Synthesis and evaluation of peptidyl Michael acceptors that inactivate human rhinovirus 3C protease and inhibit virus replication

Human rhinovirus, the chief cause of the common cold, contains a positive-sense strand of RNA which is translated into a large polyprotein in infected cells. Cleavage of the latter to produce the mature viral proteins required for replication is catalyzed in large part by a virally encoded cysteine proteinase (3C(pro)) which is highly selective for -Q similar to GP- cleavage sites. We synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g., Boc-Val-Leu-Phe-vGln-OR: R = Me, 1; R = Et, 2) and evaluated them as inhibitors of HRV-14 3C protease (3C(pro)). Compounds 1 and 2 and several related tetra- and pentapeptide analogues rapidly inactivated 3C(pro) with submicromolar IC50 values. Electrospray mass spectrometry confirmed the expected 1:1 stoichiometry of 3C(pro) inactivation by 1, 2, and several other analogues. Compound 2 also proved to be useful for active site titration of 3C(pro), which has not been possible heretofore because of the lack of a suitable reagent. In contrast to 1, 2, and congeners, peptidyl Michael accepters lacking a Pq residue have greatly reduced or negligible activity against 3C(pro), consistent with previously established structure-activity relationships for 3C(pro) substrates. Hydrolysis of the P-1 vinylogous grutamine ester to a carboxylic acid also decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic acids vs acrylic esters as Michael accepters. Incorporating a vinylogous methionine sulfone ester in place of the corresponding glutamine derivative in 1 also reduced activity substantially. Compounds 1 and 2 and several of their analogues inhibited HRV replication in cell culture by 50% at low micromolar concentrations while showing little or no evidence of cytotoxicity at 10-fold higher concentrations. Peptidyl Michael acceptors and their analogues may prove useful as therapeutic agents for pathologies involving cysteine proteinase enzymes.