Chunfu Wang

Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-α and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC50) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log10 reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.

Resistance Analysis of the Hepatitis C Virus NS5A Inhibitor BMS-790052 in an In Vitro Replicon System

ABSTRACT BMS-790052 is the most potent hepatitis C virus (HCV) inhibitor reported to date, with 50% effective concentrations (EC50s) of ≤50 pM against genotype 1 replicons. This exceptional potency translated to rapid viral load declines in a phase I clinical study. By targeting NS5A, BMS-790052 is distinct from most HCV inhibitors in clinical evaluation. As an initial step toward correlating in vitro and in vivo resistances, multiple cell lines and selective pressures were used to identify BMS-790052-resistant variants in genotype 1 replicons. Similarities and differences were observed between genotypes 1a and 1b. For genotype 1b, L31F/V, P32L, and Y93H/N were identified as primary resistance mutations. L23F, R30Q, and P58S acted as secondary resistance substitutions, enhancing the resistance of primary mutations but themselves not conferring resistance. For genotype 1a, more sites of resistance were identified, and substitutions at these sites (M28T, Q30E/H/R, L31M/V, P32L, and Y93C/H/N) conferred higher levels of resistance. For both subtypes, combining two resistance mutations markedly decreased inhibitor susceptibility. Selection studies with a 1b/1a hybrid replicon highlighted the importance of the NS5A N-terminal region in determining genotype-specific inhibitor responses. As single mutations, Q30E and Y93N in genotype 1a conferred the highest levels of resistance. For genotype 1b, BMS-790052 retained subnanomolar potency against all variants with single amino acid substitutions, suggesting that multiple mutations will likely be required for significant in vivo resistance in this genetic background. Importantly, BMS-790052-resistant variants remained fully sensitive to alpha interferon and small-molecule inhibitors of HCV protease and polymerase.

Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS‐790052 in Humans: In Vitro and In Vivo Correlations

The NS5A replication complex inhibitor, BMS‐790052, inhibits hepatitis C virus (HCV) replication with picomolar potency in preclinical assays. This potency translated in vivo to a substantial antiviral effect in a single‐ascending dose study and a 14‐day multiple‐ascending dose (MAD) monotherapy study. However, HCV RNA remained detectable in genotype 1a–infected patients at the end of the MAD study. In contrast, viral breakthrough was observed less often in patients infected with genotype 1b, and, in several patients, HCV RNA declined and remained below the level of quantitation (<25 IU/mL) through the duration of treatment. Here, we report on the results of the genotypic and phenotypic analyses of resistant variants in 24 genotype 1–infected patients who received BMS‐790052 (1, 10, 30, 60, and 100 mg, once‐daily or 30 mg twice‐daily) in the 14‐day MAD study. Sequence analysis was performed on viral complementary DNA isolated from serum specimens collected at baseline and days 1 (4, 8, and 12 hours), 2, 4, 7, and 14 postdosing. Analyses of the sequence variants (1) established a correlation between resistant variants emerging in vivo with BMS‐790052 treatment and those observed in the in vitro replicon system (major substitutions at residues 28, 30, 31, and 93 for genotype 1a and residues 31 and 93 for genotype 1b); (2) determined the prevalence of variants at baseline and the emergence of resistance at different times during dosing; and (3) revealed the resistance profile and replicative ability (i.e., fitness) of the variants. Conclusion: Although resistance emerged during monotherapy with BMS‐790052, the substantial anti‐HCV effect of this compound makes it an excellent candidate for effective combination therapy. (HEPATOLOGY 2011)

Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052

ABSTRACT BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC50s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interaction between NS5A residues 30 and 92. By using a trans-complementation assay with JFH1 replicons encoding inhibitor-sensitive and inhibitor-resistant NS5A proteins, we provide genetic evidence that NS5A performs the following two distinct functions in HCV RNA replication: a cis-acting function that likely occurs as part of the HCV replication complex and a trans-acting function that may occur outside the replication complex. The cis-acting function is likely performed by basally phosphorylated NS5A, while the trans-acting function likely requires hyperphosphorylation. Our data indicate that BMS-790052 blocks the cis-acting function of NS5A. Since BMS-790052 also impairs JFH1 NS5A hyperphosphorylation, it likely also blocks the trans-acting function.

