UC Berkeley

Identifying potent and selective small molecule binders for specific protein targets is a central challenge in drug discovery. Most bioactive compounds do not covalently bind their protein target, and instead have a transient, reversible interaction based on affinity to the binding site of the protein. To support non-covalent drug discovery efforts, chemists and chemical biologists have developed strategies using covalent small molecule probe compounds to covalently capture these small molecule-protein interactions. This dissertation focuses on two of the most commonly used current strategies for live-cell target identification: residue-agnostic Photoaffinity Labeling (PAL) and residue-specific Activity-Based Protein Profiling (ABPP).I first discuss work to develop a novel class of PAL probes based on the UV-triggered 1,2-photo-Brook rearrangement of acyl silanes. These new probes, particularly the iPr-substituted acyl silanes, demonstrated efficient photolabeling capabilities with minimal background, showcasing their potential for use in photoaffinity labeling campaigns. Building on these advancements in PAL, we later extended this technique to live cell applications using the same acyl silane-based probes. Our work highlights the effectiveness of acyl silane photoaffinity probes in live cell photoaffinity workflows and their complementarity to existing diazirine-based probes. We also developed a modular synthetic route to generate acyl silane scaffolds with improved labeling properties, expanding the toolkit available for in vivo studies of small molecule-protein interactions. In another area, we explored residue-specific protein bioconjugation by applying methionine-selective redox-activated chemical tagging (ReACT) strategies to ABPP workflows. This enabled the identification of new ligandable sites on cyclin-dependent kinase 4 (CDK4) and unveiled a potential role for the liganded methionine site as an allosteric regulator of protein phosphorylation. Lastly, we explored the role of chirality in protein regulation by developing enantiomeric oxaziridine reagents to investigate pro-chiral methionine oxidation sites. This approach revealed that chiral regulation of methionine oxidation can significantly affect protein function, as demonstrated in oxidative stress models where selective (R)-methionine sulfoxide formation influenced downstream protein modifications. Our work introduces a platform for studying asymmetric methionine modifications and their functional implications, contributing to a deeper understanding of chiral regulation in proteins.