Closed: Enabling novel E3 ligases for targeted protein degradation therapeutics

Unparalleled opportunities presented by targeted protein degradation (TPD) resulted so far in successful degradation of nearly a hundred of different targets with PROTACs and molecular glue degraders (MGD) and a fast progress of many into the clinic. Most degraders however utilize just a fraction of E3 ligases expressed in human tissues, most commonly von Hippel-Lindau (VHL) and Cereblon (CRBN). Despite good activity of those ligases in most tissues, degradation of some targets still presents a challenge, especially if selectivity is important. Importantly, identification of novel chemical binders with better physchem properties, tolerance and less tumor resistance would be very valuable (). Thus, exploring additional E3 ligases for TPD applications might offer opportunity to increase the target scope, improve target and tissue selectivity, overcome resistance, and discover ligands with better physicochemical properties and therapeutic window. The aim of this project is to unlock this untapped potential by enabling novel E3 ligases for TPD applications through biological and mechanistic characterisation in relevant oncology models and to pave the way for discovery of novel PROTACs and MGDs.

Aim 1. Characterize therapeutic potential of 1 – 3 novel E3 ligases in cancer models using cell biology and genetic manipulation techniques

Multiple tumor cells have aberrantly high expression of certain E3 ligases. Recruiting those E3s by PROTACs and MGDs can be utilized to enhance therapeutic window and for tissue selective degradation. In the first year the project will focus on assessing therapeutic potential of 2 - 3 novel E3 ligases selected based on literature and available transcriptomics and proteomics data (DepMap, TNMplot), and drug tractability score (internal bioinformatics analysis). This will be achieved by verifying differential E3 ligase expression and activity in different cancer cell lines vs. healthy tissues using WB or fluorescent immunostaining and ubiquitination assays. In the next step we will select one E3 ligase for more detailed evaluation as a therapeutic target and as a potential effector for PROTACs and MGDs, looking the effects of E3 ligase KO on cancer cell viability and downstream signalling pathway.

• Measurement of E3 ligase protein levels by WB in selected AML, MM and solid cancer cell lines and healthy cells (fibroblasts, PBMCs)
• E3 ligase activity will be tested using in vitro ELISA or TR-FRET autoubiquitination assay.
• E3 ligase KO using CRISPR-Cas9 and degradation with dTAG in selected AML, MM and solid cancer cell line. The results will be confirmed by WB and HiBit (dTAG).
• Determination of the effects of E3 ligase KO/ degradation on cell viability will be performed using CTG assay.

Aim 2. Testing E3 ligase degradation activity

The main objective of the study is to assess potential of a novel E3 ligase for TPD application. Therefore, in the next stage the project will focus on assessing if the selected E3 ligase is able to degrade different targets following recruitment with a PROTAC.

• The experiments will require generating cell line overexpressing BromoTag-GFP protein in combination with inducible dTAG-E3 ligase.
• E3 ligase degradation activity will be assessed by measuring degradation potency of dTAGBromoTag PROTAC in GFP fluorescence assay. Alternatively, a BRD4 – dTAG- or promiscuous kinase-dTAG PROTAC may be used to test degradation of BRD4 or selected endogenous kinases (BRD4-Hibit assay or WB).

Aim 3. Identify endogenous substrates for a selected E3 ligase and investigate substrate recognition mechanism

Majority of intracellular proteins are degraded via Ub-proteasome pathway. However, information about endogenous E3 ligase substrates is scarce and it is often context dependent. Identifying endogenous substrates for the selected E3 ligase will be an important starting point for designing E3 ligand discovery strategy. Characterising E3 ligase complex is critical for mechanistic understanding of E3 ligase function, design of screening assays and degraders.

Identifying therapeutically relevant endogenous substrates will be a cue for development of MGDs based on molecules enhancing the natural interaction. Endogenous substrates recruit E3 ligases via short peptide motifs called degrons, which are often shared by multiple proteins. For example, glycine loop recognized by CRBN ligase is shared by multiple Zinc Finger proteins. Hence identifying a degron recognized by a given E3 ligase is important for finding potential MGD targets. Importantly, mimicking the degron structure can also be used as a strategy for discovery chemical E3 ligase binders. For example, VHL binder was designed by mimicking hydroxyproline in HIF1 alpha degron.

Therefore, the next part of the project will focus on characterizing the interactome and endogenous substrates of the selected E3 ligase, and substrate recognition mechanism. The experiments will include:

• Proteomics profiling following E3 ligase degradation (E3 ligase-dTAG cells) to identify stabilised proteins. Rescue experiment with WB readout will include E3 ligase overexpression construct and/or inactive dTAG PROTAC.
• Identification of E3 ligase interactome and ubiquitinome (potential substrates) by proximity biotin labelling (TurboID) followed by mass spectrometry
• Substrate recognition mechanism studies will include expression of truncated E3 ligase target mutants with Hibit tag in cancer cells overexpressing E3 ligase, and degradation Hibit assay. If required, degron – E3 ligase binding will be confirmed by IP pull-down/WB.

Note: the ICR’s standard minimum entry requirement is a relevant undergraduate Honours degree (First or 2:1).

Pre-requisite qualifications of applicants:

BSc or MSc in Cell Biology, Molecular Biology, Biochemistry

Intended learning outcomes:

• Critical thinking
• Literature and data analysis and interpretation
• Experiment design
• Range of biochemical, cell biology and genetic engineering techniques
• Knowledge of drug discovery, novel therapeutic modalities, cancer biology
• Data presentation
• Scientific writing

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