Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA

Lam, Angela M.; Espiritu, Christine; Bansal, Shalini; Steuer, Holly M. Micolochick; Zennou, Véronique; Otto, Michael; Furman, Phillip A.

doi:10.1128/jvi.05639-11

Cited by 51 publications

(43 citation statements)

References 30 publications

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“…This loop reordering may be critical to the transition from de novo initiation with GTP to the elongation of the growing primer-template RNA. Intriguingly, this same loop reordering was observed in the 2.5-Å structure of an apo 2a JFH1 isolate HCV NS5B resistance-derived triple mutant (S15G/C223H/V321I) (19) in which the ␤-hairpin loop is extant (unpublished data). The comparison of the apo 2a ⌬8 structure to other RdRp ternary complexes (12,15,44) suggested that in this open conformation, HCV NS5B is able to bind primer-template RNA.…”

Section: Resultsmentioning

confidence: 69%

Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

Mosley¹,

Edwards²,

Murakami³

et al. 2012

J Virol

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108

117

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bThe replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory ␤-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory ␤-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

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Section: Resultsmentioning

confidence: 69%

Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

Mosley¹,

Edwards²,

Murakami³

et al. 2012

J Virol

Self Cite

108

117

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“…Subtype-dependent resistance has previously been reported for the NS3 protease and nonnucleoside NS5B inhibitors (27). Recently, we reported that nucleotide prodrugs of 2=-F-2=-C-methylguanosine monophosphate, PSI-352938 and PSI-353661, were able to select for resistant mutations in the JFH-1-derived replicons but not in GT 1a and 1b replicons due to differences in fitness caused by the amino acid changes (15).…”

Section: Genotype Coveragementioning

confidence: 99%

“…Among the various NS5B mutations, we included representative amino acid changes (C316Y, M414T, M423T, P495L) that conferred resistance to the four main classes of NNIs (12), the S96T/N142T substitutions that conferred resistance to the 4=-azidocytidine nucleoside R1479 (1), the S282T substitution which conferred resistance to 2=-C-methylnucleosides/tides (29) and 2=-F-2=-C-methylpyrimidines (18,40), the S15G/C223H/V321I (JFH-1 GT 2a) changes that in combination conferred resistance to 2=-F-2=-C-methylguanosine analogs (15), and the F415Y (GT 1a) alteration that has previously been found in patients treated with ribavirin (47). As summarized in Table 2, the replicons with the C316Y, M414T, M423T, and P495L changes remained fully susceptible to the inhibition of PSI-7977, while showing a 26-to 70-fold reduction in sensitivity for the corresponding class of NNIs.…”

Section: Genotype Coveragementioning

confidence: 99%

Genotype and Subtype Profiling of PSI-7977 as a Nucleotide Inhibitor of Hepatitis C Virus

Lam¹,

Espiritu²,

Bansal³

et al. 2012

Antimicrob Agents Chemother

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222

186

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PSI-7977, a prodrug of 2=-F-2=-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC 50 ) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons. Hepatitis C virus (HCV) currently infects more than 170 million people worldwide and is the leading cause of chronic liver disease and liver transplantation in the United States. HCV is a single plus-strand RNA virus about 9.6 kb in genome size and contains one open reading frame that encodes 10 structural and nonstructural (NS) proteins. The structural proteins (core, E1, and E2), which constitute the viral capsid and envelope proteins, are located at the amino terminus, followed by p7, which oligomerizes to form an ion channel critical for viral assembly, and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which are responsible for viral replication (35). Recently, two antiviral agents have been approved, in combination with pegylated alpha interferon and ribavirin (Peg-IFN/RBV), to treat patients infected with genotype (GT) 1 HCV. Both of these compounds, boceprevir (Victrelis) and telaprevir (Incivek), target the NS3/4A protease and inhibit HCV replication by blocking processing of NS3, NS4A/B, and NS5A/B (25, 33). These treatments showed improvement over Peg-IFN/RBV; however, patients may develop additional side effects corresponding to each of these antiviral compounds (4). Furthermore, viral resistance against these compounds has emerged both in vitro and in vivo as a result of the high genetic diversity of HCV and error-prone nature of the HCV NS5B RNA-dependent RNA polymerase (12). Therefore, research...

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“…In addition, variants NS5B S96T and N142T appear to be compensatory mutations for S282T in the replicon (39). The passaging of the 2=-methylguanosine inhibitor PSI-352938 did not readily select for variants in a genotype 1 replicon system; however, variants NS5B C223H/Y and V321I were selected after multiple passages in a genotype 2a replicon (J6/JFH-1), conveyed a 2.1-to 3.7-fold-decreased susceptibility, and are included in this analysis, but resistance Ͼ2-fold in genotype 1b was only conveyed by the combination of variants which had a significant impact on viral fitness (40). The NS5B S96T, N142T, C223H/Y, and S282T variants were not observed as the dominant viral population in any patients in the present study, and the NS5B V321I variant was observed in 0.19 and 2.51% of subtype 1a and 1b patients, respectively (Table 3).…”

Section: Baselinementioning

confidence: 99%

Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment

Bartels

Sullivan

Zhang

et al. 2013

J Virol

135

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The prevalence of naturally occurring hepatitis C virus (HCV) variants that are less sensitive to direct-acting antiviral (DAA) inhibitors has not been fully characterized. We used population sequence analysis to assess the frequency of such variants in plasma samples from 3,447 DAA-naive patients with genotype 1 HCV. In general, HCV variants with lower-level resistance (3-to 25-fold increased 50% inhibitor concentration [IC 50 ]) to telaprevir were observed as the dominant species in 0 to 3% of patients, depending on the specific variant, whereas higher-level resistant variants (>25-fold-increased IC 50 ) were not observed. Specific variants resistant to NS5A inhibitors were predominant in up to 6% of patients. Most variants resistant to nucleo(s/t)ide activesite NS5B polymerase inhibitors were not observed, whereas variants resistant to non-nucleoside allosteric inhibitors were observed in up to 18% of patients. The presence of DAA-resistant variants in NS5A, NS5B, or NS3 (including telaprevir-resistant variants), in baseline samples of treatment-naive patients receiving a telaprevir-based regimen in phase 3 studies did not affect the sustained viral response (SVR). Treatment-naive patients with viral populations containing the telaprevir-resistant variants NS3 V36M, T54S, or R155K at baseline achieved a 74% SVR rate, whereas patients with no resistant variants detected prior to treatment achieved a 76% SVR rate. The effect of specific resistant variant frequency on response to various DAA treatments in different patient populations, including interferon nonresponders, should be further studied.

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Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA

Cited by 51 publications

References 30 publications

Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

Genotype and Subtype Profiling of PSI-7977 as a Nucleotide Inhibitor of Hepatitis C Virus

Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment

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