UCSF

Cysteine-reactive covalent molecules have emerged as powerful therapeutic agents in the clinic and as useful chemical tools in the lab, but their successful development requires both strategies to enhance their selectivity and resources to better understand cysteine reactivity across biological contexts. This dissertation presents complementary approaches that advance both fronts. First, I describe a steric tuning strategy for electrophilic warheads that improves covalent inhibitor selectivity independent of the intrinsic electrophile reactivity. We demonstrate the utility of this approach by exchanging the acrylamide warhead on the Bruton’s tyrosine kinase (BTK) inhibitor, Ibrutinib (Ib), for fumarate-based electrophiles. The incorporation of a tert-butyl fumarate electrophile onto Ib (IbtBu) preserved on-target BTK engagement while markedly reducing time-dependent and time-independent off-target reactivity, decreasing proteome-wide labeling from 247 proteins to 7, with BTK as the sole time-independent target of IbtBu. Second, I report the creation of an integrated proteomic and cysteine reactivity atlas across the NCI-60, a pan-cancer cell line panel consisting of 59 unique cell lines which collectively represent 9 distinct tissue types, using label-free quantification (LFQ) and cysteine reactivity profiling. This resource, which represents the most comprehensive dataset of its kind, profiles over 12,000 proteins and more than 36,000 reactive cysteines, including >10,000 hyperreactive (HR) cysteines, revealing both conserved and tissue-specific patterns of cysteine reactivity. Conserved HR cysteines were enriched for annotated functionality, whereas tissue-specific HR cysteines mapped to protein families relevant to cellular signaling and cancer biology. Integration of our data with published electrophilic fragment screening data revealed a strong bias of covalent scout fragments for HR cysteines. To enable community access, we developed a publicly available, online database (cysteine.app) with interactive tools for exploring cysteine and protein coverage across the NCI-60 and for building custom cell line panels. Together, these studies introduce new approaches, resources, and tools (ART) that advance the design and discovery of cysteine-reactive covalent molecules and provide a framework for bridging fundamental insights into cysteine biology with translational opportunities in drug discovery.