Resistance Profile of a Hepatitis C Virus RNA-Dependent RNA Polymerase Benzothiadiazine Inhibitor

Nguyen, Tammy T.; Gates, Adam; Gutshall, Lester L.; Johnston, Victor K.; Gu, Baohua; Duffy, Kevin J.; Sarisky, Robert T.

doi:10.1128/aac.47.11.3525-3530.2003

Cited by 91 publications

(94 citation statements)

References 18 publications

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“…After treatment with concentrations approximating the IC 90 of each compound in combination, only a small number of resistant replicon colonies was obtained, possibly a reflection of the increased complexity of selecting replicon variants bearing multiple resistance mutations and/or of their impaired replication capability (Table 5). Our data showed that dual resistance to thumb I-and to palm-site inhibitors is mediated by mutations Met423Thr and/or Ile482Leu in the thumb domain and mutations in the palm and finger domains (related to palm-site inhibitors), in agreement with previous reports (30,33,40).…”

Section: Discussionsupporting

confidence: 93%

“…Resistant replicons were selected using methodologies described previously (33,41). For the selection of dual resistance 1.5 ϫ 10 5 replicon cells were plated in 6-cm dishes in the presence of 0.5 mg/ml of G418 and in the absence or presence of inhibitors NNI-1 and NNI-3 in a checkerboard fashion at concentrations of 1ϫ, 5ϫ, or 10ϫ the IC 50 .…”

Section: Methodsmentioning

confidence: 99%

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Selection and Characterization of Replicon Variants Dually Resistant to Thumb- and Palm-Binding Nonnucleoside Polymerase Inhibitors of the Hepatitis C Virus

Pogam

Kang

Harris

et al. 2006

J Virol

130

117

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Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV)polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb-and a palmbinding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.Hepatitis C virus (HCV), a positive-strand RNA virus, is a member of the genus Hepacivirus in the Flaviviridae family and is the leading cause of liver disease worldwide. It is estimated that over 170 million individuals are infected with HCV (43). The current standard of care provides good clinical efficacy for patients infected with genotype 2 and 3 but is less efficacious for patients infected with the most prevalent genotype, genotype 1, thereby emphasizing the urgent need for more effective HCV-specific antiviral therapies (15,27).The HCV RNA-dependent RNA polymerase is an essential enzyme for viral RNA replication and represents an attractive therapeutic target. HCV polymerase has the "right-hand" polymerase fold with finger, thumb, and palm domains (22). As with other RNA-dependent RNA polymerases, the extended "fingertips" contact a thicker thumb domain to create an encircled active site constituting the closed, active conformation of the enzyme (7,16,22,32). With the advent of the HCV replicon system there have been extensive developments supporting the discovery of new HCV polymerase nonnucleoside inhibitors (1-3, 5, 6, 11, 36). Several chemical classes of nonnucleoside inhibitors that inhibit the isolated enzyme and replication in the replicon system have been shown to bind at distinct sites on HCV polymerase. These polymerase inhibitors include benzothiadiazines, binding to the palm domain near the active site (38, 40), thiophene carboxylic acids which bind at the...

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Section: Discussionsupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Selection and Characterization of Replicon Variants Dually Resistant to Thumb- and Palm-Binding Nonnucleoside Polymerase Inhibitors of the Hepatitis C Virus

Pogam

Kang

Harris

et al. 2006

J Virol

130

117

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“…In this study, we demonstrate that the major in vitro resistant mutations against BILN 2061 remain fully susceptible to VX-950, and the dominant VX-950 resistance mutation is still sensitive to BILN 2061. In vitro resistance mutations against the HCV polymerase inhibitors have also been identified in the replicon system (39,40). These studies suggest that future hepatitis C therapy involving small molecule inhibitors of HCV enzymes might require multidrug combination, as in the case of the current HIV treatments.…”

Section: Discussionmentioning

confidence: 95%

In Vitro Resistance Studies of Hepatitis C Virus Serine Protease Inhibitors, VX-950 and BILN 2061

