The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins which are cleaved at specific sites by a cysteine 3C-like protease (3CL pro) in a post-translational processing step that is critical for coronavirus replication. The 3CL pro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CL pro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CL pro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19. (~450 kDa) and pp1ab (~750 kDa) that contain overlapping sequences and include a 3C-like cysteine protease (3CL pro). The function of this internally encoded 3CL pro is integral to the processing of these proteins and critical for viral replication. 7 The SARS CoV-1 3CL pro shares a high degree of structural homology and similar substrate specificity with the coronavirus 3C-like cysteine proteases of hCoV-229E and TGEV 8 , but is most similar to the SARS CoV-2 3CL pro. Specifically, the SARS CoV-1 and SARS CoV-2 share 96% identity between their respective 3CL pro sequences and 100% identity in the active site. 8 There are numerous reports of reversible cysteine protease inhibitors which include aldehydes 9-12 , thio-or oxymethylketones 13 , cyclic ketones 14 , amidomethylketones 15 , nitriles 16,17 or various 12-283,039 ± 22,586 13 Me 220 ± 0.5 14 cyc-propyl 182 ± 6 15 tert-butyl 230 ± 5 16 Ph 86 ± 3 17 4-OMe-Ph 79 ± 3 18 4-Me-Ph 87 ± 2 19 4-CN-Ph 53 ± 1 20 4-F-Ph 82 ± 3 21 4-Cl-Ph 97 ± 3 22 2,6-(Cl)2-Ph 62,993 ± 2,501 23 2,6-(F)2-Ph 12,776 ± 594 24 2-OH-4-Cl-Ph 11,525 ± 40 25 2-F, 4-CN-Ph 13,321 ± 2,309 26 2,6-(Me)2-Ph 74 ± 4 27 2,6-(MeO)2-Ph 205 ± 2 28 2-CN-Ph 17 ± 2 a See Experimental Section for details on assay methods, values were calculated from at least eight data points with at least two independent determinations.
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.
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 novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins which are cleaved at specific sites by a cysteine 3C-like protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.
Symptom severity in patients with human rhinovirus (HRV)-induced respiratory illness is associated with elevated levels of the inflammatory cytokines interleukin-6 (IL-6) and IL-8. AG7088 is a novel, irreversible inhibitor of the HRV 3C protease. In this study, AG7088 was tested for its antiviral activity and ability to inhibit the production of IL-6 and IL-8 in a human bronchial epithelial cell line, BEAS-2B. Infection of BEAS-2B cells with HRV 14 resulted in the production of both infectious virus and the cytokines IL-6 and IL-8. Treatment of HRV 14-infected cells with AG7088 resulted in a statistically significant (P, <0.05) dose-dependent reduction in the levels of infectious virus as well as IL-6 and IL-8 released into the cell supernatant compared to the results obtained for compound-free infected cells. AG7088 was also able to inhibit the replication of HRV 2 and 16 in BEAS-2B cells. In time-of-addition studies, AG7088 could be added as late as 14 to 26 h after HRV 14 infection of BEAS-2B cells and still result in a statistically significant (P, <0.05) reduction in the levels of infectious virus, IL-6, and IL-8 compared to the results obtained for compound-free infected cells. These findings have implications for the development of an antirhinovirus agent that may not only block virus replication but also diminish symptoms.Human rhinoviruses (HRV), which include over 100 different virus serotypes, are responsible for a significant portion of common colds experienced each year (reviewed in references 7, 26, and 34). In patients with underlying respiratory disorders, HRV infections may lead to sinusitis, otitis media, and lower-respiratory-tract illnesses and also may lead to exacerbations of asthma, cystic fibrosis, and bronchitis (3). Many of the clinical symptoms observed in patients with HRV-induced respiratory illness, such as sore throat, nasal congestion, sneezing, and runny nose, are associated with elevated cytokine levels that can be detected in nasal washings (27,31,33). In addition, experimental HRV infections have been shown to produce an increase in the levels of one or more of the inflammatory cytokines and mediators, including interleukin-6 (IL-6), IL-8, IL-1, kinins, tumor necrosis factor, histamine, and soluble intercellular adhesion molecule 1 (ICAM-1) (32,38,45; R. B. Turner, K. Weingand, C. H. Yeh, et al. Abstr. 36th Intersci. Conf. Antimicrob. Agents Chemother. abstr. H-48, 1996). Increased levels of both IL-6 (45) and IL-8 (39; Turner et al., 36th ICAAC) have been associated with symptom severity in HRV-infected patients.Although a number of compounds have shown in vitro activity against HRV, no antiviral agent has been shown to be effective against disease caused by HRV infection in vivo (1,2,8,15,17,28). To date, only one agent, pirodavir, has been shown to be effective when administered at the time of HRV challenge but not when given 24 h after HRV infection (19). Recent reports have described a new class of compounds directed toward a novel target, the HRV 3C protease (10-14, ...
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