(1980). PHARMACOKINETICS OF HYPOXIC CELL RADIOSENSITIZERS - A REVIEW. Cancer clin trials,
Vol.3
(3),
pp. 237-251.
HONESS, D.J.
WORKMAN, P.
MORGAN, J.E.
BLEEHEN, N.M.
(1980). EFFECTS OF LOCAL HYPERTHERMIA ON THE PHARMACOKINETICS OF MISONIDAZOLE IN THE ANESTHETIZED MOUSE. British journal of cancer,
Vol.41
(4),
pp. 529-12.
BROWN, J.M.
WORKMAN, P.
(1980). PARTITION-COEFFICIENT AS A GUIDE TO THE DEVELOPMENT OF RADIOSENSITIZERS WHICH ARE LESS TOXIC THAN MISONIDAZOLE. Radiation research,
Vol.82
(1),
pp. 171-20.
WHITE, R.A.
WORKMAN, P.
(1980). PHARMACOKINETIC AND TUMOR-PENETRATION PROPERTIES OF THE HYPOXIC CELL RADIOSENSITIZER DESMETHYLMISONIDAZOLE (RO 05-9963) IN DOGS. British journal of cancer,
Vol.41
(2),
pp. 268-9.
White, R.A.
Workman, P.
(1980). Pharmacokinetics of misonidazole and other potentially superior radiosensitizing drugs in the dog. Radiation sensitizers,
,
p. 262.
WHITE, R.A.
WORKMAN, P.
OWEN, L.N.
BLEEHEN, N.M.
(1979). STUDIES WITH THE HYPOXIC CELL SENSITIZER RO-05-9963 IN THE DOG. British journal of cancer,
Vol.40
(2),
pp. 304-2.
WORKMAN, P.
WILTSHIRE, C.R.
BLEEHEN, N.M.
(1979). EFFECT OF PHENOBARBITONE AND PHENYTOIN ON THE PHARMACOKINETICS AND TOXICITY OF MISONIDAZOLE. British journal of cancer,
Vol.40
(2),
pp. 316-2.
WORKMAN, P.
(1979). ANALYSIS OF THE BASIC 5-NITROIMIDAZOLE NIMORAZOLE IN BLOOD BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY, AND ITS APPLICATION TO PHARMACOKINETIC STUDIES IN INDIVIDUAL MICE. Journal of chromatography,
Vol.163
(4),
pp. 396-7.
WHITE, R.A.
WORKMAN, P.
FREEDMAN, L.S.
OWEN, L.N.
BLEEHEN, N.M.
(1979). PHARMACOKINETICS OF MISONIDAZOLE IN THE DOG. European journal of cancer,
Vol.15
(10),
pp. 1233-10.
WORKMAN, P.
(1979). EFFECTS OF PRETREATMENT WITH PHENOBARBITONE AND PHENYTOIN ON THE PHARMACOKINETICS AND TOXICITY OF MISONIDAZOLE IN MICE. British journal of cancer,
Vol.40
(3),
pp. 335-19.
full text
WHITE, R.A.
WORKMAN, P.
OWEN, L.N.
BLEEHEN, N.M.
(1979). PENETRATION OF MISONIDAZOLE INTO SPONTANEOUS CANINE TUMORS. British journal of cancer,
Vol.40
(2),
pp. 284-11.
full text
WATSON, J.V.
WORKMAN, P.
CHAMBERS, S.H.
(1979). ASSAY FOR PLASMA-MEMBRANE PHOSPHATASE-ACTIVITY IN POPULATIONS OF INDIVIDUAL CELLS. Biochemical pharmacology,
Vol.28
(6),
pp. 821-7.
BROWN, J.M.
YU, N.Y.
WORKMAN, P.
(1979). PHARMACOKINETIC CONSIDERATIONS IN TESTING HYPOXIC CELL RADIOSENSITIZERS IN MOUSE-TUMORS. British journal of cancer,
Vol.39
(3),
pp. 310-11.
full text
Workman, P.
(1979). Latent drugs for cancer chemotherapy. Cancer topics,
Vol.2,
p. 6.
CHAMBERS, S.H.
WORKMAN, P.
WATSON, J.V.
BLEEHEN, N.M.
(1978). FLOW CYTOFLUORIMETRIC ASSAY FOR CELLULAR ESTERASES IN POPULATIONS OF SINGLE CELLS. British journal of cancer,
Vol.38
(1),
pp. 188-1.
WORKMAN, P.
LITTLE, C.J.
MARTEN, T.R.
DALE, A.D.
RUANE, R.J.
FLOCKHART, I.R.
BLEEHEN, N.M.
(1978). ESTIMATION OF HYPOXIC CELL-SENSITIZER MISONIDAZOLE AND ITS O-DEMETHYLATED METABOLITE IN BIOLOGICAL-MATERIALS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY. Journal of chromatography,
Vol.145
(3),
pp. 507-6.
WORKMAN, P.
WILTSHIRE, C.R.
