Cysteine proteases are a class of proteolytic enzymes, which are involved in a series of metabolic and catabolic processes, such as protein turnover, digestion, blood coagulation, apoptosis, fertilization and cell differentiation, and the immune response system. The development of novel potent and selective inhibitors for cysteine proteases has therefore gained increasing attention among medicinal chemists. In this thesis we have reported the design, synthesis, and evaluation of several peptidyl inhibitors for clan CA and clan CD cysteine proteases. We have continued the investigation of dipeptidyl vinyl sulfones as potent and selective inhibitors for dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, which is involved in the processing of intracellular proteases, such as granzymes. We have found that DPPI tolerates negatively charged amino acid residues in the P2 position with inhibition rates of 7,600 M-1s-1. Dipeptidyl vinyl sulfones with positively charged amino acid residues at the P1 position, however, do not inhibit DPPI at all. A second project focused on the epoxidation of the double bond of the vinyl sulfone moiety of the dipeptidyl vinyl sulfones. Instead of epoxidizing the double bond, we found that an isomerization had occurred. The newly formed compounds were determined to be allyl sulfones. We tested this new class of inhibitors with clan CA proteases and obtained inhibition rates of 560 M-1s-1 for Cbz-Leu-Phe-AS-Ph with calpain I. Two new classes of compounds for the clan CD protease S. mansoni legumain were designed, synthesized, and evaluated. Aza-peptidyl epoxides were found to be potent and selective inhibitors of S. mansoni legumain with IC50’s as low as 45 nM. Aza-peptide Michael acceptors were derived from the aza-peptide epoxide design and synthesized in an analogous fashion. The aza-peptide Michael acceptors inhibited S. mansoni legumain with even lower IC50’s, as low as 10 nM. However, the aza-peptide Michael acceptors react with thioalkylating agents contained in the buffer, such as DTT. The rates of degradation were determined spectroscopically, and half-lives of 3 to 20 minutes were measured. This observation gave us insights into the enzymatic mechanism and allowed us to determine the point of attack for the legumain active site cysteine thiol.

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