Lourdes Valera

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.

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.

Development of a Cell-Based High-Throughput Specificity Screen Using a Hepatitis C Virus-Bovine Viral Diarrhea Virus Dual Replicon Assay

ABSTRACT The hepatitis C virus (HCV) replicon is a unique system for the development of a high-throughput screen (HTS), since the analysis of inhibitors requires the quantification of a decrease in a steady-state level of HCV RNA. HCV replicon replication is dependent on host cell factors, and any toxic effects may have a significant impact on HCV replicon replication. Therefore, determining the antiviral specificity of compounds presents a challenge for the identification of specific HCV inhibitors. Here we report the development of an HCV/bovine viral diarrhea virus (BVDV) dual replicon assay suitable for HTS to address these issues. The HCV reporter enzyme is the endogenous NS3 protease contained within the HCV genome, while the BVDV reporter enzyme is a luciferase enzyme engineered into the BVDV genome. The HTS uses a mixture of HCV and BVDV replicon cell lines placed in the same well of a 96-well plate and isolated in the same cell backgrounds (Huh-7). The format consists of three separate but compatible assays: the first quantitates the amount of cytotoxicity based upon the conversion of Alamar blue dye via cellular enzymes, while the second indirectly quantitates HCV replicon replication through measurement of the amount of NS3 protease activity present. The final assay measures the amount of luciferase activity present from the BVDV replicon cells, as an indicator of the specificity of the test compounds. This HCV/BVDV dual replicon assay provides a reliable format to determine the potency and specificity of HCV replicon inhibitors.

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.

Carbapenem Resistance in a Clinical Isolate of Enterobacter aerogenes Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs

ABSTRACT We investigated the mechanism of imipenem resistance in Enterobacter aerogenes strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 μg/ml and the meropenem MIC was 8 μg/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was ≤1 μg/ml and the meropenem MIC was ≤0.25 μg/ml. Cefepime MICs also decreased from 8 to 1 μg/ml; however, the MICs of ceftazidime (≥128 μg/ml), cefoxitin (≥32 μg/ml), and cefotaxime (≥64 μg/ml) remained the same. The β-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain E. aerogenes ATCC 13048 were determined. Strains 810 and 810-REV each produced two β-lactamases with pIs of 8.2 and 5.4. The β-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to Escherichia coli porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of Klebsiella pneumoniae (anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data suggest that imipenem resistance in E. aerogenes 810 is primarily associated with the lack of expression of the analogs of the OmpC (42-kDa) and OmpF (39-kDa) outer membrane proteins, which also results in decreased susceptibility to meropenem and cefepime.

Correlation between Genotype and Phenotypic Categorization of Staphylococci Based on Methicillin Susceptibility and Resistance

ABSTRACT Positive correlation between methicillin and oxacillin susceptibility test results and the detection of themecA gene was observed forStaphylococcusaureus, S. epidermidis, and S. haemolyticus as well as among mecA+ strains of other species of coagulase-negative staphylococci (CNS). However, at least 50% of themecA-negative strains of these other species of CNS were falsely classified as methicillin and oxacillin resistant.

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)