Project ID iCASE2024_06_CM-HD


Co Supervisor 1A Faculty of Life Sciences & Medicine, School of Cancer & Pharmaceutical Sciences, Comprehensive Cancer CentreWebsite

Co Supervisor 1B Faculty of Life Sciences & Medicine, School of Cancer & Pharmaceutical Sciences, Institute of Pharmaceutical ScienceWebsite

Additional Supervisor Dr Monica Rodrigo

Partner AstraZeneca

Collateral-ID: Mapping the off-target degradome of PROTACs

An area of untapped potential in drug development is the so-called undruggable space. Undruggable proteins are defined as those unable to be pharmacologically targeted via conventional small molecule drugs. In recent years, new modalities have arisen that have the potential to target these proteins. Targeted protein degradation (TPD) makes use of the cells’ ability to destroy proteins that contain a particular mark called ubiquitin. One version of TPD uses Proteolysis-targeting Chimeras (PROTACs) where the PROTAC molecule forms a bridge between an E3 ubiquitin ligase complex and protein of interest (POI) resulting in the ubiquitination and destruction of the POI. Protein degradation removes all target functions—structural/scaffolding, regulatory, enzymatic, etc.—allowing therapeutic targeting of proteins without enzymatic activity.
PROTAC-induced protein complexes are artificial and can result in protein-protein interactions that lead to collateral proteins being degraded along with the intended POI. Traditional methods used to study off-target effects of small molecule inhibitors are not well suited to interrogate the potential off-target liabilities of PROTACS.
This project will use protein proximity labelling to study PROTAC-induced protein complexes: Collateral-ID.
The project will aim to deliver a platform suitable for off-target profiling of any E3 ligase and POI. To this end we will:
1. Establish Collateral-ID as a method to identify interacting partners of E3 ligases
2. Assess how the choice of E3 ligase impacts off-target interactions
3. Assess the suitability of generic tags as POI ligands for new targets and the suitability of Collateral-ID to identify off-target proteomes

Overarching objectives and yearly plan
Year 1:
Objective 1: Chemical Synthesis of a PROTAC tool-box.
Techniques and skills: chemical synthesis, spectroscopy
Objective 2: Establishment of Collateral-ID using BRD4 targeting PROTACs as P-O-C.
Technique and skills: molecular cloning, mammalian tissue culture, cell line engineering including generation of CRISPR Cas9 knockouts, Protein proximity labelling, mass spectrometry and use of computational tools to interrogate data
Year 2:
Objective 3: Comparison of the off-target proteomes of CRBN and VHL directed BRD4 PROTACs
Techniques: continuation of techniques from year 1. High content fluorescent microscopy and automated image analysis
Year 3:
Objective 4: Use generic tags as POI ligands for new targets
Techniques and skills: continuation of techniques from previous year

Representative Publications

1 Hewitt, G. et al. Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD. Mol Cell 81, 767-783 e711, doi:10.1016/j.molcel.2020.12.006 (2021).
2 Rabanal-Ruiz, Y. et al. mTORC1 activity is supported by spatial association with focal adhesions. J Cell Biol 220, doi:10.1083/jcb.202004010 (2021).
3 Lehmann, L. C. et al. Mechanistic Insights into Regulation of the ALC1 Remodeler by the Nucleosome Acidic Patch. Cell Reports 33, doi:10.1016/j.celrep.2020.108529 (2020).

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[2] Kelly, J., Martini, S., Brownlow, N., Joshi, D., Federico, S., Jamshidi, S., Kjaer, S., Lockwood, N., Rahman, K. M., and Fraternali, F. (2020) The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition, Nature Communications 11, 1396.
[3] Corcoran, D. B., Lewis, T., Nahar, K. S., Jamshidi, S., Fegan, C., Pepper, C., Thurston, D. E., and Rahman, K. M. (2019) Effects of systematic shortening of noncovalent C8 side chain on the cytotoxicity and NF-κB inhibitory capacity of pyrrolobenzodiazepines (PBDs), Journal of Medicinal Chemistry 62, 2127-2139.