Towards subunit-specific proteasome inhibitors: synthesis and evaluation of peptide alpha ',beta '-epoxyketones

Background: The proteasome is a large multicatalytic protease complex (700 kDa) involved in a number of highly regulated processes. It has three major catalytic activities: a chymotrypsin-like activity, a trypsin-like activit y and a post-glutamyl peptide hydrolyzing (PGPH) activity. To be useful as molecular probes, which could help dissect the cellular functions of the pr oteasome, inhibitors should be specific for the proteasome, active in vivo and selectively block only one of the three catalytic activities. To date, few inhibitors fulfill these requirements so we set out to make novel prote asome inhibitors that incorporate these characteristics. Results: A panel of amino-terminally acetylated peptide alpha',beta'-epoxyk etones with leucine in P1 and various aliphatic or aromatic amino acids in P2-P4 were prepared and evaluated. Most compounds selectively inhibited the chymotrypsin-like activity, while only weakly inhibiting the trypsin-like and PGPH activities. After optimization, one inhibitor, Ac-hFLFL-epoxide, w as found to be more potent and selective for the inhibition of the chymotry psin-like activity than several previously described inhibitors. This inhib itor also exhibited strong in vivo anti-inflammatory activity. Conclusions: Optimization of amino-terminally acetylated peptide alpha',bet a'-epoxyketones furnished a potent proteasome inhibitor, Ac-hFLFL-epoxide, that has an excellent selectivity for the chymotrypsin-like activity. The i nhibitor also proved to be a potent antiproliferative and anti-inflammatory agent. The strong in vivo and in vitro activities suggest that this class of proteasome inhibitors could be both molecular probes and therapeutic age nts.