Design, synthesis, and evaluation of irreversible peptidyl inhibitors for clan CA and clan CD cysteine proteases

Abstract

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.