Inhibition of human caspases by peptide-based and macromolecular inhibitors

Studies with peptide-based and macromolecular inhibitors of the caspase fam ily of cysteine proteases have helped to define a central role for these en zymes in inflammation and mammalian apoptosis. A clear interpretation of th ese studies has been compromised by an incomplete understanding of the sele ctivity of these molecules. Here we describe the selectivity of several pep tide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVA D-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included s everal that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potenci es against these enzymes, with dissociation constants ranging from 75 pM to >10 mu M. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order in activation rates that range from 2.9 x 10(2) M-1 s(-1) for caspase-2 to 2.8 x 10(5) M-1 s(-1) for caspase-1. The results obtained with peptide-based i nhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (K-i < 20 nM ) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and m ost Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host in flammatory response.