Mechanistic Characterization and Molecular Modeling of Hepatitis B Virus Polymerase Resistance to Entecavir

BACKGROUND: Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I +/- L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. METHODS/PRINCIPAL FINDINGS: To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (K(i)) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (k(cat)) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. CONCLUSIONS: Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template

Antiviral Activity, Pharmacokinetics, and Safety of BMS-488043, a Novel Oral Small-Molecule HIV-1 Attachment Inhibitor, in HIV-1-Infected Subjects

ABSTRACT BMS-488043 is a novel and unique oral small-molecule inhibitor of the attachment of human immunodeficiency virus type 1 (HIV-1) to CD4 + lymphocytes. The antiviral activity, pharmacokinetics, viral susceptibility, and safety of BMS-488043 were evaluated in an 8-day monotherapy trial. Thirty HIV-1-infected study subjects were randomly assigned to sequential, safety-guided dose panels of 800 and 1,800 mg BMS-488043 or a matched placebo in a 4:1 ratio, and the drug was administered every 12 h with a high-fat meal for 7 days and on the morning of day 8. Dose-related, albeit less-than-dose-proportional, increases in plasma BMS-488043 concentrations were observed. Mean plasma HIV-1 RNA decreases from the baseline for the BMS-488043 800- and 1,800-mg dose groups on day 8 were 0.72 and 0.96 log 10 copies/ml, respectively, compared with 0.02 log 10 copies/ml for the placebo group. A lower baseline BMS-488043 50% effective concentration (EC 50 ) in the active-treatment groups was predictive of a greater antiviral response. Although absolute drug exposure was not associated with an antiviral response, the trough concentration ( C trough ), adjusted by the baseline EC 50 ( C trough /EC 50 ), was associated with antiviral activity. During dosing, four subjects experienced >10-fold reductions in viral susceptibility to BMS-488043, providing further support of the direct antiviral mechanism of BMS-488043. BMS-488043 was generally safe and well tolerated. These results suggest that further development of this novel class of oral HIV-1 attachment inhibitors is warranted

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

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people We designed a mechanistically unbiased approach based on chemical genetics to identify chemical starting points for interfering with HCV replication. Our differentiating strategy centred on the identification of compounds functionally distinct from those acting on the traditional targets of antiviral research in this field, the NS3 protease and the NS5B RNA-dependent RNA polymerase 10 . BMS-858 formed the basis of an extensive series of chemical refinements that focused on improving antiviral potency, broadening inhibitory activity to encompass the HCV 1a genotype, and optimizing for oral bioavailability and sustained pharmacokinetic properties. After defining symmetry as an important contributor to antiviral activity 10 , a discovery that preceded the disclosure of structural information (see below), we subsequently identified BMS-79005

Activities of Atazanavir (BMS-232632) against a Large Panel of Human Immunodeficiency Virus Type 1 Clinical Isolates Resistant to One or More Approved Protease Inhibitors

To evaluate the cross-resistance profile of the human immunodeficiency virus type 1 protease inhibitor (PI) atazanavir (BMS-232632), a panel of 551 clinical isolates exhibiting a wide array of PI resistance profiles and a variety of genotypic patterns were assayed for susceptibility to atazanavir and six other PIs: amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir. In general, reductions in atazanavir susceptibility in vitro required several amino acid changes and were relatively modest in degree, and susceptibility was retained among isolates resistant to one or two of the currently approved PIs. There was a clear trend toward loss of susceptibility to atazanavir, as isolates exhibited increasing levels of cross-resistance to multiple PIs. Atazanavir appeared to have a distinct resistance profile relative to each of the other six PIs tested based on susceptibility comparisons against this panel of resistant isolates. Analysis of the genotypic profiles of 943 PI-susceptible and -resistant clinical isolates identified a strong correlation between the presence of amino acid changes at specific residues (10I/V/F, 20R/M/I, 24I, 33I/F/V, 36I/L/V, 46I/L, 48V, 54V/L, 63P, 71V/T/I, 73C/S/T/A, 82A/F/S/T, 84V, and 90M) and decreased susceptibility to atazanavir. While no single substitution or combination of substitutions was predictive of atazanavir resistance (change, >3.0-fold), the presence of at least five of these substitutions correlated strongly with loss of atazanavir susceptibility. Mutations associated with reduced susceptibility to each of the other six PIs were also determined

