Molecular modeling and biochemical characterization reveal the mechanism of hepatitis B virus polymerase resistance to lamivudine (3TC) and emtricitabine (FTC)

Abstract

Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutations on the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitro HBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by determination of inhibition constants (K(i)). Recognition of the natural substrate, dCTP, was assessed by determination of Km values. The results from the in vitro studies were as follows: (i) dCTP substrate binding was largely unaffected by the mutations, with Km changing moderately, only in a range of 0.6 to 2.6-fold; (ii) K(i)s for 3TCTP and FTCTP against Met552Ile/Val mutant HBV polymerases were increased 8- to 30-fold; and (iii) the Leu528Met mutation had a modest effect on direct binding of these beta-L-oxathiolane ring-containing nucleotide analogs. A three-dimensional homology model of the catalytic core of HBV polymerase was constructed via extrapolation from retroviral reverse transcriptase structures. Molecular modeling studies using the HBV polymerase homology model suggested that steric hindrance between the mutant amino acid side chain and lamivudine or emtricitabine could account for the resistance phenotype. Specifically, steric conflict between the Cgamma2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring (common to both beta-L-nucleoside analogs lamivudine and emtricitabine) is proposed to account for the resistance observed upon Met552Ile/Val mutation. The effects of the Leu528Met mutation, which also occurs near the HBV polymerase active site, appeared to be less direct, potentially involving rearrangement of the deoxynucleoside triphosphate-binding pocket residues. These modeling results suggest that nucleotide analogs that are beta-D-enantiomers, that have the sulfur replaced by a smaller atom, or that have modified or acyclic ring systems may retain activity against lamivudine-resistant mutants, consistent with the observed susceptibility of these mutants to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.

Figures

FIG. 1

Chemical structures of dCTP, 3TCTP, FTCTP, ADVDP, LBVTP, and PCVTP.

FIG. 2

Schematic representation of the HBV pol gene, an HBV polymerase homology model (amino acids 325 to 699), and the HBV polymerase/HIV-1 RT sequence alignments used in constructing the model. The HBV polymerase is shown as a ribbon diagram (2) with the fingers (325 to 403 and 469 to 519), palm (404 to 440 and 520 to 613), and thumb (614 to 699) subdomains in blue, red, and green, respectively. The bound double-stranded DNA template primer is shown as a space-filled model in grey (with N and O atoms in blue and red, respectively), and dCTP is in gold. The four proposed disulfide links are represented by yellow lines. The HBV polymerase and HIV-1 RT sequence alignments are also color coded by subdomains, and sequence identities and amino acids functionally conserved between the two enzymes are in cyan.

FIG. 3

Electrostatic-potential surface diagrams of the modeled HBV polymerase (left) and of the HIV-1 RT-DNA-dNTP structure (right) plotted using the program GRASP (27). Regions in red and blue are charged negatively and positively, respectively. The locations of amino acids interacting with the DNA are labeled.

FIG. 4

The YMDD region of the modeled HBV polymerase with a docked dCTP substrate. Amino acids Met552 and Leu528 are mutated to confer resistance to 3TC and FTC. The orange molecular surface (left) corresponds to the deoxyribose of the docked dCTP. The green molecular surface of the protein around its YMDD region (left) indicated no steric hindrance between the protein and the substrate. The space between the two molecular surfaces is indicated by a white arrow. The space between the protein and substrate is reduced upon Met552Val+Leu528Met mutation (right).

FIG. 5

Binding of 3TCTP to wild-type (left) and Met552Val mutant (right) HBV polymerase. Molecular modeling suggests that steric hindrance (right), between 3TCTP and the mutated amino acid, Val552, is the primary cause of 3TCTP resistance. This steric conflict is not observed in the binding of 3TCTP to the wild-type HBV polymerase.

FIG. 6

Both wild-type (left) and Met552Val+Leu528Met mutant (right) HBV polymerase appear to have no steric conflict with a docked ADVDP.