Molecular Modeling and Biochemical Characterization Reveal the Mechanism of Hepatitis B Virus Polymerase Resistance to Lamivudine (3TC) and Emtricitabine (FTC)

doi:10.1128/JVI.75.10.4771-4779.2001

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Journal of Virology, May 2001, p. 4771-4779, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4771-4779.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Molecular Modeling and Biochemical Characterization Reveal the Mechanism of Hepatitis B Virus Polymerase Resistance to Lamivudine (3TC) and Emtricitabine (FTC)

Kalyan Das,¹ Xiaofeng Xiong,² Huiling Yang,² Christopher E. Westland,² Craig S. Gibbs,² Stefan G. Sarafianos,¹ and Edward Arnold¹^,^*

Center for Advanced Biotechnology and Medicine, Department of Chemistry, Rutgers University, Piscataway, New Jersey,¹ and Gilead Sciences, Foster City, California²

Received 8 November 2000/Accepted 19 February 2001

Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergenceof drug-resistant viral strains upon prolonged therapy. The predominantlamivudine resistance mutations in HBV-infected patients are Met552IIeand Met552Val (Met552Ile/Val), frequently in association witha second mutation, Leu528Met. The effects of Leu528Met, Met552Ile,and Met552Val mutations on the binding of HBV polymerase inhibitorsand the natural substrate dCTP were evaluated using an in vitroHBV polymerase assay. Susceptibility to lamivudine triphosphate(3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate,penciclovir triphosphate, and lobucavir triphosphate was assessedby determination of inhibition constants (K_i). Recognition ofthe natural substrate, dCTP, was assessed by determination ofK_m values. The results from the in vitro studies were as follows:(i) dCTP substrate binding was largely unaffected by the mutations,with K_m changing moderately, only in a range of 0.6 to 2.6-fold;(ii) K_is for 3TCTP and FTCTP against Met552Ile/Val mutant HBVpolymerases were increased 8- to 30-fold; and (iii) the Leu528Metmutation had a modest effect on direct binding of these $beta$ -L-oxathiolanering-containing nucleotide analogs. A three-dimensional homologymodel of the catalytic core of HBV polymerase was constructedvia extrapolation from retroviral reverse transcriptase structures.Molecular modeling studies using the HBV polymerase homology modelsuggested that steric hindrance between the mutant amino acidside chain and lamivudine or emtricitabine could account for theresistance phenotype. Specifically, steric conflict between theC $gamma$ 2-methyl group of Ile or Val at position 552 in HBV polymeraseand the sulfur atom in the oxathiolane ring (common to both $beta$ -L-nucleosideanalogs lamivudine and emtricitabine) is proposed to account forthe resistance observed upon Met552Ile/Val mutation. The effectsof the Leu528Met mutation, which also occurs near the HBV polymeraseactive site, appeared to be less direct, potentially involvingrearrangement of the deoxynucleoside triphosphate-binding pocketresidues. These modeling results suggest that nucleotide analogsthat are $beta$ -D-enantiomers, that have the sulfur replaced by a smalleratom, or that have modified or acyclic ring systems may retainactivity against lamivudine-resistant mutants, consistent withthe observed susceptibility of these mutants to adefovir, lobucavir,and penciclovir in vitro and adefovir invivo.

^* Corresponding author. Mailing address: CABM and Rutgers University, 679 Hoes Ln., Piscataway, NJ 08854. Phone: (732) 235-5323.Fax: (732) 235-5788. E-mail: arnold{at}cabm.rutgers.edu.

Journal of Virology, May 2001, p. 4771-4779, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4771-4779.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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