Caspases are a family
of cysteine-dependent proteases with important
cellular functions in inflammation and apoptosis, while also implicated
in human diseases. Classical chemical tools to study caspase functions
lack selectivity for specific caspase family members due to highly
conserved active sites and catalytic machinery. To overcome this limitation,
we targeted a non-catalytic cysteine residue (C264) unique to caspase-6
(C6), an enigmatic and understudied caspase isoform. Starting from
disulfide ligands identified in a cysteine trapping screen, we used
a structure-informed covalent ligand design to produce potent, irreversible
inhibitors (3a) and chemoproteomic probes (13-t) of C6 that exhibit unprecedented selectivity over
other caspase family members and high proteome 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.