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

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.     

In vitro resistance profile of the hepatitis C virus NS3 protease inhibitor BI 201335

The in vitro resistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.     

Pharmacodynamic analysis of a serine protease inhibitor, MK-4519, against hepatitis C virus using a novel in vitro pharmacodynamic system

The development of new antiviral compounds active against hepatitis C virus (HCV) has surged in recent years. In order for these new compounds to be efficacious in humans, optimal dosage regimens for each compound must be elucidated. We have developed a novel in vitro pharmacokinetic/pharmacodynamic system, the BelloCell system, to identify optimal dosage regimens for anti-HCV compounds. In these experiments, genotype 1b HCV replicon-bearing cells (2209-23 cells) were inoculated onto carrier flakes in BelloCell bottles and treated with MK-4519, a serine protease inhibitor. Our dose-ranging studies illustrated that MK-4519 inhibited replicon replication in a dose-dependent manner, yielding a 50% effective concentration (EC(50)) of 1.8 nM. Dose-fractionation studies showed that shorter dosing intervals resulted in greater replicon suppression, indicating that the time that the concentration is greater than the EC(50) is the pharmacodynamic parameter for MK-4519 linked with inhibition of replicon replication. Mutations associated with resistance to serine protease inhibitors were detected in replicons harvested from all treatment arms. These data suggest that MK-4519 is highly active against genotype 1b HCV, but monotherapy is not sufficient to prevent the amplification of resistant replicons. In summary, our findings show that the BelloCell system is a useful and clinically relevant tool for predicting optimal dosage regimens for anti-HCV compounds.     

Ketoamide resistance and hepatitis C virus fitness in val55 variants of the NS3 serine protease

Drug-resistant viral variants are a major issue in the use of direct-acting antiviral agents in chronic hepatitis C. Ketoamides are potent inhibitors of the NS3 protease, with V55A identified as mutation associated with resistance to boceprevir. Underlying molecular mechanisms are only partially understood. We applied a comprehensive sequence analysis to characterize the natural variability at Val55 within dominant worldwide patient strains. A residue-interaction network and molecular dynamics simulation were applied to identify mechanisms for ketoamide resistance and viral fitness in Val55 variants. An infectious H77S.3 cell culture system was used for variant phenotype characterization. We measured antiviral 50% effective concentration (EC₅₀) and fold changes, as well as RNA replication and infectious virus yields from viral RNAs containing variants. Val55 was found highly conserved throughout all hepatitis C virus (HCV) genotypes. The conservative V55A and V55I variants were identified from HCV genotype 1a strains with no variants in genotype 1b. Topology measures from a residue-interaction network of the protease structure suggest a potential Val55 key role for modulation of molecular changes in the protease ligand-binding site. Molecular dynamics showed variants with constricted binding pockets and a loss of H-bonded interactions upon boceprevir binding to the variant proteases. These effects might explain low-level boceprevir resistance in the V55A variant, as well as the Val55 variant, reduced RNA replication capacity. Higher structural flexibility was found in the wild-type protease, whereas variants showed lower flexibility. Reduced structural flexibility could impact the Val55 variant''s ability to adapt for NS3 domain-domain interaction and might explain the virus yield drop observed in variant strains.     

Preclinical profile of VX-950, a potent, selective, and orally bioavailable inhibitor of hepatitis C virus NS3-4A serine protease

VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (K(i)*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.     

Genetic screen for monitoring hepatitis C virus NS3 serine protease activity

We have developed a genetic system to monitor the activity of the hepatitis C virus (HCV) NS3 serine protease. This genetic system is based on the bacteriophage lambda regulatory circuit where the viral repressor cI is specifically cleaved to initiate the switch from lysogeny to lytic infection. An HCV protease-specific target, NS5A-5B, was inserted into the lambda phage cI repressor. The target specificity of the HCV NS5A-5B repressor was evaluated by coexpression of this repressor with a beta-galactosidase (betagal)-HCV NS3(2-181)/4(21-34) protease construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI.HCV5AB-cro and the betagal-HCV NS3(2-181)/4(21-34) protease constructs, lambda phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS3(2-181)/4(21-34) protease. This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine protease, and it has the potential to be used for screening specific inhibitors.     

Clevudine: a potent inhibitor of hepatitis B virus in vitro and in vivo

Clevudine (CLV) is a nucleoside analog of the unnatural L-configuration that has potent anti-hepatitis B virus (HBV) activity in vitro and in vivo with a favorable toxicity profile in all species tested. In cell culture, CLV is readily phosphorylated to the corresponding 5´-triphosphate form of the compound. The mechanism of action of CLV involves the inhibition of the HBV polymerase by CLV 5´-triphosphate. In vivo efficacy studies performed in the duck and woodchuck models showed marked, rapid inhibition of virus replication and no significant toxicity. In the woodchuck model, there was a dose-dependent delay in viral recrudescence and a reduction or loss of covalently closed circular DNA. In Phase II clinical studies, CLV was well tolerated and exhibited potent antiviral activity at all doses investigated. In Phase III studies in both hepatitis B e antigen (HBeAg)-positive and -negative patients, CLV 30 mg administered once daily demonstrated potent antiviral efficacy and significant biochemical improvement after only 24 weeks of therapy. These effects were sustained in a significant portion of the patients when therapy was stopped after 6 months with no viral rebound occurring in approximately 3 and 16% in HBeAg-positive and -negative patients, respectively. There have been no significant safety or tolerance issues associated with the drug in these studies. Future studies will investigate the safety and tolerance of CLV 30 mg given once daily over 48 weeks and longer.     

