Inhibition of Hepatitis B Virus Polymerase by Entecavir

ABSTRACT Entecavir (ETV; Baraclude) is a novel deoxyguanosine analog with activity against hepatitis B virus (HBV). ETV differs from the other nucleoside/tide reverse transcriptase inhibitors approved for HBV therapy, lamivudine (LVD) and adefovir (ADV), in several ways: ETV is >100-fold more potent against HBV in culture and, at concentrations below 1 μM, displays no significant activity against human immunodeficiency virus (HIV). Additionally, while LVD and ADV are obligate DNA chain terminators, ETV halts HBV DNA elongation after incorporating a few additional bases. Three-dimensional homology models of the catalytic center of the HBV reverse transcriptase (RT)-DNA-deoxynucleoside triphosphate (dNTP) complex, based on the HIV RT-DNA structure, were used with in vitro enzyme kinetic studies to examine the mechanism of action of ETV against HBV RT. A novel hydrophobic pocket in the rear of the RT dNTP binding site that accommodates the exocyclic alkene moiety of ETV was predicted, establishing a basis for the superior potency observed experimentally. HBV DNA chain termination by ETV was accomplished through disfavored energy requirements as well as steric constraints during subsequent nucleotide addition. Validation of the model was accomplished through modeling of LVD resistance substitutions, which caused an eightfold decrease in ETV susceptibility and were predicted to reduce, but not eliminate, the ETV-binding pocket, in agreement with experimental observations. ADV resistance changes did not affect the ETV docking model, also agreeing with experimental results. Overall, these studies explain the potency, mechanism, and cross-resistance profile of ETV against HBV and account for the successful treatment of naive and LVD- or ADV-experienced chronic HBV patients.

[1]  J. Marrero,et al.  Adefovir-resistant hepatitis B can be associated with viral rebound and hepatic decompensation. , 2005, Journal of hepatology.

[2]  Samuel H. Wilson,et al.  Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism. , 1997, Biochemistry.

[3]  T. Steitz,et al.  Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. , 1998, Current opinion in structural biology.

[4]  M. Sherman,et al.  80 Entecavir results in continued virologic and biochemical improvement and HBeAg seroconversion through 96 weeks of treatment in lamivudine-refractory, HBeAg(+) chronic hepatitis B patients (ETV-026) , 2006 .

[5]  A. D. Clark,et al.  Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Colin W Shepard,et al.  Hepatitis B virus infection: epidemiology and vaccination. , 2006, Epidemiologic reviews.

[7]  S. Sarafianos,et al.  Molecular Modeling and Biochemical Characterization Reveal the Mechanism of Hepatitis B Virus Polymerase Resistance to Lamivudine (3TC) and Emtricitabine (FTC) , 2001, Journal of Virology.

[8]  M. Manns,et al.  Mutational pattern of hepatitis B virus on sequential therapy with famciclovir and lamivudine in patients with hepatitis B virus reinfection occurring under hbig immunoglobulin after liver transplantation , 1999, Hepatology.

[9]  G. Vriend,et al.  Prediction of protein residue contacts with a PDB-derived likelihood matrix. , 2002, Protein engineering.

[10]  J. Barnard,et al.  Kinetic Analysis of Wild-Type and YMDD Mutant Hepatitis B Virus Polymerases and Effects of Deoxyribonucleotide Concentrations on Polymerase Activity , 2002, Antimicrobial Agents and Chemotherapy.

[11]  C. Mazzucco,et al.  Two-Year Assessment of Entecavir Resistance in Lamivudine-Refractory Hepatitis B Virus Patients Reveals Different Clinical Outcomes Depending on the Resistance Substitutions Present , 2006, Antimicrobial Agents and Chemotherapy.

[12]  A. Cross,et al.  Entecavir for treatment of lamivudine-refractory, HBeAg-positive chronic hepatitis B. , 2006, Gastroenterology.

[13]  Angeline Bartholomeusz,et al.  HBV drug resistance: mechanisms, detection and interpretation. , 2006, Journal of hepatology.

[14]  C. K. Chu,et al.  Molecular mechanisms of adefovir sensitivity and resistance in HBV polymerase mutants: a molecular dynamics study. , 2004, Bioorganic & medicinal chemistry letters.

[15]  R. Schinazi,et al.  The polymerase L528M mutation cooperates with nucleotide binding-site mutations, increasing hepatitis B virus replication and drug resistance. , 2001, The Journal of clinical investigation.

[16]  F. Diederich,et al.  Interactions with aromatic rings in chemical and biological recognition. , 2003, Angewandte Chemie.

[17]  C. Gibbs,et al.  Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient. , 2003, Journal of hepatology.

[18]  P. Tiollais,et al.  Hepatitis B virus. , 1991, Scientific American.

[19]  Lennart Nilsson,et al.  Empirical energy functions for energy minimization and dynamics of nucleic acids , 1986 .

[20]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[21]  P. Egli,et al.  Metabolic Studies on BMS-200475, a New Antiviral Compound Active against Hepatitis B Virus , 1999, Antimicrobial Agents and Chemotherapy.

[22]  R. Hamatake,et al.  In Vitro Inhibition of Hepadnavirus Polymerases by the Triphosphates of BMS-200475 and Lobucavir , 1998, Antimicrobial Agents and Chemotherapy.

