In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease

AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C pro tease {inactivation rate constant (k(obs)/[I]} = 1,470,000 +/- 440,000 M-1 s(-1) for HRV 14) that was discovered by protein structure-based drug desig n methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibi ted the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC50) of 0.023 mu M (range, 0.003 to 0.081 mu M) a nd a mean EC90 of 0.082 mu M (range, 0.018 to 0.261 mu M) as well as that o f related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus Il. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of alpha (1)-acid glycoprotein or mucin, proteins present in nasal secretions. The 5 0% cytotoxic concentration of AG7088 was >1,000 mu M, yielding a therapeuti c index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection . In contrast, a compound targeting viral attachment and/or uncoating was e ffective only when added at the initiation of virus infection. Direct inhib ition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a nalysis of radiolabeled proteins, which showed a dose-dependent accumulatio n of viral precursor polyproteins and reduction of processed protein produc ts. The broad spectrum of antiviral activity of AG7088, combined with its e fficacy even when added late in the virus life cycle, highlights the advant ages of 3C protease as a target and suggests that AG7088 will be a promisin g clinical candidate.