Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes Original Research Article

Background The 20S proteasome is a multicatalytic protease complex that exhibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the β2, β5 and β1 subunits, respectively. Modulation of these activities using inhibitors is essential for a better understanding of the proteasomeʹs mechanism of action. Although there are highly selective inhibitors of the proteasomeʹs chymotryptic activity, inhibitors of similar specificity have not yet been identified for the other activities. Results The X-ray structure of the yeast proteasome reveals that the sidechain of CyResults18 of the β3 subunit protrudes into the S3 subsite of the β2 active site. The location of this residue was exploited for the rational 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 spacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activity to the β2 active site. X-ray crystallographic analysis of a yeast proteasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite. Conclusions Maleoyl-β-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes. Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting