Discovery of a Potent and Selective Noncovalent Linear Inhibitor of the Hepatitis C Virus NS3 Protease (BI 201335)

Llinàs‐Brunet, Montse; Bailey, Murray; Goudreau, Nathalie; Bhardwaj, Punit Kumar; Bordeleau, Josée; Bös, Michael; Bousquet, Yves; Cordingley, Michael G.; Duan, Jiamin; Forgione, Pat; Garneau, Michel; Ghiro, Élise; Gorys, Vida; Goulet, Sylvie; Halmos, Ted; Kawai, Stephen H.; Naud, Julie; Poupart, Marc‐André; White, Peter W.

doi:10.1021/jm100690x

Cited by 87 publications

(46 citation statements)

References 32 publications

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“…Similarly, this signal was not detected using an NS4A-NS3 tethered protein, even though the catalytic triad was shown to be properly aligned in the presence of the NS4A peptide (33). Consistent with these results, we were also unable to detect a specific signal for the His-57 imidazole NH protons in a 1 H jump-return spectrum recorded in the free state using a uniformly 15 N-labeled NS4A peptide-NS3 protease-tethered protein ( 15 N-sc-protease) (Fig. 4a, bottom).…”

Section: Resultssupporting

confidence: 82%

“…4a, bottom). In contrast, binding of BI 201335 to 15 N-sc-protease led to the observation of two distinct signals in the 1 H spectrum, at 17.2 ppm (singlet) and 13.5 ppm (doublet, J ϭ 95.6 Hz) (Fig. 4a, top).…”

Section: Resultsmentioning

confidence: 98%

“…Inhibitors, Peptides, and Proteins-BI 201335 was synthesized as described (15), and analog 1 was synthesized by similar procedures. The HCV genotype 1b Con1 strain NS3-NS4A protein (FL-protease), the HCV genotype 1b J strain NS3 protease domain, and the same domain with an NS4A peptide fused to its N terminus (sc-protease) were expressed and purified as described previously (16).…”

Section: Methodsmentioning

confidence: 99%

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Combined X-ray, NMR, and Kinetic Analyses Reveal Uncommon Binding Characteristics of the Hepatitis C Virus NS3-NS4A Protease Inhibitor BI 201335

Lemke

Goudreau

Zhao

et al. 2011

Journal of Biological Chemistry

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Hepatitis C virus infection, a major cause of liver disease worldwide, is curable, but currently approved therapies have suboptimal efficacy. Supplementing these therapies with directacting antiviral agents has the potential to considerably improve treatment prospects for hepatitis C virus-infected patients. The critical role played by the viral NS3 protease makes it an attractive target, and despite its shallow, solvent-exposed active site, several potent NS3 protease inhibitors are currently in the clinic. BI 201335, which is progressing through Phase IIb trials, contains a unique C-terminal carboxylic acid that binds noncovalently to the active site and a bromo-quinoline substitution on its proline residue that provides significant potency. In this work we have used stopped flow kinetics, x-ray crystallography, and NMR to characterize these distinctive features. Key findings include: slow association and dissociation rates within a singlestep binding mechanism; the critical involvement of water molecules in acid binding; and protein side chain rearrangements, a bromine-oxygen halogen bond, and profound pK a changes within the catalytic triad associated with binding of the bromoquinoline moiety.Worldwide, 130 -170 million people are chronically infected with hepatitis C virus (HCV) 2 (1). Infection can progress to cirrhosis, which can lead to hepatic decompensation or hepatocellular carcinoma. HCV infection can be cured by administering a combination of pegylated interferon and the nonspecific antiviral drug ribavirin for 24 -48 weeks. Unfortunately, this treatment is only 40 -50% successful in clearing genotype 1 infections, the most common type in most regions of the world (2). Clearly there is considerable need for more efficacious therapies.HCV is a small, positive-strand RNA virus with a 9600-nucleotide genome encoding a 3000-amino acid polyprotein that is subsequently processed into individual proteins by cellular and viral proteases. Four of the five cleavage sites between the NS (nonstructural) proteins are cleaved by the viral NS3 serine protease (EC 3.4.21.98). HCV NS3 is a bifunctional protein comprised of an N-terminal 180-amino acid serine protease domain and a C-terminal 420-amino acid helicase domain (3). The central portion of the 54-amino acid NS4A protein is structurally integrated into the NS3 protease domain (4) and is necessary for full protease activity (5).The search for specific, direct-acting, small molecule inhibitors of HCV replication began shortly after the virus was discovered in 1989 (6); the fact that no such drug has yet made it to the market attests to the challenging nature of this research. Throughout this period, the NS3-NS4A protease has remained a major focus of HCV drug discovery efforts. Beginning with the protease inhibitor BILN 2061 in 2002 (7, 8), a number of specific antivirals have been tested in human trials and have shown very promising results (9). The most advanced compounds in the clinic are NS3-NS4A protease inhibitors, with two, telaprevir and boceprevir, cu...

