Sonal Rajyaguru

The Hepatitis C Virus Replicon Presents a Higher Barrier to Resistance to Nucleoside Analogs than to Nonnucleoside Polymerase or Protease Inhibitors

ABSTRACT Specific inhibitors of hepatitis C virus (HCV) replication that target the NS3/4A protease (e.g., VX-950) or the NS5B polymerase (e.g., R1479/R1626, PSI-6130/R7128, NM107/NM283, and HCV-796) have advanced into clinical development. Treatment of patients with VX-950 or HCV-796 rapidly selected for drug-resistant variants after a 14-day monotherapy treatment period. However, no viral resistance was identified after monotherapy with R1626 (prodrug of R1479) or NM283 (prodrug of NM107) after 14 days of monotherapy. Based upon the rapid selection of resistance to the protease and nonnucleoside inhibitors during clinical trials and the lack of selection of resistance to the nucleoside inhibitors, we used the replicon system to determine whether nucleoside inhibitors demonstrate a higher genetic barrier to resistance than protease and nonnucleoside inhibitors. Treatment of replicon cells with nucleoside inhibitors at 10 and 15 times the 50% effective concentration resulted in clearance of the replicon, while treatment with a nonnucleoside or protease inhibitor selected resistant colonies. In combination, the presence of a nucleoside inhibitor reduced the frequency of colonies resistant to the other classes of inhibitors. These results indicate that the HCV replicon presents a higher barrier to the selection of resistance to nucleoside inhibitors than to nonnucleoside or protease inhibitors. Furthermore, the combination of a nonnucleoside or protease inhibitor with a nucleoside polymerase inhibitor could have a clear clinical benefit through the delay of resistance emergence.

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

ABSTRACT 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 palm-binding 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.

GT-1a or GT-1b Subtype-Specific Resistance Profiles for Hepatitis C Virus Inhibitors Telaprevir and HCV-796

ABSTRACT In vitro, telaprevir selects subtype-specific resistance pathways for hepatitis C virus GT-1a and GT-1b, as described to have occurred in patients. In GT-1a, the HCV-796 resistance mutation C316Y has low replication capacity (7%) that can be compensated for by the emergence of the mutation L392F or M414T, resulting in an increase in replication levels of ≥10-fold.

2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2′-α-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of α-hydroxy moieties. 2′-Deoxy-2′-β-fluoro-4′-azidocytidine (RO-0622) and 2′-deoxy-2′-β-hydroxy-4′-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC50 = 171 ± 12 nm and 24 ± 3 nm for RO-9187 and RO-0622, respectively; CC50 >1 mm for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4′-azidocytidine) or 2′-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC50 values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2′-α-deoxy-4′-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.

Slow binding inhibition and mechanism of resistance of non-nucleoside polymerase inhibitors of hepatitis C virus.

The binding affinity of four palm and thumb site representative non-nucleoside inhibitors (NNIs) of HCV polymerase NS5B to wild-type and resistant NS5B polymerase proteins was determined, and the influence of RNA binding on NNI binding affinity was investigated. NNIs with high binding affinity potently inhibited HCV RNA polymerase activity and replicon replication. Among the compounds tested, HCV-796 showed slow binding kinetics to NS5B. The binding affinity of HCV-796 to NS5B increased 27-fold over a 3-h incubation period with an equilibrium Kd of 71 ± 2 nm. Slow binding kinetics of HCV-796 was driven by slow dissociation from NS5B with a koff of 4.9 ± 0.5 × 10−4 s−1. NS5B bound a long, 378-nucleotide HCV RNA oligonucleotide with high affinity (Kd = 6.9 ± 0.3 nm), whereas the binding affinity was significantly lower for a short, 21-nucleotide RNA (Kd = 155.1 ± 16.2 nm). The formation of the NS5B-HCV RNA complex did not affect the slow binding kinetics profile and only slightly reduced NS5B binding affinity of HCV-796. The magnitude of reduction of NNI binding affinity for the NS5B proteins with various resistance mutations in the palm and thumb binding sites correlated well with resistance -fold shifts in NS5B polymerase activity and replicon assays. Co-crystal structures of NS5B-Con1 and NS5B-BK with HCV-796 revealed a deep hydrophobic binding pocket at the palm region of NS5B. HCV-796 interaction with the induced binding pocket on NS5B is consistent with slow binding kinetics and loss of binding affinity with mutations at amino acid position 316.

2'-deoxy-4'-azido nucleoside analogs are highly potent inhibitors of HCV replication despite the lack of 2'-alpha hydroxyl groups

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2′-α-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of α-hydroxy moieties. 2′-Deoxy-2′-β-fluoro-4′-azidocytidine (RO-0622) and 2′-deoxy-2′-β-hydroxy-4′-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC50 = 171 ± 12 nm and 24 ± 3 nm for RO-9187 and RO-0622, respectively; CC50 >1 mm for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4′-azidocytidine) or 2′-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC50 values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2′-α-deoxy-4′-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.

Modulation of alpha interferon anti-hepatitis C virus activity by ISG15

ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-alpha) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-alpha, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-alpha-specific effect, which was not observed with IFN-gamma, correlated with an increase in expression of the IFN-alpha-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-alpha-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-alpha after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-alpha-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-alpha treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-alpha pathway and its antiviral effect.

Cyclic amide bioisosterism: strategic application to the design and synthesis of HCV NS5B polymerase inhibitors.

Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.

3-Hydroxyisoquinolines as inhibitors of HCV NS5b RNA-dependent RNA polymerase.

Isoquinoline-based non-nucleoside inhibitors of HCV NS5b RNA-dependent RNA-polymerase are described. The synthesis and structure-activity relationships are detailed, along with enzyme and cellular activity.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 2: Synthesis and structure―activity relationships of benzothiazine-substituted quinolinediones

A new series of benzothiazine-substituted quinolinediones were evaluated as inhibitors of HCV polymerase NS5B. SAR studies on this series revealed a methyl sulfonamide group as a high affinity feature. Analogues with this group showed submicromolar potencies in the HCV cell based replicon assay. Pharmacokinetic and toxicology studies were also performed on a selected compound (34) to evaluate in vivo properties of this new class of inhibitors of HCV NS5B polymerase.