Clevudine: a potent inhibitor of hepatitis B virus in vitro and in vivo

Clevudine (CLV) is a nucleoside analog of the unnatural L-configuration that has potent anti-hepatitis B virus (HBV) activity in vitro and in vivo with a favorable toxicity profile in all species tested. In cell culture, CLV is readily phosphorylated to the corresponding 5'-triphosphate form of the compound. The mechanism of action of CLV involves the inhibition of the HBV polymerase by CLV 5'-triphosphate. In vivo efficacy studies performed in the duck and woodchuck models showed marked, rapid inhibition of virus replication and no significant toxicity. In the woodchuck model, there was a dose-dependent delay in viral recrudescence and a reduction or loss of covalently closed circular DNA. In Phase II clinical studies, CLV was well tolerated and exhibited potent antiviral activity at all doses investigated. In Phase III studies in both hepatitis B e antigen (HBeAg)-positive and -negative patients, CLV 30 mg administered once daily demonstrated potent antiviral efficacy and significant biochemical improvement after only 24 weeks of therapy. These effects were sustained in a significant portion of the patients when therapy was stopped after 6 months with no viral rebound occurring in approximately 3 and 16% in HBeAg-positive and -negative patients, respectively. There have been no significant safety or tolerance issues associated with the drug in these studies. Future studies will investigate the safety and tolerance of CLV 30 mg given once daily over 48 weeks and longer.     

Activity of a potent hepatitis C virus polymerase inhibitor in the chimpanzee model

A-837093 is a potent and specific nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase. It possesses nanomolar potencies in both enzymatic and replicon-based cell culture assays. In rats and dogs this compound demonstrated an oral plasma half-life of greater than 7 h, and its bioavailability was >60%. In monkeys it had a half-life of 1.9 h and 15% bioavailability. Its antiviral efficacy was evaluated in two chimpanzees infected with HCV in a proof-of-concept study. The design included oral dosing of 30 mg per kg of body weight twice a day for 14 days, followed by a 14-day posttreatment observation. Maximum viral load reductions of 1.4 and 2.5 log(10) copies RNA/ml for genotype 1a- and 1b-infected chimpanzees, respectively, were observed within 2 days after the initiation of treatment. After this initial drop in the viral load, a rebound of plasma HCV RNA was observed in the genotype 1b-infected chimpanzee, while the genotype 1a-infected chimpanzee experienced a partial rebound that lasted throughout the treatment period. Clonal analysis of NS5B gene sequences derived from the plasma of A-837093-treated chimpanzees revealed the presence of several mutations associated with resistance to A-837093, including Y448H, G554D, and D559G in the genotype 1a-infected chimpanzee and C316Y and G554D in the genotype 1b-infected chimpanzee. The identification of resistance-associated mutations in both chimpanzees is consistent with the findings of in vitro selection studies, in which many of the same mutations were selected. These findings validate the antiviral efficacy and resistance development of benzothiadiazine HCV polymerase inhibitors in vivo.     

In vitro development of resistance to human immunodeficiency virus protease inhibitor GW640385

Development of in vitro resistance to GW640385, a new human immunodeficiency virus type 1 protease inhibitor, was studied. Variants characterized included one with <4-fold resistance and amino acid substitutions Q58E/A71V (protease) and P452K (Gag) and one with >50-fold resistance and amino acid substitutions L10F/G16E/E21K/A28S/M46I/F53L/A71V (protease) and L449F/P453T (Gag). The A28S substitution substantially reduced replication capacity.     

Resistance analysis of the hepatitis C virus NS3 protease inhibitor asunaprevir

Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensive in vitro genotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.     

Viral kinetics in patients with chronic hepatitis C treated with the serine protease inhibitor BILN 2061

We analysed viral kinetics from a 2-day treatment with BILN 2061, a serine protease inhibitor of hepatitis C virus, in patients chronically infected with genotype 1 hepatitis C virus. The efficiency (E), describing inhibition of viral production, was above 99.45% in all patients with minor or moderate fibrosis receiving doses of 200mg and 500 mg twice daily and larger than in previous studies for interferon-based treatments. However, epsilon was slightly smaller in patients with cirrhosis given 200mg and markedly smaller in patients given 25 mg. Estimates of viral clearance and infected-cell loss support conclusions on these rates and on treatment mechanisms from previous studies on interferon-alpha-based treatments.     

