Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have e volved a highly complex cascade of proteolytic processing events to control viral gene expression and replication, Most maturation cleavages within th e precursor polyprotein are mediated by rhinovirus 3C protease (or its imme diate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral ser otypes have been used for the design and elaboration of 3C protease inhibit ors representing different structural and chemical classes. Inhibitors havi ng a,b-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophili c addition of the enzyme's catalytic Cys-147, resulting in covalent-bond fo rmation and irreversible inactivation of the viral protease. Direct inhibit ion of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precu rsor polyproteins as determined by SDS/PAGE analysis of radiolabeled protei ns. Cocrystal-structure-assisted optimization of 3C-pretease-directed Micha el accepters has yielded molecules having extremely rapid in vitro inactiva tion of the viral protease, potent antiviral activity against multiple rhin ovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.