Potent Efficacy of Entecavir (BMS-200475) in a Duck Model of Hepatitis B Virus Replication

The ability of entecavir (ETV) to inhibit Duck hepatitis B virus (DHBV) infection in duck hepatocytes and ducklings was examined using lamivudine (3TC) as a comparator drug. ETV exhibited antiviral activity (50% effective concentration [EC(50)], 0.13 nM) in DHBV-infected duck hepatocytes that was >1,000-fold more potent than that of 3TC (EC(50), 138 nM). A 21-day treatment of ducklings with 1 mg of ETV per kg of body weight per day by oral gavage resulted in a mean reduction of log(10) 3.1 in serum DHBV DNA levels. Daily treatment with 0.1 mg of ETV/kg was nearly as effective, achieving an average viral DNA level decrease of log(10) 2.1. Reducing the daily dose of ETV to only 0.01 mg/kg resulted in an average viral DNA level decrease of log(10) 0.97. Daily treatment with 25 mg of 3TC/kg resulted in an average viral DNA level decrease of log(10) 0.66, compared to the log(10) 0.20 drop seen for ducklings given the vehicle alone. ETV was also more effective in decreasing the DHBV DNA levels in duck livers after 21 days of treatment, causing average drops of log(10) 1.41, log(10) 0.76, and log(10) 0.26 for dose levels of 1.0, 0.1, and 0.01 mg/kg, respectively, compared to a decrease of log(10) 0.06 for 3TC at a dose level of 25 mg/kg. Levels of viral covalently closed circular DNA in the treatment group receiving 1 mg of ETV/kg were reduced compared to those in the vehicle-treated group. ETV and 3TC were both well tolerated in all treated animals. These results show that ETV is a highly potent and effective antiviral in the DHBV duck model

Entecavir for Treatment of Hepatitis B Virus Displays No In Vitro Mitochondrial Toxicity or DNA Polymerase Gamma Inhibition▿

Therapy with nucleoside reverse transcriptase inhibitors (NRTIs) can be associated with mitochondrial toxicity. In vitro studies have been used to predict the predisposition for and characterize the mechanisms causing mitochondrial toxicity. Entecavir (ETV) is an approved deoxyguanosine nucleoside for the treatment of chronic hepatitis B virus (HBV) infection that exhibits potent activity against viral reverse transcriptase. We assessed the potential for mitochondrial toxicity of ETV in long-term cultures of HepG2 hepatoma cells by measuring mitochondrial function (through lactate secretion), levels of mitochondrial DNA (mtDNA), and levels of mitochondrial proteins COX II and COX IV. Furthermore, we tested the activity of ETV-triphosphate (ETV-TP) against mitochondrial DNA polymerase γ (Pol γ) in vitro. ETV concentrations as high as 100 times the maximal clinical exposure (Cmax) did not affect cell proliferation, levels of lactate, mitochondrial DNA, or mitochondrial proteins throughout the 15-day culture. The lack of mitochondrial toxicity was consistent with the finding that ETV-TP was not recognized by mitochondrial DNA Pol γ and failed to be incorporated into DNA or inhibit the polymerase assay at the highest levels tested, 300 μM. Combinations of ETV with each of the other HBV NRTI antivirals, adefovir, tenofovir, and lamivudine at 10 times their respective Cmax levels also failed to result in cellular or mitochondrial toxicity. In summary, cell culture and enzymatic studies yielded no evidence that would predict mitochondrial toxicity of ETV at exposure levels in excess of those expected to be achieved clinically