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

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

Specific Inhibition of Bovine Viral Diarrhea Virus Replicase

Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is ∼2.2 μM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 μM compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 μM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase

Entecavir Therapy Combined with DNA Vaccination for Persistent Duck Hepatitis B Virus Infection

This study was designed to test the efficacy of antiviral treatment with entecavir (ETV) in combination with DNA vaccines expressing duck hepatitis B virus (DHBV) antigens as a therapy for persistent DHBV infection in ducks. Ducks were inoculated with 10(9) DHBV genomes at 7 days of age, leading to widespread infection of the liver and viremia within 7 days, and were then treated orally with either ETV (0.1 mg/kg of body weight/day) or distilled water from 21 days posthatch for 244 days. Treatment with ETV caused a 4-log drop in serum DHBV DNA levels within 80 days and a slower 2- to 3-log drop in serum DHBV surface antigen (DHBsAg) levels within 120 days. Following withdrawal of ETV, levels of serum DHBV DNA and DHBsAg rebounded to match those in the water-treated animals within 40 days. Sequential liver biopsy samples collected throughout the study showed that ETV treatment reduced DHBV DNA replicative intermediates 70-fold in the liver, while the level of the stable, template form, covalently closed circular DNA decreased only 4-fold. ETV treatment reduced both the intensity of antigen staining and the percentage of antigen-positive hepatocytes in the liver, but the intensity of antigen staining in bile duct cells appeared not to be effected. Intramuscular administration of five doses of a DNA vaccine expressing the DHBV presurface, surface, precore, and core antigens, both alone and concurrently with ETV treatment, on days 50, 64, 78, 127, and 141 did not result in any significant effect on viral markers

Effect of Antiviral Treatment with Entecavir on Age- and Dose-Related Outcomes of Duck Hepatitis B Virus Infection

Entecavir (ETV), a potent inhibitor of the hepadnaviral polymerases, prevented the development of persistent infection when administered in the early stages of duck hepatitis B virus (DHBV) infection. In a preliminary experiment, ETV treatment commenced 24 h before infection showed no significant advantage over simultaneous ETV treatment and infection. In two further experiments 14-day-old ducks were inoculated with DHBV-positive serum containing 10(4), 10(6), 10(8), or 5 × 10(8) viral genomes (vge) and were treated orally with 1.0 mg/kg of body weight/day of ETV for 14 or 49 days. A relationship between virus dose and infection outcome was seen: non-ETV-treated ducks inoculated with 10(4) vge had transient infection, while ducks inoculated with higher doses developed persistent infection. ETV treatment for 49 days did not prevent initial infection of the liver but restricted the spread of infection more than ∼1,000-fold, a difference which persisted throughout treatment and for up to 49 days after withdrawal. Ultimately, three of seven ETV-treated ducks resolved their DHBV infection, while the remaining ducks developed viremia and persistent infection after a lag period of at least 63 days. ETV treatment for 14 days also restricted the spread of infection, leading to marked and sustained reductions in the number of DHBV-positive hepatocytes in 7 out of 10 ducks. In conclusion, short-term suppression with ETV provides opportunity for the immune response to successfully control DHBV infection. Since DHBV infection of ducks provides a good model system for HBV infection in humans, it seems likely that ETV may be useful in postexposure therapy for HBV infection aimed at preventing the development of persistent infection

Incorporation of [³H]-ETV into HBV nucleocapsid DNA in culture.

<p>[³H]-ETV was added to cultures of HepG2 cells transfected with an HBV expression construct, as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#s4" target="_blank">Materials and Methods</a>. HBV nucleocapsids were isolated from cell lysates, as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#s4" target="_blank">Materials and Methods</a>, and radiolabeled HBV DNA was quantified through scintillation counting. The levels of nucleocapsid-associated [³H] from cells grown in [³H]-ETV are presented as percent wildtype control values ± standard deviation. Yields of HBV nucleocapsid DNA were standardized according to real-time PCR quantification of HBV DNA within isolated nucleocapsids, as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#s4" target="_blank">Materials and Methods</a>. Similar results were obtained by standardizing total HBV nucleocapsid DNA levels with nucleocapsids from parallel cultures metabolically labeled with [³H]-thymidine (data not shown). WT, wildtype nucleocapsids; LVDr, M204V+L180M substituted HBV nucleocapsids, LVDr+M250V, M204V+L180M+M250V substituted nucleocapsids; LVDr+T184G+S202I, M204V+L180M+T184G+S202I substituted nucleocapsids. HBVs were tested independently 3 to 4 times, except the LVDr+M250V, which was tested twice.</p

The T184-S202-204 hydrogen bonding network stabilizes the YMDD loop.

<p>The YMDD loop is shown with residues M204V+L180M and the H-bonding between residues T184, S202 and M204V are shown as dotted white lines.</p

Molecular homology model of resistant HBV RTs.

<p>The ETV-TP binding pocket of HBV RT. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#pone.0009195-Langley1" target="_blank">[3]</a>. (A) HBV RT with LVDr substitutions, M204V+L180M, (B) HBV RT with LVDr + S202G. HBV RT, ETV-TP and primer-template DNAs are labeled. The residues lining the pocket are orange, changes from LVDr are red, from ETVr are blue, and the M250, S202, and T184 residue positions (panel B) are pink. Panel A was essential reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#pone.0009195-Langley1" target="_blank">[3]</a> with permission.</p

Analysis of ETVr through strand-specific DNA synthesis in culture.

<p>Cell culture ETV EC₅₀ determinations were made for wildtype and various resistant HBVs. The levels of HBV DNA synthesized in the presence of ETV was determined by HBV-specific probe hybridization to HBV nucleocapsid DNA from the cultures. The probes used were the typical double-stranded DNA probe, or strand-specific riboprobes which hybridized to a single HBV DNA strand. Comparison of strand-specific EC₅₀ versus double stranded EC₅₀ for wildtype polymerase (WT) or LVDr M204V+L180M HBV, or the LVDr substitutions with ETVr substitutions (+T184, +S202, +M250) as indicated. Values at 4000 nM indicate that 50% inhibition was not observed at the highest ETV concentration tested.</p

Kinetic Parameters for HBV Polymerases.

a<p>Values are the mean ± standard deviation from at least three independent experiments.</p>b<p>Fold change from wildtype value.</p