A Continuous Spectrophotometric Assay for the Hepatitis C Virus Serine Protease

Zhang, Rumin; Beyer, Brian M.; Durkin, James; Ingram, Richard; Njoroge, F. George; Windsor, William; Malcolm, Bruce A.

doi:10.1006/abio.1999.4109

Cited by 106 publications

(74 citation statements)

References 34 publications

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“…Recombinant single-chain HCV NS3 and NS4A proteases from genotypes 1 to 3 were prepared as described previously (2,23,26). HCV protease assays were performed by use of a 200-l reaction mixture, as described previously (29). Typically, 100 l protease was added to 100 l of assay buffer containing the chromogenic substrate acetyl-DTEDVVP(norvaline)-O-4-phenylazophenol.…”

Section: Methodsmentioning

confidence: 99%

Preclinical Characterization of the Antiviral Activity of SCH 900518 (Narlaprevir), a Novel Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease

Tong¹,

Arasappan

Bennett

et al. 2010

Antimicrob Agents Chemother

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Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K i * ) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC 90 ) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5؋ EC 90 of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Despite recent advances, the eradication of chronic HCV infection remains challenging. Approximately 50% of patients infected with the most prevalent form of the virus (genotype 1) fail to respond to treatment with the current standard of care of pegylated alpha interferon in combination with ribavirin (4, 13). Thus, there is a great unmet medical need for the development of new agents with activity against HCV to improve the sustained virological response (SVR). The HCV genome is translated as a single polyprotein precursor which is processed by cellular and viral proteases into structural proteins (the C, E1, and E2 proteins), followed by nonstructural (NS) proteins (the p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins). The NS3 protein is multifunctional, with the N-terminal region (amino acids 1 to 181) encoding a serine protease and the remaining polypeptide encoding a helicase. The NS3 protease noncovalently binds to the cofactor NS4A, which contributes to the formation and stability of the active site and helps to anchor the NS3/NS4A complex to the membrane. The NS3 protease is responsible for the processing of the HCV polyp...

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Section: Methodsmentioning

confidence: 99%

Preclinical Characterization of the Antiviral Activity of SCH 900518 (Narlaprevir), a Novel Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease

Tong¹,

Arasappan

Bennett

et al. 2010

Antimicrob Agents Chemother

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“…The production and characterization of the single-chain NS3 protease domain, NS4A 21-32 -GSGS-NS3 3-181 (plasmid p24), has been previously described (38). Single-chain NS3 protease inhibition was evaluated using the chromogenic assay previously described (45). The assays were performed at 30°C in a 96-well microtiter plate.…”

Section: Methodsmentioning

confidence: 99%

SCH 503034, a Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease, Suppresses Polyprotein Maturation and Enhances the Antiviral Activity of Alpha Interferon in Replicon Cells

Malcolm

Liu

Lahser

et al. 2006

Antimicrob Agents Chemother

293

189

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). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored by immunofluorescence and real-time PCR analysis. Continuous exposure of repliconbearing cell lines to six times the 90% effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.

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“…Kinetic Studies-Kinetic constants of peptide substrates, APP, and KL-1, were obtained by using the HPLC assay described above and analysis as reported previously (8,41,42). In brief, ADAM33cat (80 nM) was incubated with peptide substrate (30 -2000 M) in assay buffer for 10 -60 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%