Resistance analysis of hepatitis C virus genotype 1 prior treatment null responders receiving daclatasvir and asunaprevir

In a sentinel cohort, hepatitis C virus (HCV) patients (primarily genotype [GT] 1a) were treated with daclatasvir (NS5A inhibitor) and asunaprevir (NS3 protease inhibitor). Preexistence, emergence, and persistence of resistance variants in patients who failed this treatment are described. HCV‐infected null responders received daclatasvir (60 mg once daily) and asunaprevir (600 mg twice daily) alone (Group A, 11 patients) or with peginterferon alfa‐2a and ribavirin (Group B, 10 patients) for 24 weeks. Resistance testing was performed on baseline samples and samples with HCV RNA ≥1,000 IU/mL at Week 1 through posttreatment Week 48. Resistance substitution susceptibility to inhibition by asunaprevir and daclatasvir was assessed using HCV replicon assays. In Group A, six GT1a patients experiencing viral breakthrough and one GT1a patient who relapsed had detectable NS5A (Q30E/R, L31V/M, Y93C/N) and NS3 (R155K, D168A/E/V/Y) resistance‐associated variants at failure. Two of six viral breakthrough patients achieved SVR48 after treatment intensification with peginterferon alfa‐2a and ribavirin. For 2/4 viral breakthrough patients not responding to treatment intensification, NS3 resistance variants changed (D168Y to D168T; R155K to V36M‐R155K). At posttreatment Week 48, daclatasvir‐resistant variants persisted while asunaprevir‐resistant variants were generally replaced by wild‐type sequences. The NS3 sequence remained unchanged in the one patient with NS3‐R155K at baseline, relapse, and posttreatment Week 48. In Group B, no viral breakthrough was observed. Conclusion: The treatment failure of daclatasvir and asunaprevir in HCV GT1a patients was associated with both NS5A and NS3 resistance variants in prior null responders. NS5A resistance variants persisted while NS3 resistance variants generally decayed, suggesting a higher relative fitness of NS5A variants. (Hepatology 2013;53:902–911)

Persistence of Resistant Variants in Hepatitis C Virus-Infected Patients Treated with the NS5A Replication Complex Inhibitor Daclatasvir

ABSTRACT Daclatasvir (DCV; BMS-790052) is a hepatitis C virus (HCV) NS5A replication complex inhibitor (RCI) with picomolar to low nanomolar potency and broad genotypic coverage in vitro. Viral RNA declines have been observed in the clinic for both alpha interferon-ribavirin (IFN-α–RBV) and IFN-RBV-free regimens that include DCV. Follow-up specimens (up to 6 months) from selected subjects treated with DCV in 14-day monotherapy studies were analyzed for genotype and phenotype. Variants were detected by clonal sequencing in specimens from baseline and were readily detected by population sequencing following viral RNA breakthrough and posttreatment. The major amino acid substitutions generating resistance in vivo were at residues M28, Q30, L31, and Y93 for genotype 1a (GT-1a) and L31 and Y93 for GT-1b, similar to the resistance substitutions observed with the in vitro replicon system. The primary difference in the resistance patterns observed in vitro and in vivo was the increased complexity of linked variant combinations observed in clinical specimens. Changes in the percentage of individual variants were observed during follow-up; however, the overall percentage of variants in the total population persisted up to 6 months. Our results suggest that during the 14-day monotherapy, most wild-type virus was eradicated by DCV. After the end of DCV treatment, viral fitness, rather than DCV resistance, probably determines which viral variants emerge as dominant in populations.

Discovery and development of hepatitis C virus NS5A replication complex inhibitors.

Lead inhibitors that target the function of the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein have been identified by phenotypic screening campaigns using HCV subgenomic replicons. The demonstration of antiviral activity in HCV-infected subjects by the HCV NS5A replication complex inhibitor (RCI) daclatasvir (1) spawned considerable interest in this mechanistic approach. In this Perspective, we summarize the medicinal chemistry studies that led to the discovery of 1 and other chemotypes for which resistance maps to the NS5A protein and provide synopses of the profiles of many of the compounds currently in clinical trials. We also summarize what is currently known about the NS5A protein and the studies using NS5A RCIs and labeled analogues that are helping to illuminate aspects of both protein function and inhibitor interaction. We conclude with a synopsis of the results of notable clinical trials with HCV NS5A RCIs.

