New chemical approaches for the development of targeted protein degradation

Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2019
Thesis identifier
  • T15981
Person Identifier (Local)
  • 201594314
Qualification Level
Qualification Name
Department, School or Faculty
  • Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules which induce targeted protein degradation by redirecting the ubiquitin-proteasome system. PROTACs simultaneously bind to both a protein of interest and an E3 ubiquitin ligase. The proximity of the target protein and the E3 ligase complex allows transfer of ubiquitin onto the target protein, after which the protein can be recognised and then degraded by the proteasome. The PROTAC mechanism-of-action offers a number of potential advantages over small molecule inhibition for the development of new medicines. Efficacy maybe achieved from low doses, extended duration of action is possible, arising from pharmacokinetic pharmacodynamic disconnects, and challenging targets may become tractable through the identification of suitable affinity binders. In this research, the development of PROTAC technology is explored. In order to expand the breadth of E3 ligases that are currently recruited using this approach, a promiscuous toolbox was established to prosecute new chemical matter for E3 ligases. In order to further elaborate the promiscuous toolbox already known for the degradation of kinases, investigation of a promiscuous bromodomain PROTAC was explored. After assessment of bromosporine derived PROTACs in multiple cell lines with two validated E3 ligases, it was deemed an unsuitable binder for the toolbox. The PROTACs were unable to induce potent nor promiscuous bromodomain degradation. As a result, a known BET bromodomain binder was selected for the promiscuous toolbox in addition to the known promiscuous kinase binder and a RIPK2 binder for new E3 ligase validation. With a promiscuous toolbox in hand, a new E3 ligase was evaluated. Indisulam, a small molecule “molecular glue”, was found to bind to the E3 ligase DCAF15. Indisulam derived PROTACs were synthesised and evaluated with a range of linker lengths and multiple protein binders. These studies conclusively demonstrated that protein degradation was not achieved using these PROTACs. The indisulam derived binder was subsequently found not to be suitable for the PROTAC approach without further investigation to determine DCAF15 recruitment. Given the lack of degradation with the initial, empirically selected E3 ligases from the literature, a distinct E3 ligase agnostic approach to protein degradation was developed. A high throughput phenotypic screen was established using green fluorescent protein (GFP) as the protein of interest, where cellular fluorescence levels correlate with protein degradation. High throughput chemistry techniques were implemented and optimised to synthesise thousands of HaloCompounds in-situ by amide coupling. The compounds were tested directly in cells to find new chemical matter for the induction of protein degradation. This strategy allowed identification of several potential hits from a 3000-amine screen, with one high-confidence hit currently being further evaluated. For this effort the screen was optimised successfully and can potentially be employed for a target-agnostic high-throughput screening campaign of hundreds of thousands of compounds for new E3 ligases to employ in future protein degradation strategies.
Advisor / supervisor
  • Queisser, Markus
  • Smith, Ian E. D.
  • Burley, Glenn A.
Resource Type
  • Previously held under moratorium in Chemistry department (GSK) from 10th September 2019 until 18th June 2021
Embargo Note
  • This thesis is restricted to Strathclyde users only until 10th September 2024.