A quantum mechanics/molecular mechanics study of the reaction mechanism of the hepatitis C virus NS3 protease with the NS5A/5B substrate

ABSTRACTAsunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensivein vitrogenotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

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Phenylglycine as a novel P2 scaffold in hepatitis C virus NS3 protease inhibitors

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2006.11.003 ◽

2007 ◽

Vol 15 (3) ◽

pp. 1448-1474 ◽

Cited By ~ 34

Author(s):

Pernilla Örtqvist ◽

Shane D. Peterson ◽

Eva Åkerblom ◽

Thomas Gossas ◽

Yogesh A. Sabnis ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitors ◽

Ns3 Protease Inhibitors ◽

Ns3 Protease

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Inhibition and Binding Kinetics of the Hepatitis C Virus NS3 Protease Inhibitor ITMN-191 Reveals Tight Binding and Slow Dissociative Behavior

Biochemistry ◽

10.1021/bi900038p ◽

2009 ◽

Vol 48 (11) ◽

pp. 2559-2568 ◽

Cited By ~ 47

Author(s):

Ravi Rajagopalan ◽

Shawn Misialek ◽

Sarah K. Stevens ◽

David G. Myszka ◽

Barbara J. Brandhuber ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitor ◽

Tight Binding ◽

Binding Kinetics ◽

Ns3 Protease ◽

Kinetics Of

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Identification of Hepatitis C Virus NS5A Inhibitors

Journal of Virology ◽

10.1128/jvi.01360-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 482-491 ◽

Cited By ~ 137

Author(s):

Julie A. Lemm ◽

Donald O'Boyle ◽

Mengping Liu ◽

Peter T. Nower ◽

Richard Colonno ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Therapeutic Index ◽

Dna Viruses ◽

Genotype 1A ◽

A Cell ◽

Ns3 Protease ◽

Derivatives Of ◽

Rna And Dna

ABSTRACT Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ∼5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.

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