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

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Combined X-ray, NMR, and Kinetic Analyses Reveal Uncommon Binding Characteristics of the Hepatitis C Virus NS3-NS4A Protease Inhibitor BI 201335

Lemke

Goudreau

Zhao

et al. 2011

Journal of Biological Chemistry

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“…To this end, we developed a high-throughput amplicon pyrosequencing strategy, which we utilised to monitor the evolutionary stability of the viral NS3 protease coding-region in two chronically infected patients over a 10 year period. In addition, both patients failed to achieve viral clearance after therapy with NS3 PI Faldeprevir (Llinas-Brunet et al, 2010) in combination with PEG-IFNα/RBV. Consequently, characterised RAV frequencies pre-and post-therapy were examined, to determine whether undectable baseline RAVs contributed to treatment failure, and assess the extent of long-term RAV persistence post-DAA therapy failure.…”

Section: Introductionmentioning

confidence: 97%

Development of a high-throughput pyrosequencing assay for monitoring temporal evolution and resistance associated variant emergence in the Hepatitis C virus protease coding-region

Irving¹,

Rupp²,

McClure³

et al. 2014

Antiviral Research

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A new generation of drugs targeting the non-structural (NS) proteins of the Hepatitis C virus (HCV) will substantially increase treatment success rates, reducing global infections. Amongst the NS proteins, the NS3 protease represents an important drug target, responsible for liberation of mature NS proteins from the nascent HCV polyprotein and suppression of host innate immunity. Despite this, the evolutionary stability of the genomic locus encoding the NS3 protease is poorly characterized in chronic HCV infection. To address this shortfall, we developed a high-throughput amplicon pyrosequencing protocol and utilised it to monitor NS3 protease coding-sequence evolution for over a decade in two patients. Although patient-specific evolutionary trends were apparent, the protease amino acid population consensus remained stable with a massive excess of synonymous mutations observed, confirming this locus is under strong purifying selection during chronic infection within individual patients. No evidence for continuous immune escape was detected. Additionally, both patients failed protease inhibitor (PI) therapy and protease sequence diversity pre-and post-therapy were also assessed. No baseline resistance associated variants (RAVs) contributed to treatment failure. Significant reductions in viral diversity were observed post-PI therapy, indicating a population bottleneck occurred. The genetic vestiges of this bottleneck were still detectable 18 months after therapy discontinuation. Although significant enrichment of the Q80L mutation was observed in one patient, genetic and phenotypic data reveal no detectable RAV persistence post-therapy failure. Together this investigation provides a sensitive and reproducible high-throughput framework to interrogate viral sequence diversity at high-resolution, with potential applications for routine monitoring of treatment regimens. This study also reveals novel insights into the evolutionary processes that shape NS3 sequence divergence in both chronic HCV infection and post PI-therapy failure.3

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“…Faldaprevir (BI 201335) is a potent HCV NS3/4A PI administered once daily [10][11][12]. Four phase 2 studies evaluated the efficacy and safety of faldaprevir with PegIFN alfa-2a plus RBV [13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

STARTVerso1: A randomized trial of faldaprevir plus pegylated interferon/ribavirin for chronic HCV genotype-1 infection

Ferenci

Asselah

Foster

et al. 2015

Journal of Hepatology

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Background & Aims:The efficacy and tolerability of faldaprevir, a potent hepatitis C virus (HCV) NS3/4A protease inhibitor, plus peginterferon (PegIFN) and ribavirin (RBV) was assessed in a double-blind, placebo-controlled phase 3 study of treatment-naïve patients with HCV genotype-1 infection. Methods: Patients were randomly assigned (1:2:2) to PegIFN/ RBV plus: placebo (arm 1, n = 132) for 24 weeks; faldaprevir (120 mg, once daily) for 12 or 24 weeks (arm 2, n = 259); or faldaprevir (240 mg, once daily) for 12 weeks (arm 3, n = 261). In arms 2 and 3, patients with early treatment success (HCV-RNA <25 IU/ml at week 4 and undetectable at week 8) stopped all treatment at week 24. Other patients received PegIFN/RBV until week 48 unless they met futility criteria. The primary endpoint was sustained virologic response 12 weeks post-treatment (SVR12). Results: SVR12 was achieved by 52%, 79%, and 80% of patients in arms 1, 2, and 3, respectively (estimated difference for arms 2 and 3 vs. arm 1: 27%, 95% confidence interval 17%-36%; and 29%, 95% confidence interval, 19%-38%, respectively; p <0.0001 for both). Early treatment success was achieved by 87% (arm 2) and 89% (arm 3) of patients, of whom 86% and 89% achieved SVR12. Adverse event rates were similar among groups; few adverse events led to discontinuation of all regimen components. Conclusions: Faldaprevir plus PegIFN/RBV significantly increased SVR12, compared with PegIFN/RBV, in treatment-naïve patients with HCV genotype-1 infection. No differences were seen in responses of patients given faldaprevir once daily at 120 or 240 mg. Ó

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Discovery of a Potent and Selective Noncovalent Linear Inhibitor of the Hepatitis C Virus NS3 Protease (BI 201335)

Cited by 87 publications

References 32 publications

Combined X-ray, NMR, and Kinetic Analyses Reveal Uncommon Binding Characteristics of the Hepatitis C Virus NS3-NS4A Protease Inhibitor BI 201335

Combined X-ray, NMR, and Kinetic Analyses Reveal Uncommon Binding Characteristics of the Hepatitis C Virus NS3-NS4A Protease Inhibitor BI 201335

Development of a high-throughput pyrosequencing assay for monitoring temporal evolution and resistance associated variant emergence in the Hepatitis C virus protease coding-region

STARTVerso1: A randomized trial of faldaprevir plus pegylated interferon/ribavirin for chronic HCV genotype-1 infection

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