Mutations conferring zanamivir resistance in human influenza virus N2 neuraminidases compromise virus fitness and are not stably maintained in vitro

BACKGROUND: Viruses resistant to zanamivir have been generated in vitro, but no resistant virus has yet been isolated from a zanamivir-treated immunocompetent patient. In contrast most resistant viruses isolated from oseltamivir-treated patients correspond to those selected in vitro. However, despite mutations being in conserved residues in the neuraminidase (NA) they do not confer resistance in all NA subtypes. OBJECTIVES AND METHODS: We have used reverse genetics and the recombinant baculovirus expression system for investigating reasons for the lack of isolation of zanamivir-resistant H3N2 viruses and for further exploring subtype-specific oseltamivir resistance. RESULTS: H3N2 viruses generated by reverse genetics with H274Y, R292K E119V and E119D mutations were rescued. Those with E119G, E119A or R152K mutations could only be rescued in the presence of exogenous NA and after passage in the absence of exogenous NA only isolates that had reverted to the wild-type NA or, surprisingly, E119G/A to E119V NA were isolated. Mutations conferring zanamivir resistance significantly affected enzyme activity, virus replication or NA thermal stability. E119V viruses were stable and grew to similar titres as wild-type virus, consistent with their isolation from oseltamivir-treated patients. Mutations conferring oseltamivir resistance in N1 (H274Y) and B (R152K) NAs also conferred resistance in recombinant G70C N9 NA expressed in insect cells. CONCLUSIONS: These data suggest that zanamivir-resistant H3N2 viruses may not readily arise in vivo due to their poor viability. The G70C N9 NA may also provide a useful model for understanding the structural basis of subtype-specific drug resistance.     

NIM811, a cyclophilin inhibitor, exhibits potent in vitro activity against hepatitis C virus alone or in combination with alpha interferon

Host factors involved in viral replication are potentially attractive antiviral targets that are complementary to specific inhibitors of viral enzymes, since resistant mutations against the latter are likely to emerge during long-term treatment. It has been reported recently that cyclosporine, which binds to a family of cellular proteins, cyclophilins, inhibits hepatitis C virus (HCV) replication in vitro. Here, the activities of various cyclosporine derivatives were evaluated in the HCV replicon system. There was a strong correlation between the anti-HCV activity and cyclophilin-binding affinity of these compounds. Of these, NIM811 has been selected as a therapeutic candidate for HCV infection, since it binds to cyclophilins with higher affinity than cyclosporine but is devoid of the significant immunosuppressive activity associated with cyclosporine. NIM811 induced a concentration-dependent reduction of HCV RNA in the replicon cells with a 50% inhibitory concentration of 0.66 microM at 48 h. Furthermore, a greater than three-log(10) viral RNA reduction was achieved after treating the cells with as little as 1 microM of NIM811 for 9 days. In addition, the combination of NIM811 with alpha interferon significantly enhanced anti-HCV activities without causing any increase of cytotoxicity. Taken together, these promising in vitro data warrant clinical investigation of NIM811, an inhibitor of novel mechanism, for the treatment of hepatitis C.     

In vitro resistance study of AG-021541, a novel nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase

A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.     

In vitro resistance study of rupintrivir, a novel inhibitor of human rhinovirus 3C protease

Rupintrivir (formerly AG7088) is an irreversible inhibitor of the human rhinovirus (HRV) 3C protease that has been demonstrated to have in vitro activity against all HRVs tested, consistent with its interaction with a strictly conserved subset of amino acids in the 3C protease. The potential for resistance was studied following in vitro serial passage of HRV serotypes 14, 2, 39, and Hanks in the presence of increasing rupintrivir concentrations. HRV variants with reduced susceptibilities to rupintrivir (sevenfold for HRV 14) or with no significant reductions in susceptibility but genotypic changes (HRV 2, 39, and Hanks) were initially isolated following 14 to 40 cumulative days in culture (three to six passages). Sequence analysis of the 3C protease identified one to three substitutions in diverse patterns but with common features (T129T/A, T131T/A, and T143P/S in HRV 14; N165T in HRV 2; N130N/K and L136L/F in HRV 39; T130A in HRV Hanks). Notably, three of the four HRV variants contained a substitution at residue 130 (residue 129 in HRV 14). Continued selection in the presence of escalating concentrations of rupintrivir (40 to 72 days) resulted in the accumulation of additional mutations (A121A/V and Y139Y/H in HRV 14, E3E/G and A103A/V in HRV 2, S105T in HRV 39), with only minimal further reductions in susceptibility (up to fivefold). The ability of specific substitutions to confer resistance was examined by susceptibility testing of HRV 14 variants constructed to contain 3C protease mutations. In summary, the slow accumulation of multiple amino acid substitutions with only minimal to moderate reductions in susceptibility highlight the advantages of 3C protease as an antiviral target.     

The hepatitis C virus replicon presents a higher barrier to resistance to nucleoside analogs than to nonnucleoside polymerase or protease inhibitors

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.     

VX-950, a novel hepatitis C virus (HCV) NS3-4A protease inhibitor, exhibits potent antiviral activities in HCv replicon cells

The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log(10) was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log(10) reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.