Optimization and Chemoproteomic Profiling of a Selective, Covalent Bfl-1-Targeting Cellular Tool

We describe herein the discovery and optimization of a potent and irreversible cellular probe for selective labeling of Bfl-1, a member of the Bcl-2 family. This chemical series demonstrates robust selectivity for Bfl-1 over other related antiapoptotic proteins and exhibits favorable cellular potency as well as promising in vivo pharmacokinetics. Notably, compound 25 achieves a k_inact/K_I value of 9300 M^-1s^-1 and elicits caspase activation at submicromolar concentrations in cellular assays. To comprehensively profile proteome-wide selectivity, we performed chemoproteomic analyses on compound 25 alongside our previously reported Bfl-1 inhibitors. This enabled critical insights into potential off-target interactions and facilitated direct comparison of off-target profiles among distinct chemotypes targeting Bfl-1.