Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes

Background: The 20S proteasome is a multicatalytic protease complex that ex hibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the beta 2, beta 5 and pr s ubunits, respectively. Modulation of these activities using inhibitors is e ssential for a better understanding of the proteasome's mechanism of action . Although there are highly selective inhibitors of the proteasome's chymot ryptic activity, inhibitors of similar specificity have not yet been identi fied for the other activities. Results: The X-ray structure of the yeast proteasome reveals that the sidec hain of Cys118 of the beta 3 subunit protrudes into the S3 subsite of the b eta 2 active site. The location of this residue was exploited for the ratio nal design of bidentated inhibitors containing a maleinimide moiety at the P3 position for covalent linkage to the thiol group and a carboxy-terminal aldehyde group for hemiacetal formation with the Thr1 hydroxyl group of the active site. Structure-based modelling was used to determine the optimal s pacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activit y to the beta 2 active site. X-ray crystallographic analysis of a yeast pro teasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite. Conclusions: Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor t hat is highly selective for the trypsin-like activity of eukaryotic proteas omes. Despite the reactivity of the maleinimide group towards thiols, and t herefore the limited use of this inhibitor for in vitro studies, it might r epresent an interesting new biochemical tool.