Chemoproteomic profiling studies of compound selectivity and target protein ligandability

Thesis

Chemical probes are valuable tools to study compound selectivity and target engagement. These qualities are immensely important for drug discovery, as compounds lacking efficacy and safety are a major reason for phase II failures in clinical trials. It is highly important to study lead compounds, not only in biophysical and biochemical assays but also in the cellular context, revealing selectivity as well as potential unwanted off-targets. In cells, the protein is preserved in its native form and environment, which allows for the consideration of physiological co-factor effects and post-translational modifications, which may be absent in assays performed with recombinant protein. The chemoproteomic techniques referred to within this work thus present a useful example of deconvoluting the targets of small bioactive molecules in the cellular proteome that can enable and simultaneously help de-risk drug discovery projects.

The introduction describes chemoproteomic techniques coupled to mass spectrometry as well as complementary techniques for studying target engagement in cellulo. The second chapter delves into the family of E3 ligases and their role as tools in targeted protein degradation. It presents a bioinformatic analysis and a compound library selection to maximise the number of ligandable E3 ligases. The third chapter reports how an isoTOP ABPP screening platform was established and optimised to enable the creation of an E3-ligase-centric druggability map by profiling covalent fragments. Followingly, the fourth chapter describes utilising the isoTOP ABPP for profiling not only fragments but also lead compounds and chemical probes for KEAP1 and NUDT7. The fifth chapter characterises the clinical compound GSK3326595 using mass spectrometry-based proteomic methods supplemented by a newly developed cellular NanoBRET assay. In the sixth chapter, a NanoBRET PPI assay is introduced to evaluate fragments as pharmacological modulators of the DENND1A and RAB35 interactions.

Authors: Rothweiler, EM

• DOI: 10.5287/ora-2zkkdxwvb
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Chemical Biology