Lin

Luong

et al. 2004

Journal of Biological Chemistry

289

298

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We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3⅐4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3⅐4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3⅐4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. It is estimated that 170 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) 1 (1). The majority of acute HCV infections become chronic, some of which progress toward liver cirrhosis or hepatocellular carcinoma (2, 3). The current standard of care is pegylated interferon ␣ in combination with ribavirin, which has a sustained viral response rate of 40 -50% in genotype 1 HCV-infected patients, which accounts for the majority of the hepatitis C population in the United States and Japan, and of 80 -90% in patients infected with genotype 2 or 3 HCV (4, 5) (for a review, see Ref. 6). Thus, more effective therapeutic drugs with fewer side effects and shorter treatment durations are needed for patients infected with HCV.HCV is an enveloped, single-stranded RNA virus with a 9.6-kb positive-polarity genome, which encodes a polyprotein precursor of about 3,000 amino acids. The HCV polyprotein is proteolytically processed by cellular and HCV proteases into at least 10 distinct products, in the order of NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (for a review, see Ref. 7). NS3 serine protease and helicase as well as NS5B RNA-dependent RNA polymerase are believed to be components of a replication complex responsible for viral RNA replication and have been shown to be essential for the HCV replication in chimpanzees (8). These HCV enzymes have been the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 9). However, successful discovery of a new HCV-specific drug candidate has been hampered by the lack of a robust, reproducible infectious virus cell culture system. The development of a HCV replicon system by Lohmann et al. (10) and subsequent optimization by several laboratories (11, 12) has enabled quantitative evaluation of the antiviral potency of HCV inhibitors.The HCV NS3⅐4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (13-17). It has been shown that the central region (amino acids 21-30) of the 54-residue NS4A protein is essentia...

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“…The benzothiadiazine series, including NNI-1, was reported to select for major resistance mutations at amino acid positions Met 414 and Tyr 448 in the palm domain, which lies near the enzyme active site (35,36). Mapping of replicon cells resistant to HCV-796 identified specific mutations in NS5B involving changes at residues Cys 316 and Ser 365 at the palm binding site (8,24).…”

Section: Correlation Of Binding Affinity and Inhibition Potency Of Nomentioning

confidence: 99%

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

Hang

Yang

Harris

et al. 2009

Journal of Biological Chemistry

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Hepatitis C virus (HCV)4 constitutes a global health problem. Current therapies are unable to effectively eliminate viral infection in a significant number of patients. The RNA-dependent RNA polymerase (RdRp) of HCV NS5B is an attractive target for the development of orally bioavailable small molecule inhibitors (1, 2). The structure of the NS5B apoenzyme and the NS5B-RNA complex reveals the characteristic right hand architecture of polymerase enzymes, comprising three distinct domains (palm, thumb, and finger) encircling the enzyme active site located in the palm domain (3-6). The structural and biochemical characterization of HCV NS5B polymerase can provide a basis for drug design efforts, and the elucidation of the mechanism of inhibition can guide the optimization of inhibitor efficiency against wild-type and resistant mutants.Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction. The benzofuran compound HCV-796 has been shown to have significant antiviral effects in patients chronically infected with HCV (9, 10). In addition, two series of compounds based on the thiophene and benzimidazole scaffolds have been reported to inhibit NS5B by binding to two different binding pockets in the thumb domain of NS5B (11,12). The thumb domain is connected to the palm domain by a ␤-hairpin termed the primer grip motif. The C-terminal region of the thumb protrudes toward the active site (3). The thumb binding inhibitors have been proposed to inhibit the RdRp activity of NS5B, perhaps by interfering with template/primer interaction and conformational dynamics of the protein (13,14).Despite the elucidation of a number of NNIs that bind to the thumb and palm binding sites, the mechanism by which NNIs cause inhibition of RNA synthesis is unclear. Also, our understanding of the kinetics of NNI interaction with NS5B, the role of NNI binding and kinetics for inhibition, and the inhibitor efficacy on NS5B-resistant mutations remains incomplete. The four representative palm-and thumb-binding NNIs selected in this study have been reported to effectively inhibit replication of subgenomic replicons with low toxicity. Noncompetitive inhibition of NS5B polymerase activity with respect to NTPs has been reported (2, 15, 16). Based on co-crystallization studies with NS5B, it has been proposed that allosteric inhibitors may lock the NS5B protein in an inactive formation by binding tightly to the protein (16,17). It is important to understand how the binding affinity relates to inhibition potency and resistance to HCV inhibition. Because the intrinsic potency of slowly binding compounds can be underestimated in the short time □ S The on-line version of this article (available at htt...

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Resistance Profile of a Hepatitis C Virus RNA-Dependent RNA Polymerase Benzothiadiazine Inhibitor

Cited by 91 publications

References 18 publications

Selection and Characterization of Replicon Variants Dually Resistant to Thumb- and Palm-Binding Nonnucleoside Polymerase Inhibitors of the Hepatitis C Virus

Selection and Characterization of Replicon Variants Dually Resistant to Thumb- and Palm-Binding Nonnucleoside Polymerase Inhibitors of the Hepatitis C Virus

In Vitro Resistance Studies of Hepatitis C Virus Serine Protease Inhibitors, VX-950 and BILN 2061

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

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