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 M (range, 0.003 to 0.081 M) and a mean EC 90 of 0.082 M (range, 0.018 to 0.261 M) 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 ␣ 1 -acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 M, 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 sulfatepolyacrylamide 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. MATERIALS AND METHODSCompounds. AG7088 and pleconaril (17) were synthesized at Agouron Pharmaceuticals, Inc. Pirodavir (1) was kindly provided by Janssen Research Foundation (Beerse, Belgium), and WIN 51711 (40) was kindly provided by Sterling Winthrop Research Institute (Collegeville, Pa.). Ganciclovir (Syntex Corp., Palo Alto, Calif.) was obtained from a local pharmacy, and acyclovir was purchased from Sigma (St. Louis, Mo.).Cells and virus strains. All numbered HRV serotypes, echovirus type 11 (EV 11), enterovirus type 70 (ETV 70), coxsackievirus types A21 (CAV 21) and B3 strain Nancy (CVB 3), human cytomegalovirus (HCMV) strain AD169, and herpes simplex virus type 1 (HSV-1) strain McIntyre were purchased from the American Type Culture Collection (ATCC; Manassas, Va.). HRV Hanks and a nasal lavage from a patient challenged with HRV Hanks were kindly provided by Ronald Turner from the Medical University of South Carolina, Charleston, S.C. HRV and coxsackievirus stocks were propagated, and antiviral assays were performed, in H1-HeLa cells (ATCC) incubated at 34 and 37°C, respectively. ETV 70, EV 11, and HCMV stocks were propagated, and antiviral assays were performed, in MRC-5 (ATCC) cells at 37°C. HSV-1 stocks were propagated, and antiviral assays were performed, in Vero (ATCC) cells incubated at 37°C. Vero cells ...

Three-dimensional structure of foot-and-mouth disease virus and its biological functions

“…These results have shed new light on the mechanism and function of FMDV 3C pro , which will drive the development of effective antiviral drugs [37,38]. Just as rupintrivir, a specific, irreversible human rhinovirus (HRV) 3C pro inhibitor targeting the active site of the enzyme, was discovered by structureassisted drug design methodologies [103,116], based on the complex structures of EV71/CVA16 3C pro with rupintrivir, modifications of rupintrivir at the P2 and P1 0 positions will make it more effective in inhibiting EV71 and CVA16 [91]. Moreover, the interaction between FMDV 3C pro and the 5 0 terminus of the RNA could also be a potential target for drug discovery [107,109].…”

The future of antivirals

“…These viruses all have 3C or 3C-like proteases with a typical chymotrypsin-like fold and a catalytic triad (or dyad) containing a cysteine residue as a nucleophile, making them interesting targets for BSAA development. Rupintrivir for instance is an irreversible 3C-protease inhibitor that was originally developed for the treatment of human rhinovirus infections [22] and antiviral activity has also been shown against other picornaviruses, coronaviruses, and norovirus [23][24][25]. Other inhibitors of 3C-like proteases were reported with broad-spectrum antiviral activity against both feline coronaviruses and caliciviruses [26].…”

Characterization and Inhibition of SARS-Coronavirus Main Protease

“…However, these are not SARS protease inhibitors and their targets are not currently known. an EC 50 of 0.013 µM [54][55][56]. The structure of AG7088 (G = CH 2 and R = 4F-C 6 H 4 as shown in Fig.…”