Thesis

Photoaffinity labelling for the study of ligand-protein interactions

Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2021
Thesis identifier
  • T17440
Person Identifier (Local)
  • 201775831
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • The sequencing of the human genome has led to a vast increase in the number of protein targets that are known to be associated with human disease. Modulation of a protein offers the potential for a therapeutic strategy to treat the disease. However, the validation of these targets can prove challenging due to the limited portion of the proteome that has been liganded. Therefore, new technologies are required for the rapid identification of tool molecules to enable the study of protein targets of interest, and their interaction with small molecule ligands. Target-based drug discovery has typically relied on screening of drug-like compound libraries for hit identification. However, the combination of covalent capture approaches and fragment-based drug discovery now offers a new paradigm within chemical biology for the discovery of tool compounds. The work in this thesis focusses on two innovations: i) the development of a chemical proteomic tool compound for the cyclin-dependent kinase family and ii) a fragment-based photoaffinity platform for the efficient identification of protein ligands. A series of photoaffinity probes were designed to target the cyclin-dependent kinase family in order to investigate the crosslinking profile of commonly used photoreactive groups. Chemical proteomic studies enabled the evaluation of the optimal probe as a tool to study the protein family. The covalent capture of small molecule-protein interactions was also investigated through the development of a photoaffinity-based fragment screening platform. This enabled fragment hit identification to protein targets of interest, including human bromodomain BRD4-BD1 and KRas, which is a notoriously undruggable target. The platform was further extended to screen parasitic bromodomains implicated in neglected diseases. The photoaffinity fragments identified in each of these studies not only provided a starting point for evolution into more potent binders, but additionally led to the discovery of tool compounds for further profiling studies. A photoaffinity-based competition assay was developed using both fully functionalised probes, and fragments, as reporters to screen potential inhibitors. This approach enabled a site-selective evaluation of competitor affinity, providing Ki values. Overall, the work in this thesis exemplifies the utility of photoaffinity labelling in the study of ligand-protein interactions. The results illustrate that photoaffinity probes can be developed to enable the study of protein families in chemical proteomic workflows and photoaffinity-based fragments offer an important approach to expanding the druggable proteome.
Advisor / supervisor
  • Bush, Jacob T.
  • Tomkinson, Nicholas C. O.
Resource Type
Note
  • Previously held under moratorium in Chemistry department (GSK) from 19th July 2021 until 15th August 2025. Add second Stax note: The confidentiality statement on each page of this thesis DOES NOT apply
DOI
Funder
Embargo Note
  • The digital version of this thesis is restricted to Strathclyde users only until 19th July 2026.

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