Global chemoproteomic profiling of bromodomain inhibitors

Thesis

Bromodomain-containing proteins (BCPs) are epigenetic readers that regulate gene expression and play key roles in many diseases including cancer, making them prime targets for drug discovery. While several high-quality chemical probes and inhibitors have been developed for various bromodomains, mainly BET inhibitors have advanced to clinical trials albeit with limited success due toxicity. Despite the potential of proteome-wide selectivity assessments to reveal mechanisms of action for endogenously expressed BCPs, which often function within large multi-protein complexes, this approach has not been widely adopted.

In this thesis, we profiled 17 bromodomain inhibitors using chemoproteomics to map direct and indirect drug-protein interactions across the proteome. Building on extensive optimization efforts, we were able to enrich all chemically accessible human BCPs and demonstrate the co-purification of complex partners as well as unexpected non-bromodomain off-targets. By leveraging our highly characterised selection of affinity probes, we developed a generic bromodomain profiling matrix, termed “bromobeads”, that allows for the profiling of unmodified compounds against a broad array of BCPs in parallel. The matrix composition was optimised to create a suitable assay window and robust detection was achieved with S-Trap-based sample processing combined with DIA LC-MS/MS.

Using the bromobeads platform, we characterized 10 clinical BET inhibitors in dose-dependent experiments, determining apparent binding constants and revealing differential behaviour in complex partner binding. To demonstrate the platform’s versatility, we extended our experiments to parasites whose bromodomain proteins are emerging drug targets. Additionally, we utilised our set of bespoke affinity probes to expand the bromodomain chemical biology toolbox by developing NanoBRET tracers and PROTACs. The workflow described herein also enabled us to explore how BRD4 inhibitor target occupancy is affected by cellular perturbations, suggesting our chemoproteomic platform as versatile platform for drug discovery and tool to enhance our understanding of bromodomain inhibitor interactions in cells.

Authors: Bauer, LG

• DOI: 10.5287/ora-g7zmyk1ky
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: chemical proteomics; bromodomain inhibitors; bromobeads; chemical probes
• Subjects: Chemical biology