Comparison of Daclatasvir Resistance Barriers on NS5A from Hepatitis C Virus Genotypes 1 to 6: Implications for Cross-Genotype Activity

ABSTRACT A comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b > 4a ≥ 5a > 6a ≅ 1a > 2a JFH > 3a > 2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC50s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC50s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC50s ranging from 2 to 250 nM). The in vitro data suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy.

Hepatitis C Virus RNA Elimination and Development of Resistance in Replicon Cells Treated with BMS-790052

ABSTRACT BMS-790052, a first-in-class hepatitis C virus (HCV) replication complex inhibitor, targeting nonstructural protein 5A (NS5A), displays picomolar to nanomolar potency against genotypes 1 to 5. This exceptional potency translated into robust anti-HCV activity in clinical studies with HCV genotype 1-infected subjects. To date, all BMS-790052-associated resistance mutations have mapped to the N-terminal region of NS5A. To further characterize the antiviral activity of BMS-790052, HCV replicon elimination and colony formation assays were performed. Replicon was cleared from genotype 1a and 1b replicon cells in a time- and dose-dependent manner. Elimination of the genotype 1a replicon required longer treatment durations and higher concentrations of BMS-790052 than those for the genotype1b replicon. Single amino acid substitutions that conferred relatively low levels of resistance were observed at early time points and at low doses. Higher doses and longer treatment durations yielded mutations that conferred greater levels of resistance, including linked amino acid substitutions. Replicon cells that survived inhibitor treatment remained fully sensitivity to pegylated alpha interferon (pegIFN-α) and other HCV inhibitors. Moreover, genotype 1a replicon elimination was markedly enhanced when pegIFN-α and BMS-790052 were combined. Resistant variants observed in this study were very similar to those observed in a multiple ascending dose (MAD) monotherapy trial of BMS-790052, validating replicon elimination studies as a model to predict clinical resistance. Insights gained from the in vitro anti-HCV activity and resistance profiles of BMS-790052 will be used to help guide the clinical development of this novel HCV inhibitor.

Impact of a baseline polymorphism on the emergence of resistance to the hepatitis C virus nonstructural protein 5a replication complex inhibitor, BMS‐790052

The influence of naturally occurring polymorphisms on the potency of the HCV nonstructural protein 5A (NS5A) replication complex inhibitor, BMS‐790052, was investigated by evaluating hybrid replicons in which the entire NS5A coding region of genotype (GT) la and 1b laboratory (lab) strains (H77c and Con1) were replaced with the corresponding regions of specimens collected from 10 GT‐1a‐ and 6 GT‐1b‐infected subjects. For baseline (BL) specimens, with no previously observed resistance variants identified by population sequencing, the median 50% effective concentration (EC50) values for BMS‐790052 were similar for the clinically derived and lab strains. A Q30R variant was observed at viral breakthrough (VBT) in one of the GT‐1a‐infected subjects. Because the lowest plasma exposure of BMS‐790052 observed in this subject was 117 nM and the median 50% effective concentration value for a GT‐1a H77c replicon containing a Q30R substitution is ∼7 nM, a rigorous investigation was initiated to determine the basis for resistance. Three approaches were used: (1) replacement of the entire H77c NS5A or (2) replacement of the N‐terminal region of NS5A, with sequence from BL and day 14, and (3) substitution of specific amino acids. A BL polymorphism (E62D) did not contribute resistance to BMS‐790052; however, the linked variant, Q30R‐E62D, conferred high‐level resistance in vitro and is likely responsible for VBT in vivo. Conclusion: Our data show that a BL polymorphism with minimal effect on the anti‐HCV effect of BMS‐790052 can affect the emergence of resistance and significantly affect clinical outcome. This work establishes a clear, systematic approach to monitor resistance to NS5A inhibitors in the clinic. (HEPATOLOGY 2012;55:1692–1699)