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~GP− cleavage sites. We synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g., Boc-Val-Leu-Phe-vGln-OR:  R = Me, <BO>1</BO>; R = Et, <BO>2</BO>) and evaluated them as inhibitors of HRV-14 3C protease (3C^pro). Compounds <BO>1</BO> and <BO>2</BO> and several related tetra- and pentapeptide analogues rapidly inactivated 3C^pro with submicromolar IC<INF>50</INF> values. Electrospray mass spectrometry confirmed the expected 1:1 stoichiometry of 3C^pro inactivation by <BO>1</BO>, <BO>2</BO>, and several other analogues. Compound <BO>2</BO> 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 <BO>1</BO>, <BO>2</BO>, and congeners, peptidyl Michael acceptors lacking a P<INF>4</INF> 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<INF>1</INF> vinylogous glutamine ester to a carboxylic acid also decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic acids vs acrylic esters as Michael acceptors. Incorporating a vinylogous methionine sulfone ester in place of the corresponding glutamine derivative in <BO>1</BO> also reduced activity substantially. Compounds <BO>1</BO> and <BO>2</BO> 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.