Design, Synthesis, and Evaluation of Cysteine Protease Inhibitors

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Campbell, Amy
Powers, James C.
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Both clan CA and clan CD proteases have a variety of physiological and pathological roles. In particular, both clans have members who have been implicated in cell death pathways, including apoptosis. Caspases are members of clan CD. Many of the caspase inhibitors used in apoptotic studies have shown cross reactivity with clan CA proteases. Thus, the anti-apoptotic effect of these inhibitors could be due to the broad-spectrum inhibition of a variety of cysteine proteases. Recently, the Powers laboratory designed a new class of inhibitors highly specific for clan CD proteases, aza-peptide epoxides. Initial data showed that this high selectivity could be due to the presence of the aza-residue, and not simply an artifact of substrate specificities. E-64c, an epoxysuccinyl inhibitor, is known to be a highly potent inhibitor of cathepsin B and calpain I. Thus, to determine if these clan CA proteases could tolerate an aza-residue, aza-E-64c and its analogues were synthesized. These inhibitors, termed epoxysuccinyl aza-peptides, were found to be significantly less potent for cathepsin B, calpain I, and papain than their non-aza counterparts, including E-64c. Previous findings have shown that the reactivity and selectivity of aza-peptide epoxides with caspases were significantly influenced by epoxide stereochemistry and the prime side substituent. Thus, this second project involved the systematic study of epoxide stereochemistry effects, prime side substituent effects, and the combined effect of these two variables. All inhibitors were tested with the seven apoptotic caspases: caspases-2, -3, -6, -7, -8, -9, and -10. We found that epoxide stereochemistry, prime side substituent, and also the peptidyl sequence have combined effects on potency and selectivity. In general, the (S,S) stereoisomer is the most potent relative to the (R,R) and (cis) stereochemistries. Modeling studies were done to determine why this is true. Aza-peptide epoxides were also briefly compared to aza-peptide Michael acceptors, another class of inhibitors highly specific for clan CD proteases
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