Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. Highresolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having ␣,␤-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS͞PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

The design, synthesis, and biological evaluation of reversible, nonpeptidic inhibitors of human rhinovirus (HRV) 3C protease (3CP) are reported. A novel series of 2,3-dioxindoles (isatins) were designed that utilized a combination of protein structure-based drug design, molecular modeling, and structure-activity relationship (SAR). The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis, thus forming a stabilized transition state mimic. Molecular-modeling experiments using the apo crystal structure of human rhinovirus-serotype 14 (HRV-14) 3CP and a peptide substrate model allowed us to design recognition features into the P1 and P2 subsites, respectively, from the 5- and 1-positions of isatin. Attempts to optimize recognition properties in the P1 subsite using SAR at the 5-position were performed. In addition, a series of ab initio calculations were carried out on several 5-substituted isatins to investigate the stability of sulfide adducts at C-3. The inhibitors were prepared by general synthetic methods, starting with commercially available 5-substituted isatins in nearly every case. All compounds were tested for inhibition of purified HRV-14 3CP. Compounds 8, 14, and 19 were found to have excellent selectivity for HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B. Selected compounds were assayed for antiviral activity against HRV-14-infected HI-HeLa cells. A 2.8 A cocrystal structure of derivative 19 covalently bound to human rhinovirus-serotype 2 (HRV-2) 3CP was solved and revealed that the isatin was situated in essentially the same conformation as modeled.

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 ...

The picornavirus 3C protease is required for the majority of proteolytic cleavages that occur during the viral life cycle. Comparisons of published amino acid sequences from 6 human rhinoviruses (HRV) and 20 human enteroviruses (HEV) show considerable variability in the 3C protease-coding region but strict conservation of the catalytic triad residues. Rupintrivir (formerly AG7088) is an irreversible inhibitor of HRV 3C protease with potent in vitro activity against all HRV serotypes (48 of 48), HEV strains (4 of 4), and untyped HRV field isolates (46 of 46) tested. To better understand the relationship between in vitro antiviral activity and 3C protease-rupintrivir binding interactions, we performed nucleotide sequence analyses on an additional 21 HRV serotypes and 11 HRV clinical isolates. Antiviral activity was also determined for 23 HRV clinical isolates and four additional HEV strains. Sequence comparison of 3C proteases (n ‫؍‬ 58) show that 13 and 11 of the 14 amino acids that are involved in side chain interactions with rupintrivir are strictly conserved among HRV and HEV, respectively. These sequence analyses are consistent with the comparable in vitro antiviral potencies of rupintrivir against all HRV serotypes, HRV isolates, and HEV strains tested (50% effective concentration range, 3 to 183 nM; n ‫؍‬ 125). In summary, the conservation of critical amino acid residues in 3C protease and the observation of potent, broad-spectrum antipicornavirus activity of rupintrivir highlight the advantages of 3C protease as an antiviral target.The picornavirus family consists of over 200 medically important viruses, including human rhinoviruses (HRV) and human enteroviruses (HEV). HRV, comprising over 100 different serotypes, are a major cause of mild upper respiratory infections (reviewed in references 4, 8, and 40). Although HRV infections are usually mild and self-limiting, they can also be associated with exacerbation of disease in individuals with underlying respiratory disorders (4, 48). The HEV include over 70 viruses that are associated with diverse clinical syndromes ranging from mild, self-limiting infections to fulminant and potentially fatal disease (3,52,53). Earlier clinical studies with agents that inhibit virus attachment and/or uncoating (e.g., tremacamra, a soluble intracellular adhesion molecule-1, pirodavir, and pleconaril) and nonspecific antiviral agents such as ␣-2␤ interferon have demonstrated that prevention and early treatment of HRV colds could provide clinical benefit (3, 18-23, 53). Recently, a retrospective analysis of two multicenter clinical trials demonstrated that pleconaril, a capsid-function inhibitor (17), significantly reduced the duration and severity of picornavirus-induced colds (24). To date, however, no antiviral agents have been approved for the prevention or treatment of HRV infection.We have focused our antiviral strategy on the inhibition of 3C protease, a viral enzyme that is absolutely required for the proteolytic cleavage of viral precursor polyproteins to functional p...

The structure-based design, chemical synthesis, and biological evaluation of peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds incorporate various Michael acceptor moieties and are shown to irreversibly bind to HRV serotype 14 3CP with inhibition activities (kobs/[I]) ranging from 100 to 600 000 M-1 s-1. These inhibitors are also shown to exhibit antiviral activity when tested against HRV-14-infected H1-HeLa cells with EC50's approaching 0.50 microM. Extensive structure-activity relationships developed by Michael acceptor alteration are reported along with the evaluation of several compounds against HRV serotypes other than 14. A 2.0 A crystal structure of a peptide-derived inhibitor complexed with HRV-2 3CP is also detailed.