Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Mo, Hongmei; Lu, Liangjun; Pilot‐Matias, Tami; Pithawalla, Ron; Mondal, Rubina; Masse, Sherie; Dekhtyar, Tatyana; Ng, Teresa I.; Koev, Gennadiy; Stoll, V.S.; Stewart, Kent D.; Pratt, John K.; Donner, Pam; Rockway, Todd W.; Maring, Clarence J.; Molla, Akhteruzzaman

doi:10.1128/aac.49.10.4305-4314.2005

Cited by 121 publications

(124 citation statements)

References 55 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: 82%

“…In the presence of NNI-3 alone, substitutions Asn411Ser, Met414Thr, and Ser556Asn were selected. Mutations at residues 411 and 414 after prolonged treatment of replicon cells with benzothiadiazine derivatives have been previously described (30,40). We also observed that mutation Ser556Asn confers resistance to NNI-3 (S. Le Pogam, H. Kang, and I. Najera, unpublished results).…”

Section: Resultssupporting

confidence: 57%

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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

129

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: 82%

Section: Resultssupporting

confidence: 57%

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

129

117

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“…The S282T mutation is the only substitution reported to confer resistance to the nucleoside MK-608 and has significantly impaired fitness (11,29); accordingly, this compound showed the greatest barrier to resistance for any of the direct acting antivirals we tested. In contrast, multiple mutations can be selected by both A-782759 (H95Q, N411S, Y448H, and M414L/T) and HCV-796 (C316S/F/Y/N, S365A/T, L392F, and M414I/T/V), and these mutations range in fitness from significantly impaired to highly fit (17,18,23,29). Consistent with this knowledge, the resistance barrier curves for these nonnucleoside inhibitors demonstrate significant numbers of resistant foci persisting at high multiples of EC 50 .…”

Section: Discussionmentioning

confidence: 99%

“…Despite different mechanisms of action, these molecules have similar EC 50 values (271 and 378 nM, for A-782759 and CsA, respectively, in 51C-RFP-1a cells). A-782759 selects highly fit mutations at NS5B residue M414 that confer a large decrease in drug susceptibility (17,18). In contrast, a single HCV mutation that confers a more than fivefold shift in potency has not been reported despite a large body of literature on CsA (19)(20)(21).…”

Section: Quantification Of Relative Frequencies Of Preexisting Drug-rmentioning

confidence: 99%

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

Robinson

Yang

Delaney

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Clinical trials of direct-acting antiviral agents in patients chronically infected with hepatitis C virus (HCV) have demonstrated that viral resistance is detected rapidly during monotherapy. In patients, HCV does not exist as a single, genetically homogenous virus but rather as a population of variants termed "quasispecies." Preexisting variants resistant to specific antiviral drugs, overlooked in traditional hit-to-lead discovery efforts, may be responsible for these poor clinical outcomes. To enable real-time studies of resistance emergence in live cells, we established fluorescent protein-labeled HCV replicon cell lines. We validated these cell lines by demonstrating that antiviral susceptibility and the selection of signature resistance mutations for various drug classes are similar to traditional replicon cell lines. By quantifying the kinetics and uniformity of replication within colonies of drug-resistant fluorescent replicon cells, we showed that resistance emerged from a single cell and preexisted in a treatment-naive replicon population. Within this population, we determined the relative frequency of preexisting replicons capable of establishing foci during treatment with distinct antivirals. By measuring relative frequency as a function of dose, we quantitatively ranked distinct antiviral molecules on the basis of their distinct barriers to resistance. These insights into RNA virus quasispecies structure provide guidance for selecting clinical drug concentrations and selecting antiviral drug combinations most likely to suppress resistance.evolution | virology R esistance to antiviral drugs is a significant worldwide health problem. Although we typically discuss drug resistance in language suggesting that resistance "emerges" after administration of an antiviral drug, classical microbial genetics suggests that drug-resistant mutants preexist in viral populations. The Luria and Delbruck experiment (1) provided the first evidence that drug-resistance mutations could preexist. In infected patients, hepatitis C virus (HCV), a positive-strand RNA virus, is composed of a swarm of genetically heterogeneous variants termed "quasispecies." A central question is whether this diversity is so pronounced that drug-resistance mutations preexist in a drugnaïve HCV population. Despite the importance of drug resistance in driving therapeutic outcomes, identification of rare variants within polymorphic virus populations has been fundamentally difficult in vitro and in vivo.Although many new classes of direct-acting anti-HCV drugs are in clinical development, viral resistance to these agents generally is detected within a few days of monotherapy (2, 3). Further supporting the model of preexisting resistance, ultradeep sequencing experiments identified mutations, before drug exposure, that confer resistance to NS3 protease inhibitors (4, 5). Although drugdiscovery campaigns traditionally have prioritized leads and lead classes on the basis of potency and toxicity, quantitative comparisons of the frequency of drug-resista...

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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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Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Cited by 121 publications

References 55 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

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

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

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