Professor Paul Workman
Group Leader: Computational Biology and Chemogenomics, Signal Transduction and Molecular Pharmacology
OrcID: 0000-0003-1659-3034
Phone: +44 20 3437 6783
Email: [email protected]
Also on: PaulWorkmanICR
Location: Sutton
OrcID: 0000-0003-1659-3034
Phone: +44 20 3437 6783
Email: [email protected]
Also on: PaulWorkmanICR
Location: SuttonBiography
Professor Paul Workman FMedSci, FRS was Chief Executive and President of The Institute of Cancer Research (ICR) from 2014 to 2021.
Professor Workman is a passionate advocate of personalised molecular medicine and is an enthusiastic practitioner of multidisciplinary cancer drug discovery and development approaches to 'drugging the cancer genome'. He also conceptualised the 'Pharmacological Audit Trail' approach.
As well as establishing and successfully leading drug discovery project groups yielding clinical candidates, Professor Workman’s personal and collaborative research utilises molecular pharmacology and chemical biology approaches, including high-throughput and genome-wide as well as hypothesis-driven strategies, to interrogate cancer biology, identify and validate new drug targets, discover and develop chemical tools and drugs acting on these targets, identify predictive and mechanism of action biomarkers, and elucidate mechanisms of drug sensitivity of resistance.
He is especially interested in exploiting the addictions, vulnerabilities and dependencies of cancer cells using a combination of small molecule tools and drugs alongside molecular genetic techniques.
Professor Workman has successfully built a series of multidisciplinary drug discovery and development groups in the academic, large pharma and biotechnology company sectors. Through this experience he has been able to combine the best elements of each of these environments. He has been responsible for the discovery of a number of drug development candidates, including in particular pathfinding inhibitors of the HSP90 molecular chaperone and the PI3 kinase family of signalling enzymes.
Professor Workman completed his BSc in Biochemistry at the University of Leicester UK (1973) and his PhD in Cancer Pharmacology at the University of Leeds UK (1977). In 1976 he moved to the Medical Research Council's Clinical Oncology Unit in the MRC Centre, University of Cambridge UK where he established a preclinical and clinical cancer pharmacology laboratory. He developed his early career in Cambridge from 1976 to 1990, focusing mainly on drugs designed to exploit tumour hypoxia and contributing to the discovery and clinical development of several hypoxia-targeted agents, leading to a tenured professorial level appointment.
Following a brief sabbatical at Stanford University and Stanford Research International in Palo Alto, California USA in 1990, Professor Workman was appointed as Cancer Research Campaign Professor of Experimental Cancer Therapy and Director of Laboratory Research in the Department of Medical Oncology, Beatson Laboratories, University of Glasgow UK.
In 1993, Professor Workman moved to a senior scientific leadership position in AstraZeneca Pharmaceuticals, where he was Head of the Cancer Research Bioscience Section at the Alderley Park site in Cheshire UK. He also initiated and led AZ’s biotechnology collaboration with Sugen, San Francisco USA.
In 1997, Professor Workman moved to ICR to take over and build up what is now the Centre for Cancer Drug Discovery, and held the position of unit Director until January 2016 - a period of over 18 years. Under his leadership, the Unit has identified 20 clinical drug candidates since 2005, has progressed 11 of its drugs into Phase I clinical trials in ICR’s partner Royal Marsden Hospital, and has seen its prostate cancer drug approved by the US FDA, European Medicines Agency and NICE and successfully launched.
Many of the Unit’s drug discovery projects have been partnered with biotech companies. In addition, Professor Workman was a scientific founder of Chroma Therapeutics in 2000 and of Piramed Pharma in 2002, the latter being acquired by Roche in 2008.
Honours and prizes include: Fellow of the Royal Society (elected in 2016), UICC ICRETT Fellow, Cancer Research UK Life Fellow, Fellow of the Academy of Medical Sciences, Fellow of the Royal Society of Chemistry, Fellow of the Royal Society of Medicine, Fellow of the Society of Biology and Member of the Faculty of 1000. Professor Workman has received the European School of Oncology Award for Excellence in Oncology Research, NDDO Award for Cancer Drug Development, ICON Distinguished Lecture, Bruce Cain Memorial Lecture of the New Zealand Cancer Society and NCRI/BACR Tom Connors Award Lecture.
In 2009 he received an Honorary DSc from the University of Leicester and the Royal Society of Chemistry George and Christine Sosnovsky Award in Cancer Therapy for “his seminal research on the role of chaperone proteins in cellular processes and the application of this knowledge at the forefront of anti-cancer drug discovery”. In 2012, Professor Workman was once again honoured by the Royal Society of Chemistry, this time with the organisation’s Chemistry World Entrepreneur of the Year Award in recognition of his success at taking pioneering drugs out of the laboratory and into commercial development for the ultimate benefit of patients worldwide. RSC President Professor David Phillips cited Professor Workman for his “work as a scientific pioneer and serial entrepreneur whose numerous commercialised discoveries and academic research led to his founding two successful chemical companies: Piramed Pharma and Chroma Therapeutics".
