Engaging a Non-Catalytic Cysteine Residue Drives Unprecedented Selectivity of Caspase Inhibition

The caspases are a family of cysteine dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human disease. Classical chemical tools to study caspase function lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we tar-geted a non-catalytic cysteine residue (C264) unique to Caspase-6, an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used structure-informed covalent ligand design to produce potent, irreversible inhibitors (e.g., 3a) and chemoproteomic probes (e.g., 13-t) of Caspase-6 that exhibit unprecedented se-lectivity over other caspase family members and high proteomic selectivity. This approach and the new tools described will enable rigorous interrogation of the role of Caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases