Drug Targets and Molecular Mechanisms of Drug Resistance in Chronic Hepatitis B
2007; Elsevier BV; Volume: 132; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2007.02.039
ISSN1528-0012
Autores Tópico(s)Liver Disease Diagnosis and Treatment
ResumoChronic hepatitis B continues to be a major cause of end-stage liver disease and hepatocellular carcinoma worldwide. Nucleos(t)ide analogues have proven to be effective in controlling the disease and perhaps decreasing the incidence of hepatocellular carcinoma. However, development of drug resistance is a major limitation to their long-term effectiveness. Understanding the mechanisms of drug resistance are important for designing new agents and devising strategies to manage and prevent the development of antiviral drug resistance. The development of resistance is determined by an interplay of viral, host, and drug characteristics Homology of the HBV polymerase to the human immunodeficiency virus-1 reverse transcriptase has allowed predictions to be made on the effect mutations have on HBV polymerase structure. In vitro functional studies provide complementary information. Several broad principles on the mechanism of resistance have emerged from these studies. First, most of the primary mutations cluster in the vicinity of the incoming nucleotide and act by directly affecting the position or stability of the bound substrate, template, or primer. In contrast, secondary mutations tend to occur away from the nucleotide-binding pocket. Finally, the structural and functional consequences of mutations are quite variable among the different agents. This paper reviews the key mutations and mechanisms associated with resistance to the nucleos(t)ide analogues approved for clinical use and discuss new targets for drug development. Chronic hepatitis B continues to be a major cause of end-stage liver disease and hepatocellular carcinoma worldwide. Nucleos(t)ide analogues have proven to be effective in controlling the disease and perhaps decreasing the incidence of hepatocellular carcinoma. However, development of drug resistance is a major limitation to their long-term effectiveness. Understanding the mechanisms of drug resistance are important for designing new agents and devising strategies to manage and prevent the development of antiviral drug resistance. The development of resistance is determined by an interplay of viral, host, and drug characteristics Homology of the HBV polymerase to the human immunodeficiency virus-1 reverse transcriptase has allowed predictions to be made on the effect mutations have on HBV polymerase structure. In vitro functional studies provide complementary information. Several broad principles on the mechanism of resistance have emerged from these studies. First, most of the primary mutations cluster in the vicinity of the incoming nucleotide and act by directly affecting the position or stability of the bound substrate, template, or primer. In contrast, secondary mutations tend to occur away from the nucleotide-binding pocket. Finally, the structural and functional consequences of mutations are quite variable among the different agents. This paper reviews the key mutations and mechanisms associated with resistance to the nucleos(t)ide analogues approved for clinical use and discuss new targets for drug development. One third of the world's population has been exposed to hepatitis B virus (HBV).1Lee W.M. Hepatitis B virus infection.N Engl J Med. 1997; 337: 1733-1745Crossref PubMed Scopus (2200) Google Scholar The prevalence of hepatitis B surface antigen (HBsAg), the serologic marker of chronic infection, has declined recently, in endemic areas of the world, mostly as a result of successful vaccine programs.2Chen H.L. Chang M.H. Ni Y.H. Hsu H.Y. Lee P.I. Lee C.Y. Chen D.S. Seroepidemiology of hepatitis B virus infection in children: ten years of mass vaccination in Taiwan.JAMA. 1996; 276: 906-908Crossref PubMed Google Scholar, 3Ni Y.H. Chang M.H. Huang L.M. Chen H.L. Hsu H.Y. Chiu T.Y. Tsai K.S. Chen D.S. Hepatitis B virus infection in children and adolescents in a hyperendemic area: 15 years after mass hepatitis B vaccination.Ann Intern Med. 2001; 135: 796-800Crossref PubMed Scopus (317) Google Scholar However, there still remains an enormous reservoir of subjects (>350 million) who have chronic HBV infection. These individuals are at risk for chronic liver disease, cirrhosis, and hepatocellular carcinoma. Antiviral therapy is the only option to control and prevent progression of disease in patients with chronic HBV infection. There are currently 6 drugs approved for management of chronic hepatitis B (CHB) (see accompanying review by Lok.4Lok A.S.-F. Navigating the maze of hepatitis B treatments.Gastroenterology. 2007; 132: 1586-1594Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) Broadly, these drugs can be grouped into 2 classes of agents: cytokines and nucleos(t)ide analogues (NAs). Interferon is a naturally occurring cytokine that possesses antiviral and immunomodulatory properties and was the first agent approved for therapy of CHB. It is effective in approximately one third of patients but must be administered by subcutaneous injection and is associated with numerous adverse effects. NAs target the reverse transcriptase of HBV and are potent inhibitors of HBV replication. They have the advantage of oral administration and are relatively free of adverse effects. However, they generally must be used for long periods to maintain effectiveness. The development of drug resistance is emerging as a major limitation to their long-term efficacy. An understanding of the mechanisms of drug resistance is therefore important for rational drug design and managing patients with existing drug resistance. This review will focus on our current knowledge of the molecular mechanisms of drug resistance and discuss new targets for drug therapy in CHB. HBV is a small DNA virus. The infectious virion circulates as a 42-nm Dane particle that comprises a nucleocapsid surrounded by a lipid envelope.5Ganem D. Varmus H.E. The molecular biology of the hepatitis B viruses.Annu Rev Biochem. 1987; 56: 651-693Crossref PubMed Scopus (927) Google Scholar, 6Seeger C. Mason W.S. Hepatitis B virus biology.Microbiol Mol Biol Rev. 2000; 64: 51-68Crossref PubMed Scopus (1245) Google Scholar The nucleocapsid contains a partially double-stranded ∼3.2-kilobase genome. The compact genome encodes 4 overlapping reading frames and 4 RNA species that encode for 7 viral proteins: 3 envelope proteins—large, middle, and small that form the HBsAg, the nucleocapsid core protein, the secretory hepatitis B e antigen, the viral reverse transcriptase/polymerase, and the X protein (Figure 1). Although HBV is a DNA virus, replication of the viral DNA occurs via a RNA intermediate. HBV replication occurs predominantly in hepatocytes. Whether the virus can replicate in other cell types is still a matter of debate. The mechanism for attachment and entry into the hepatocyte is not known. The related duck hepatitis B virus (DHBV) has been shown to use carboxypeptidase D as its receptor.7Breiner K.M. Urban S. Schaller H. Carboxypeptidase D (gp180), a Golgi-resident protein, functions in the attachment and entry of avian hepatitis B viruses.J Virol. 1998; 72: 8098-8104Crossref PubMed Google Scholar, 8Tong S. Li J. Wands J.R. Carboxypeptidase D is an avian hepatitis B virus receptor.J Virol. 1999; 73: 8696-8702PubMed Google Scholar Upon entry into the cell, the virus uncoats, and the relaxed circular genome is transported to the nucleus. In the nucleus, host and viral polymerases repair the partially relaxed circular genome to a fully double-stranded covalent closed circular genome or cccDNA (Figure 2). The cccDNA serves as the template for the transcription of all the viral messenger RNA (mRNA). The viral RNAs include the pregenomic RNA, which serves as both the template for reverse transcription and for the core and polymerase synthesis, as well as the 3 subgenomic mRNAs necessary for the translation of the envelope proteins and the X protein.5Ganem D. Varmus H.E. The molecular biology of the hepatitis B viruses.Annu Rev Biochem. 1987; 56: 651-693Crossref PubMed Scopus (927) Google Scholar, 6Seeger C. Mason W.S. Hepatitis B virus biology.Microbiol Mol Biol Rev. 2000; 64: 51-68Crossref PubMed Scopus (1245) Google Scholar The viral mRNAs are transported to the cytoplasm at which translation of viral proteins, nucleocapsid assembly, and viral replication occur. Replication occurs within a nucleocapsid that consists of the core protein, the pregenomic RNA, and the polymerase.5Ganem D. Varmus H.E. The molecular biology of the hepatitis B viruses.Annu Rev Biochem. 1987; 56: 651-693Crossref PubMed Scopus (927) Google Scholar, 6Seeger C. Mason W.S. Hepatitis B virus biology.Microbiol Mol Biol Rev. 2000; 64: 51-68Crossref PubMed Scopus (1245) Google Scholar Nucleocapsid formation requires the coordinated binding of the polymerase to a RNA stem-loop structure at the 5′ end of the pregenomic RNA called epsilon, which triggers encapsidation by core particles (Figure 2). The polymerase bound to epsilon serves as a protein primer for DNA synthesis with epsilon serving as the template for this reaction. After completion of the negative-strand DNA synthesis, the RNA is degraded by the viral RNase H that is part of the polymerase, followed by positive-strand synthesis and circularization of the viral genome. Once replication is completed, the viral nucleocapsid interacts with the envelope proteins in the endoplasmic reticulum to form mature virions that are secreted from the cell. Viral nucleocapsids can also be transported back to the nucleus at which a pool of cccDNA is maintained. This pathway is thought to be regulated by the large surface protein.9Lenhoff R.J. Summers J. Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus.J Virol. 1994; 68: 4565-4571PubMed Google Scholar The small genome and the requirement of host cellular enzymes for many stages of the HBV life cycle suggest that relatively few targets are available for antiviral development. Because the HBV polymerase carries out the primary enzymatic function of viral replication, it has been the main target of anti-HBV drug development. NAs have been the major class of antiviral agents developed for this purpose. Four of the currently approved treatments for CHB belong to this class of compounds. Many other NAs are in preclinical or clinical trials. These compounds are very active against HBV replication, but the major problem is the emergence of resistance, which will be discussed later. Most of the current effort in anti-HBV development has focused on developing drugs that either offer low rates of resistance or little cross-resistance with other NAs. However, several promising molecular targets other than the HBV polymerase have been developed (Figure 2). Recently, a group of compounds has been identified that specifically targets the encapsidation step, in which the viral RNA, polymerase, and core are assembled into the nucleocapsid before viral replication occurs.10Deres K. Schroder C.H. Paessens A. Goldmann S. Hacker H.J. Weber O. Kramer T. Niewohner U. Pleiss U. Stoltefuss J. Graef E. Koletzki D. Masantschek R.N. Reimann A. Jaeger R. Gross R. Beckermann B. Schlemmer K.H. Haebich D. Rubsamen-Waigmann H. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids.Science. 2003; 299: 893-896Crossref PubMed Scopus (469) Google Scholar These compounds belong to the class of heteroaryldihydropyrimidines (HAPs). In comparison with lamivudine in a cell-based HBV replication assay, HAPs were more potent.10Deres K. Schroder C.H. Paessens A. Goldmann S. Hacker H.J. Weber O. Kramer T. Niewohner U. Pleiss U. Stoltefuss J. Graef E. Koletzki D. Masantschek R.N. Reimann A. Jaeger R. Gross R. Beckermann B. Schlemmer K.H. Haebich D. Rubsamen-Waigmann H. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids.Science. 2003; 299: 893-896Crossref PubMed Scopus (469) Google Scholar The HAP was shown to bind to the core protein and inhibit nucleocapsid formation.10Deres K. Schroder C.H. Paessens A. Goldmann S. Hacker H.J. Weber O. Kramer T. Niewohner U. Pleiss U. Stoltefuss J. Graef E. Koletzki D. Masantschek R.N. Reimann A. Jaeger R. Gross R. Beckermann B. Schlemmer K.H. Haebich D. Rubsamen-Waigmann H. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids.Science. 2003; 299: 893-896Crossref PubMed Scopus (469) Google Scholar The binding of the HAP to the core protein leads to degradation of the core protein. Furthermore, the HAP has also been shown to inhibit HBV replication in a transgenic mouse model with a similar efficacy as lamivudine.11Weber O. Schlemmer K.H. Hartmann E. Hagelschuer I. Paessens A. Graef E. Deres K. Goldmann S. Niewoehner U. Stoltefuss J. Haebich D. Ruebsamen-Waigmann H. Wohlfeil S. Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model.Antiviral Res. 2002; 54: 69-78Crossref PubMed Scopus (158) Google Scholar Another group of compounds that appear to inhibit the encapsidation step are the phenopropenamides.12King R.W. Ladner S.K. Miller T.J. Zaifert K. Perni R.B. Conway S.C. Otto M.J. Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (−)β-L-2',3'-dideoxy-3'-thiacytidine.Antimicrob Agents Chemother. 1998; 42: 3179-3186PubMed Google Scholar Their mechanism of action appears to be different from that of the HAPs. These compounds directly inhibit the formation of the nucleocapsid. In the cell-based replication system, the phenopropenamides are not as potent as lamivudine in inhibiting HBV replication. These compounds are specific for HBV and have no activity against related viruses such as woodchuck hepatitis virus and DHBV. Various steps of the assembly process including glycosylation of HBV envelope proteins and HBsAg-nucleocapsid interaction have been the targets of antiviral development.13Block T.M. Lu X. Mehta A.S. Blumberg B.S. Tennant B. Ebling M. Korba B. Lansky D.M. Jacob G.S. Dwek R.A. Treatment of chronic hepadnavirus infection in a woodchuck animal model with an inhibitor of protein folding and trafficking.Nat Med. 1998; 4: 610-614Crossref PubMed Scopus (141) Google Scholar Gene therapy with nucleic acid-based technology, such as antisense,14Offensperger W.B. Offensperger S. Blum H.E. Antisense therapy of hepatitis B virus infection.Mol Biotechnol. 1998; 9: 161-170Crossref PubMed Scopus (18) Google Scholar ribozyme,15Cech T.R. Ribozymes, the first 20 years.Biochem Soc Trans. 2002; 30: 1162-1166Crossref PubMed Scopus (92) Google Scholar and short interfering RNA (siRNA),16Wu H.L. Huang L.R. Huang C.C. Lai H.L. Liu C.J. Huang Y.T. Hsu Y.W. Lu C.Y. Chen D.S. Chen P.J. RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant.Gastroenterology. 2005; 128: 708-716Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 17Wu J. Nandamuri K.M. Inhibition of hepatitis viral replication by siRNA.Expert Opin Biol Ther. 2004; 4: 1649-1659Crossref PubMed Scopus (24) Google Scholar is a promising approach and has demonstrated favorable results in vitro and in animal models. The major obstacle with these approaches is the issue of delivery into the appropriate site in which the virus can replicate in vivo. Recent advances in delivery technology may render solutions to this critical issue. Other areas of anti-HBV development focus on immunotherapy, which targets the host immune response to overcome chronic HBV infection.18Loomba R. Liang T.J. Novel approaches to new therapies for hepatitis B virus infection.Antivir Ther. 2006; 11: 1-15PubMed Google Scholar However, whether any of these strategies can be ultimately developed into clinically useful therapy remains unclear. The HBV polymerase is a multifunctional protein that has 4 domains: a priming region, a spacer region of unknown function, a catalytic region that functions as a RNA-dependent RNA polymerase/DNA polymerase, and a carboxy terminal region that has ribonuclease H activity (Figure 3A).6Seeger C. Mason W.S. Hepatitis B virus biology.Microbiol Mol Biol Rev. 2000; 64: 51-68Crossref PubMed Scopus (1245) Google Scholar Although the crystal structure of HBV polymerase is unknown, much of its structure has been deduced from the human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) based on their homology.19Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1864) Google Scholar, 20Huang H. Chopra R. Verdine G.L. Harrison S.C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1366) Google Scholar, 21Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr, A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. et al.Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.Proc Natl Acad Sci U S A. 1993; 90: 6320-6324Crossref PubMed Scopus (1129) Google Scholar Regardless of their amino acid sequences and differences in domain structure, all polymerases appear to have a common right-handed configuration with a thumb, a palm, and a fingers domain (Figure 3B).22Steitz T.A. DNA polymerases: structural diversity and common mechanisms.J Biol Chem. 1999; 274: 17395-17398Crossref PubMed Scopus (720) Google Scholar, 23Doublie S. Sawaya M.R. Ellenberger T. An open and closed case for all polymerases.Structure. 1999; 7: R31-R35Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar The palm domain appears to be the active site and catalyzes the phosphoryl transfer reaction; the fingers domain facilitates interactions with the incoming dNTPs as well as the template base to which it is paired; and the thumb domain may play a role in positioning the duplex DNA, processivity, and translocation.22Steitz T.A. DNA polymerases: structural diversity and common mechanisms.J Biol Chem. 1999; 274: 17395-17398Crossref PubMed Scopus (720) Google Scholar NAs and dNTPs bind at a site that is located in the palm subdomain adjacent to the 3′ terminus of the primer strand.24Tantillo C. Ding J. Jacobo-Molina A. Nanni R.G. Boyer P.L. Hughes S.H. Pauwels R. Andries K. Janssen P.A. Arnold E. Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase Implications for mechanisms of drug inhibition and resistance.J Mol Biol. 1994; 243: 369-387Crossref PubMed Scopus (500) Google Scholar An interesting property of the HBV polymerase seems to be its preference for nucleotides with the L-configuration in contrast to other polymerases that prefer nucleotides with the D-configuration.25Davis M.G. Wilson J.E. VanDraanen N.A. Miller W.H. Freeman G.A. Daluge S.M. Boyd F.L. Aulabaugh A.E. Painter G.R. Boone L.R. DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs.Antiviral Res. 1996; 30: 133-145Crossref PubMed Scopus (39) Google Scholar The catalytic region can be subdivided into 7 domains: A–G (Figure 3A).19Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1864) Google Scholar Domain A of HIV-1 RT is in close proximity to the 2 aspartic acid residues in domain C and forms part of the dNTP binding pocket.19Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1864) Google Scholar, 21Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr, A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. et al.Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.Proc Natl Acad Sci U S A. 1993; 90: 6320-6324Crossref PubMed Scopus (1129) Google Scholar Residues in this domain are involved in the coordination of the incoming triphosphate moiety of the dNTP and the magnesium ions. Domain B for HBV RT forms an α helix with a loop region and is involved with positioning of the primer-template strand to the catalytic region.21Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr, A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. et al.Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.Proc Natl Acad Sci U S A. 1993; 90: 6320-6324Crossref PubMed Scopus (1129) Google Scholar Domain C contains a sequence of 4 amino acids, tyrosine, methionine, aspartate, aspartate (YMDD), which is highly conserved among viral polymerases/reverse transcriptases that binds 2 magnesium ions and represents the active site of the enzyme.21Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr, A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. et al.Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.Proc Natl Acad Sci U S A. 1993; 90: 6320-6324Crossref PubMed Scopus (1129) Google Scholar, 26Poch O. Sauvaget I. Delarue M. Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.EMBO J. 1989; 8: 3867-3874Crossref PubMed Scopus (1030) Google Scholar, 27Beese L.S. Steitz T.A. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism.EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (943) Google Scholar Residues within domain D lie outside but may contribute to the dNTP binding site of HIV-1 RT. Mutations in this domain may indirectly affect the geometry of the dNTP binding site.24Tantillo C. Ding J. Jacobo-Molina A. Nanni R.G. Boyer P.L. Hughes S.H. Pauwels R. Andries K. Janssen P.A. Arnold E. Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase Implications for mechanisms of drug inhibition and resistance.J Mol Biol. 1994; 243: 369-387Crossref PubMed Scopus (500) Google Scholar In HIV-1 RT, domain E forms part of the template-primer binding site.19Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1864) Google Scholar, 21Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr, A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. et al.Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.Proc Natl Acad Sci U S A. 1993; 90: 6320-6324Crossref PubMed Scopus (1129) Google Scholar The primer strand contacts the loops between the palm and the thumb at rtM230 and rtG231.21Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr, A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. et al.Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.Proc Natl Acad Sci U S A. 1993; 90: 6320-6324Crossref PubMed Scopus (1129) Google Scholar The methionine and glycine residues present in domain E are conserved in all HBV isolates. Domains F and G are upstream of domain A. This region may be involved in interactions with the incoming dNTP and also with the template nucleotide. HBV has a high rate of replication, with 1012King R.W. Ladner S.K. Miller T.J. Zaifert K. Perni R.B. Conway S.C. Otto M.J. Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (−)β-L-2',3'-dideoxy-3'-thiacytidine.Antimicrob Agents Chemother. 1998; 42: 3179-3186PubMed Google Scholar virions produced per day and a high mutational rate of approximately 10−5 substitution/base/cycle.28Nowak M.A. Bonhoeffer S. Hill A.M. Boehme R. Thomas H.C. McDade H. Viral dynamics in hepatitis B virus infection.Proc Natl Acad Sci U S A. 1996; 93: 4398-4402Crossref PubMed Scopus (879) Google Scholar This translates to approximately 1010Deres K. Schroder C.H. Paessens A. Goldmann S. Hacker H.J. Weber O. Kramer T. Niewohner U. Pleiss U. Stoltefuss J. Graef E. Koletzki D. Masantschek R.N. Reimann A. Jaeger R. Gross R. Beckermann B. Schlemmer K.H. Haebich D. Rubsamen-Waigmann H. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids.Science. 2003; 299: 893-896Crossref PubMed Scopus (469) Google Scholar, 11Weber O. Schlemmer K.H. Hartmann E. Hagelschuer I. Paessens A. Graef E. Deres K. Goldmann S. Niewoehner U. Stoltefuss J. Haebich D. Ruebsamen-Waigmann H. Wohlfeil S. Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model.Antiviral Res. 2002; 54: 69-78Crossref PubMed Scopus (158) Google Scholar point mutations produced per day in individuals with active replication. Because the HBV genome is only ∼3200 base pairs, all possible single-base changes can be produced per day. The HBV reverse transcriptase does not have a proofreading function to repair incorrectly incorporated nucleotides. Therefore, mutations can arise very rapidly. Prior to therapy, a diverse swarm of viruses (quasispecies), including mutants with single and double mutations potentially associated with drug resistance, probably exists. The probability of a mutation being selected during therapy depends on the ability of a drug to suppress viral replication. Hence, a drug with low antiviral activity does not exert substantial selection pressure on the virus, and the chance of drug resistance is not high. Conversely, complete suppression of viral replication allows little opportunity for resistance to emerge because mutagenesis is replication dependent. NAs inhibit viral replication but do not eliminate existing virus or affect cccDNA in any major way. Monotherapy exerting modest antiviral activity and directed at 1 single target site would result in the highest probability of selecting drug resistance. The ideal treatment regimen should have antiviral activities targeted at different sites to reduce the risk of selecting out drug-resistant species. Resistance emerges when replication occurs in the presence of the drug selection pressure. Therefore, if we could achieve complete suppression of replication, resistance would not be an issue. Other factors contributing to the emergence of drug resistance are genetic barriers to the development of mutations, mechanism of drug resistance, viral replication space, and various host factors involved in controlling viral replication. NAs can be viewed as prodrugs because they need to be activated for their antiviral activity via a phosphorylation process to their nucleoside triphosphates or nucleotide diposphate that functions as the inhibitor of polymerase. The NAs are first phosphorylated by cellular kinases to nucleoside monophosphates, which are then further phosphorylated by cellular enzymes to the diphosphates and triphosphates.29De Clercq E. Strategies in the design of antiviral drugs.Nat Rev Drug Discov. 2002; 1: 13-25Crossref PubMed Scopus (609) Google Scholar The initial phosphorylation step is usually the rate-limiting step in the activation process and may account for some of the differences in potency among the various NAs.29De Clercq E. Strategies in the design of antiviral drugs.Nat Rev Drug Discov. 2002; 1: 13-25Crossref PubMed Scopus (609) Google Scholar Three categories of agents are currently available: L-nucleosides, acyclic phosphonate nucleotides, and cyclopentane deoxyguanosine analogues (Figure 4). Lamivudine was the first NA to be approved for therapy of CHB and belongs to the class of L-nucleosides (Figure 4A). Lamivudine resistance develops at a rate of 14%–24% per year and is approximately 70% by years 4 to 5.30Lok A.S. Lai C.L. Leung N. Yao G.B. Cui Z.Y. Schiff E.R. Dienstag J.L. Heathcote E.J. Little N.R. Griffiths D.A. Gardner S.D. Castiglia M. Long-term safety of lamivudine treatment in patients with chronic hepatitis B.Gastroenterology. 2003; 125: 1714-1722Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar There are 2 primary mutations associated with antiviral resistance to lamivudine: rtM204V and rtM204I (Table 1).31Allen M.I. Deslauriers M. Andrews C.W. Tipples G.A. Walters K.A. Tyrrell D.L. Brown N. Condreay L.D. Lamivudine Clinical Investigation GroupIdentification and characterization of mutations in hepatitis B virus resistant to lamivudine.Hepatology. 1998; 27: 1670-1677Crossref PubMed Scopus (790) Google Scholar The rtM204V mutation is usually associated with the compensatory rtL180M mutation. A molecular mechanism of resistance has been predicted based on the crystal structure of the HIV polymerase.19Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1864) Google Scholar, 20Huang H. Chopra R. Verdine G.L. Harrison S.C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1366) Google Scholar Lamivudine binds at a pocket in the surface of the polymerase (palm region) formed in part by residue rt204.31Allen M.I. Deslauriers M. Andrews C.W. Tipples G.A. Walters K.A. Tyrrell D.L. Brown N. Condreay L.D. Lamivudine Clinical Investigation GroupIdentif
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