Professor Workman also led the group that received the American Association for Cancer Research’s 6th Team Science Award 2012. The citation for this prestigious Award said: “This team’s research is an outstanding example of how innovative cancer research conducted by a highly functioning translational team can start with biologic hypotheses and ultimately lead to much-needed cancer therapeutics.” The Award citation also highlighted the group’s discovery of 16 therapeutic drug candidates over the past six years. Six of these candidate drugs, which include highly innovative inhibitors of the molecular chaperone heat shock protein 90 (HSP90), phosphatidylinositol 3-kinase (PI3K), protein kinase B/AKT, and cyclin-dependent kinases (CDKs), have now entered clinical trials. The 16-member group also carried out pioneering preclinical work on BRAF and its inhibitors, and discovered CHK1 and dual Aurora/FLT3 inhibitors.” Further highlighted in the citation was that “this team’s many research accomplishments include the discovery and development of abiraterone.” The AACR Award citation went on to say: “Overall, the work carried out by this multidisciplinary team over the last six years provides an outstanding example of the non-profit cancer drug discovery and development model that they have pioneered, as well as exemplifying a meritorious ability to collaborate productively with industry to accelerate patient benefit.”
In December 2015, the ICR was presented with the British Pharmacological Society’s UK Pharmacology on the Map award for institutions that have made a significant contribution to improving human health through drug discovery and pharmacology research. Professor Workman was presented the award by Stephen Metcalfe MP, Chair of the Parliamentary and Scientific Committee. Professor Workman said: “We are delighted to be given this accolade from the British Pharmacological Society in recognition of our work in the discovery and pharmacology of cancer drugs. The ICR has been at the forefront of discovering innovative new cancer treatments for decades, with many of our drugs in use worldwide today."
In 2017, Professor Workman was recognised by his alma mater, the University of Leeds, with an Honorary DSc. He was also awarded an Honorary Fellowship from the University of Cumbria in Carlisle. Professor Workman serves as a member of the Athena SWAN steering group.
Professor Workman has published over 470 research articles and edited several books and journal issues on cancer drug development. He has also chaired a number of significant committees and been an adviser to many research institutions as well as pharma and biotechnology companies.
Professor Workman also has a personal biography.
BSc (Hons) Biological Sciences, University of Leicester.
PhD Cancer Pharmacology, University of Leeds.
FSB, Fellow of the Society of Biology.
FMedSci, Fellow of the Academy of Medical Sciences.
DSc (Hon), University of Leicester.
FRSC, Fellow of the Royal Society of Chemistry.
PhD Molecular Pharmacology, The Institute of Cancer Research.
Award for Excellence in Oncology Research, European School of Oncology, 1985.
ICRETT Fellowship, UICC, 1990.
Life Fellowship, Cancer Research Campaign, 1991.
Bruce Cain Memorial Award Lecturer, New Zealand Society for Oncology, 2003.
Honorary Award for Cancer Drug Discovery and Development, Dutch New Drug Development Office, 2006.
ICON Distinguished Lecturer, University of Manchester, 2007.
Tom Connors Award Lecturer, British Association for Cancer Research, National Cancer Research Institute, 2009.
George & Christine Sosnovsky Award, Royal Society of Chemistry, 2010.
Bruce Cain Memorial Lecturer, New Zealand Society for Oncology, 2011.
AACR Team Science Award (Team Leader), American Association for Cancer Research, 2012.
Chemistry World Entrepreneur of the Year, Royal Society of Chemistry, 2012.
Cancer Research UK Translational Cancer Research Prize (Joint Team Leader), Cancer Research UK, 2013.
Fellow of The Royal Society (FRS), The Royal Society, 2016.
Fellow of the European Academy of Cancer Sciences, The European Academy of Cancer Sciences, 2014.
Fellow of the Academy of Medical Sciences (FMedSci), The Academy of Medical Sciences, 2002.
Fellow of the Royal Society of Medicine, The Royal Society of Medicine, 2007.
Editorial BoardsCancer Cell.
Oncotarget.
Molecular Oncology.
Current Cancer Drug Targets.
BBA Reviews on Cancer.
Cancer Science.
Molecular Cancer Therapeutics, 2002-2012.
British Journal of Cancer.
Cell Cycle.
Committee on the Welfare of Animals in Cancer Research, Chair, National Cancer Research Institute.
Related pages
Types of Publications
Journal articles
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.
Types of Publications
Journal articles
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.
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.
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.
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.
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.
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).
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.