[23]  C. Chu,et al.  Clearance of the original hepatitis B virus YMDD‐motif mutants with emergence of distinct lamivudine‐resistant mutants during prolonged lamivudine therapy , 2000, Hepatology.

[24]  E. De Clercq,et al.  Antiretrovirus Activity of a Novel Class of Acyclic Pyrimidine Nucleoside Phosphonates , 2002, Antimicrobial Agents and Chemotherapy.

[25]  R. Colonno,et al.  Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus , 1997, Antimicrobial agents and chemotherapy.

[26]  E. Schiff,et al.  A dose-ranging study of the efficacy and tolerability of entecavir in Lamivudine-refractory chronic hepatitis B patients. , 2005, Gastroenterology.

[27]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[28]  Y. Cheng,et al.  Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. , 1973, Biochemical pharmacology.

[29]  F. Momany,et al.  Validation of the general purpose QUANTA ®3.2/CHARMm® force field , 1992 .

[30]  J. Villeneuve,et al.  Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir , 2005, Hepatology.

[31]  Angeline Bartholomeusz,et al.  Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. , 2003, Gastroenterology.

[32]  L. Verlet.  Computer "Experiments" on Classical Fluids. I. Thermodynamical Properties of Lennard-Jones Molecules , 1967 .

[33]  J. Marrero,et al.  Virologic response and resistance to adefovir in patients with chronic hepatitis B. , 2006, Journal of hepatology.

[34]  M Karplus,et al.  Dynamics of DNA oligomers. , 1983, Journal of biomolecular structure & dynamics.

[35]  Ching-Lung Lai,et al.  Entecavir versus lamivudine for patients with HBeAg-negative chronic hepatitis B. , 2006, The New England journal of medicine.

[36]  Huiling Yang,et al.  In vitro antiviral susceptibility of full-length clinical hepatitis B virus isolates cloned with a novel expression vector. , 2004, Antiviral research.

[37]  M. Manns,et al.  In vitro susceptibilities of wild-type or drug-resistant hepatitis B virus to (-)-beta-D-2,6-diaminopurine dioxolane and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil. , 2001, Antimicrobial agents and chemotherapy.

[38]  P. Angus,et al.  Clinical Emergence of Entecavir-Resistant Hepatitis B Virus Requires Additional Substitutions in Virus Already Resistant to Lamivudine , 2004, Antimicrobial Agents and Chemotherapy.

[39]  B. Dominy,et al.  Development of a generalized Born model parameterization for proteins and nucleic acids , 1999 .

[40]  P. Boyer,et al.  Fixed conformation nucleoside analogs effectively inhibit excision-proficient HIV-1 reverse transcriptases. , 2005, Journal of molecular biology.

[41]  Graeme Currie,et al.  Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B. , 2005, The New England journal of medicine.

[42]  K. Walters,et al.  Identification and characterization of mutations in hepatitis B virus resistant to lamivudine , 1998 .

[43]  M. Otto,et al.  Mechanism of Antiviral Activities of 3′-Substituted L-Nucleosides against 3Tc-Resistant HBV Polymerase: A Molecular Modelling Approach , 2003, Antiviral chemistry & chemotherapy.

[44]  R. J. Colonno,et al.  Efficacies of Entecavir against Lamivudine-Resistant Hepatitis B Virus Replication and Recombinant Polymerases In Vitro , 2002, Antimicrobial Agents and Chemotherapy.

[45]  C. Mazzucco,et al.  Entecavir resistance is rare in nucleoside naïve patients with hepatitis B , 2006, Hepatology.

[46]  A. Cross,et al.  A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B. , 2006, The New England journal of medicine.

[47]  G L Verdine,et al.  Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. , 1998, Science.

[48]  S. Goff,et al.  Mutations in the epsilon sequences of human hepatitis B virus affect both RNA encapsidation and reverse transcription , 1995, Journal of virology.

[49]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

[50]  H. Ren,et al.  Entecavir for the treatment of lamivudine-refractory chronic hepatitis B patients in China , 2007, Hepatology international.

[51]  C. Seeger,et al.  Hepatitis B Virus Biology , 2000, Microbiology and Molecular Biology Reviews.

[52]  A. D. Clark,et al.  Sarafianos, S.G. et al. Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with -branched amino acids. Proc. Natl. Acad. Sci. USA 96, 10027-10032 , 1999 .

[53]  G. Makar.  Entecavir versus Lamivudine for Patients with HBeAg-Negative Chronic Hepatitis BLai C-L, for the BEHoLD Al463027 Study Group (Queen Mary Hosp, Hong Kong; et al) N Engl J Med 354:1011–1020, 2006§ , 2007 .

[54]  B. Tehan,et al.  Comparisons of the Hbv and HIV Polymerase, and Antiviral Resistance Mutations , 2003, Antiviral therapy.

[55]  W. Lam,et al.  Reverse transcriptase activity of hepatitis B virus (HBV) DNA polymerase within core capsid: interaction with deoxynucleoside triphosphates and anti-HBV L-deoxynucleoside analog triphosphates. , 2004, Molecular pharmacology.

[56]  M. Manns,et al.  In Vitro Susceptibilities of Wild-Type or Drug-Resistant Hepatitis B Virus to (−)-β-d-2,6-Diaminopurine Dioxolane and 2′-Fluoro-5-Methyl-β-l-Arabinofuranosyluracil , 2001, Antimicrobial Agents and Chemotherapy.