WATSON, J.V.
BLEEHEN, N.M.
(1978). STUDIES ON PHARMACOKINETICS OF MISONIDAZOLE (RO 07-0582) IN MAN USING HIGH-PRESSURE LIQUID-CHROMATOGRAPHY (HPLC) ANALYSIS. British journal of cancer,
Vol.37
(3),
pp. 474-2.
WORKMAN, P.
WILTSHIRE, C.R.
PLOWMAN, P.N.
BLEEHEN, N.M.
(1978). MONITORING SALIVARY MISONIDAZOLE IN MAN - POSSIBLE ALTERNATIVE TO PLASMA MONITORING. British journal of cancer,
Vol.38
(6),
pp. 709-10.
WORKMAN, P.
DOUBLE, J.A.
(1978). DRUG LATENTIATION IN CANCER CHEMOTHERAPY. Biomedicine,
Vol.28
(5),
pp. 255-8.
WILTSHIRE, C.R.
WORKMAN, P.
WATSON, J.V.
BLEEHEN, N.M.
(1978). CLINICAL STUDIES WITH MISONIDAZOLE. British journal of cancer,
Vol.37,
pp. 286-4.
full text
WATSON, J.V.
WORKMAN, P.
CHAMBERS, S.H.
(1978). DIFFERENT ESTERASE-ACTIVITIES OF EXPONENTIAL AND PLATEAU PHASES OF EMT6 CELLS MONITORED BY FLOW CYTOFLUORIMETRY. British journal of cancer,
Vol.37
(3),
pp. 397-6.
full text
Workman, P.
(1978). Inhibition of human prostatic tumour acid phosphatases by N,N-p-di-2-chloroethylaminophenol,N,N-p-di-2-chloroethylamino-phenyl phosphate and other disfunctional nitrogen mustards. Chem biol interactions,
Vol.20,
pp. 103-112.
Workman, P.
(1978). Enzyme-activated antitumor agents - IV Comparative kinetics of N,pN-p-di-2-chloroethylaminophenyl phosphate hydrolysis catalysed by phosphatases of normal and neoplastic tissues. Biochem pharmacol,
Vol.27,
pp. 199-202.
BLEEHEN, N.M.
HONESS, D.J.
MORGAN, J.E.
WORKMAN, P.
(1977). COMBINED ACTION OF HYPOXIC SENSITIZERS AND HYPERTHERMIA ON EMT6 TUMOR. International journal of radiation biology,
Vol.32
(4),
pp. 382-1.
WATSON, J.V.
CHAMBERS, S.H.
WORKMAN, P.
HORSNELL, T.S.
(1977). FLOW CYTOFLUORIMETRIC METHOD FOR MEASURING ENZYME REACTION-KINETICS IN INTACT-CELLS. Febs letters,
Vol.81
(1),
pp. 179-4.
Double, J.A.
Workman, P.
(1977). A new high-glucuronidase tumour curable by aniline mustard therapy. Cancer treat rep,
Vol.61,
pp. 909-911.
WORKMAN, P.
DOUBLE, J.A.
WILMAN, D.E.
(1976). ENZYME ACTIVATED ANTI-TUMOUR AGENTS 3 HYDROLYSIS OF CONJUGATES OF PARA-HYDROXYANILINE MUSTARD IN AQUEOUS-SOLUTION. Biochemical pharmacology,
Vol.25
(21),
pp. 2347-4.
Workman, P.
Double, J.A.
(1976). Enzyme-activated antitumour agents - II The role of alkaline phosphatase in the release of p-hydroxyaniline mustard from its phosphate conjugate in cells in culture. Biochem pharmacol,
Vol.25,
pp. 1139-1144.
Workman, P.
Clarke, P.A.
Guillard, S.
Raynaud, F.I.
Drugging the PI3 kinome. Nat biotechnol.,
Vol.24
(7),
pp. 794-3.
Benson, C.
Kaye, S.
Workman, P.
Garrett, M.
De Bono, J.S.
Clinical anticancer drug development: targeting the cyclin dependent kinases. Br j cancer 2005,
(92),
pp. 7-12.
Folkes, A.J.
Ahmadi, K.
Alderton, W.K.
Alix, S.
Baker, S.J.
Box, G.
Chuckowree, I.S.
Clarke, P.A.
Depledge, P.
Eccles, S.A.
Friedman, L.S.
Hayes, A.
Hancox, T.C.
Kugendradas, A.
Lensun, L.
Moore, P.
Olivero, A.G.
Pang, J.
Patel, S.
Pergl-Wilson, G.H.
Raynaud, F.I.
Robson, A.
Saghir, N.
Salphati, L.
Sohal, S.
Ultsch, M.H.
Valenti, M.
Wallweber, H.J.
Wan, N.C.
Wiesmann, C.
Workman, P.
Zhyvoloup, A.
Zvelebil, M.J.
Shuttleworth, S.J.
The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer. J med chem,
Vol.51
(18),
pp. 5522-10.
show abstract
Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer..