Application of targeted protein degradation strategies to deubiquitinating enzymes (DUBs)

Abstract

Ubiquitination is an important post translational modification which is a regulated reversible process involving a cascade of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ligases which transfer ubiquitin covalently onto proteins. The reverse of this process is carried out by deubiquitinating enzymes (DUBs) which remove ubiquitin and cleave poly-ubiquitin chains. Dysregulation of DUBs has been implicated as a driver in various diseases including, cancer, neurodegenerative diseases, and immune disorders. Inhibition of DUBs has attracted significant interest but proven quite challenging, not least due to the variable conformational flexibility and complex biology across DUBs as a class. Alternative strategies which overcome some of the challenges raised by small molecule inhibition would be useful to decouple the structural and functional roles of DUBs. Targeted protein degradation (TPD) is a rapidly expanding field which aims to co-opt the UPS by recruiting E3 ligases to ubiquitinate a particular protein of interest (POI) leading to its degradation. PROTACs (Proteolysis Targeting Chimeras) have emerged as an important method of achieving TPD. PROTACs are bifunctional molecules which consist of a POI binder and an E3 ligase binder joined via a linker which enable a ternary complex to form between the POI and E3 leading to ubiquitination and subsequent degradation.

In this work, targeted protein degradation strategies have been applied to DUBs as a therapeutic strategy. To this end, a range of PROTACs were synthesised using proprietary DUB-inhibitor recruiters against three DUB targets (USP1, USP7 and USP15) and evaluated in various cellular assays including chemo-proteomic analysis. From this work, lead PROTACs have been identified which show rapid degradation of USP15 with a long-lasting effect and the mechanism of action was confirmed to be through the proteasome. This provides a valuable tool to further probe USP15 biology and allow investigation into the effects of target degradation in disease relevant models.

Thesis is embargoed until 31st July 2028.

Date of Award	Jul 2025
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Almac Discovery Ltd
Supervisor	Richard Williams (Supervisor) & Tim Harrison (Supervisor)