PMID- 20169198 OWN - NLM STAT- MEDLINE DCOM- 20100930 LR - 20211020 IS - 1932-6203 (Electronic) IS - 1932-6203 (Linking) VI - 5 IP - 2 DP - 2010 Feb 12 TI - Mechanistic characterization and molecular modeling of hepatitis B virus polymerase resistance to entecavir. PG - e9195 LID - 10.1371/journal.pone.0009195 [doi] LID - e9195 AB - 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. FAU - Walsh, Ann W AU - Walsh AW AD - Research and Development, Bristol-Myers Squibb Inc., Wallingford, Connecticut, United States of America. FAU - Langley, David R AU - Langley DR FAU - Colonno, Richard J AU - Colonno RJ FAU - Tenney, Daniel J AU - Tenney DJ LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20100212 PL - United States TA - PLoS One JT - PloS one JID - 101285081 RN - 0 (Antiviral Agents) RN - 0 (Viral Proteins) RN - 2T8Q726O95 (Lamivudine) RN - 5968Y6H45M (entecavir) RN - 5Z93L87A1R (Guanine) RN - 86-01-1 (Guanosine Triphosphate) RN - EC 2.7.7.49 (RNA-Directed DNA Polymerase) SB - IM MH - Amino Acid Sequence MH - Amino Acid Substitution MH - Antiviral Agents/pharmacology MH - Binding Sites/genetics MH - Drug Resistance, Viral/*genetics MH - Guanine/*analogs & derivatives/pharmacology MH - Guanosine Triphosphate/chemistry/metabolism MH - Hep G2 Cells MH - Hepatitis B virus/enzymology/*genetics MH - Humans MH - Hydrogen Bonding MH - Kinetics MH - Lamivudine/pharmacology MH - Models, Molecular MH - Protein Binding MH - Protein Structure, Tertiary MH - RNA-Directed DNA Polymerase/chemistry/*genetics/metabolism MH - Substrate Specificity MH - Viral Proteins/chemistry/*genetics/metabolism MH - Virus Replication/drug effects/genetics PMC - PMC2820545 COIS- Competing Interests: All authors were paid employees of Bristol-Myers Squibb at the time of the work and all hold stock or stock options in the company. Entecavir, the subject of the paper, is a marketed product of Bristol-Myers Squibb. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials in the paper, as detailed in the guide to authors. EDAT- 2010/02/20 06:00 MHDA- 2010/10/01 06:00 PMCR- 2010/02/12 CRDT- 2010/02/20 06:00 PHST- 2009/10/22 00:00 [received] PHST- 2010/01/06 00:00 [accepted] PHST- 2010/02/20 06:00 [entrez] PHST- 2010/02/20 06:00 [pubmed] PHST- 2010/10/01 06:00 [medline] PHST- 2010/02/12 00:00 [pmc-release] AID - 09-PONE-RA-13739R1 [pii] AID - 10.1371/journal.pone.0009195 [doi] PST - epublish SO - PLoS One. 2010 Feb 12;5(2):e9195. doi: 10.1371/journal.pone.0009195.