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

Abstract

AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease (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 design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC(50)) of 0.023 microM (range, 0.003 to 0.081 microM) and a mean EC(90) of 0.082 microM (range, 0.018 to 0.261 microM) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. 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 50% cytotoxic concentration of AG7088 was >1,000 microM, yielding a therapeutic 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 effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate.

FIG. 1

Chemical structure of AG7088.

FIG. 2

In vitro activity of AG7088 against HRV serotypes. EC₅₀ (A) and EC₉₀ (B) of AG7088, pleconaril, and pirodovir for 48, 45, and 47 HRV serotypes, respectively, were determined by measuring XTT dye reduction following 2 to 5 days of infection of H1-HeLa cells as described in Materials and Methods.

FIG. 3

Antiviral activity of AG7088 when added at various times after virus infection. H1-HeLa cells were infected with HRV 14 at an MOI of 15. AG7088 (0.5 μM) and WIN 51711 (3.0 μM) were added at various times after infection. Virus control represents infected cells incubated with medium only. Eight hours after infection, cell lysates and supernatants were collected and the level of infectious virus was determined as described in Materials and Methods.

FIG. 4

Inhibition of HRV 14 3C-mediated proteolytic processing by AG7088. SDS-solubilized lysates were prepared from uninfected cells (cell ctrl; 0.1× indicates 1/10 the amount of uninfected cell lysate analyzed in lane designated 1×) or infected cells treated with AG7088, pleconaril, or medium only (virus ctrl), and equal amounts of protein were analyzed by PAGE as described in Materials and Methods. P2-P3, P1, 3CD, VP0, 2C, and VP1 designate HRV-specific polypeptides. MWT, molecular weight (in thousands).