Caspase prime-side active-site characterization with non-hydrolyzable peptides assists in the design of a caspase-7-selective irreversible probe

Abstract

AbstractLimited structural information and biochemical studies are available that demonstrate how the prime side of cysteine protease active sites, such as for human caspases, are used for substrate recognition and how these binding regions can be exploited in the design of inhibitors and probes. Reported small molecules that interact with, and are optimized for, the prime side of caspase active sites are limited to methylketone leaving groups and other nonpeptidic inhibitor moieties, such as aza-Michael acceptors. We present the design, synthesis, and co-complex x-ray structures of the first prime-side elongated non-hydrolyzable peptidomimetic ketomethylene inhibitors designed to interrogate the S4-S4’ active-site binding pockets of the executioner caspases-3 and -7. In addition to our structures depicting the first examples of an active-site cysteine in complex with a P1 residue trapped in a non-covalent tetrahedral intermediate, we elucidated prime-side binding interactions for pockets S1’ through S4’ with our biologically relevant peptide Ac-DEVD-Propionate-AAA. Despite the substantial homology among the caspase active sites, we identified a key difference in the prime-side architecture within binding distance to the P2’ inhibitor alanine whereby caspase-3 F128 is substituted for caspase-7 Y151. We exploited this prime-side difference in side chains and their reactivities in the design of non-hydrolyzable ketomethylene-based probes bearing a C-terminal tyrosine-reactive 4-phenyl-1,2,4-triazole-3,5-dione moiety. Our probe selectively labels caspase-7 over caspase-3 and we posit that further characterization of protease active-site prime sides with similar non-hydrolyzable molecules will yield additional tool-like compounds that will assist in establishing the non-redundant roles of caspase family members and other cysteine proteases.