Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interferon-alpha is the most widely used antiviral drug in chronic hepatitis B and C. Tolerability is usually good and serious adverse effects are rare. Most of the adverse effects are mild or transient and do not necessitate drug withdrawal. More than 90% of patients who are given interferon-alpha achieve 6 months to 1 year of treatment without serious adverse effects. The serious adverse effects usually occur in predisposed patients with pre-existing organ dysfunction. Nevertheless, careful selection of patients for therapy and observation during therapy are recommended. Nucleoside analogues are promising drugs in the treatment of chronic hepatitis B through inhibition of viral DNA polymerase. Lamivudine has been licensed for use in this indication. Its tolerability is excellent even when used for periods of 1 year or more. The main concern is the relatively high incidence of viral resistance resulting in breakthrough during or relapse after therapy. In the treatment of chronic hepatitis C, ribavirin, in combination with interferon-alpha is currently the reference therapy. The main adverse effect is haemolytic anaemia, which necessitates careful monitoring and adjustment of dosage in many cases. Recently, large trials showed the better efficacy of pegylated interferons as compared with standard interferon. The combination of pegylated interferon with ribavirin is under evaluation.
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PMID:Tolerability of treatments for viral hepatitis. 1141 64

Among the AIDS drugs approved by the US Food and Drug Administration (FDA) for clinical use, two are modified cytosine analogues, zalcitabine (ddC) and lamivudine [(-)3TC]. (-)3TC is the only analogue containing an unnatural L(-)nucleoside configuration. Similar to other dideoxynucleosides, these analogues are metabolically activated to the triphosphate that is incorporated into DNA by HIV-1 reverse transcriptase (RT), resulting in DNA chain termination and ultimately cessation of viral replication. The natural d(+)3TC isomer also acts in a similar manner to inhibit HIV-1 RT. In cell culture (-)3TC is less toxic than its d(+)isomer (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric 3TC and ddC compounds is not completely understood, although a number of factors may clearly come into play. We have previously investigated the mechanistic basis for the differential stereoselective inhibition and toxicity of these three cytosine analogues by comparing the effects of ddCTP (+)3TC-TP and (-)3TC-TP on the HIV-1 RT, as well as a recombinant form of the human mitochondrial DNA polymerase (Polgamma), the holoenzyme polymerase responsible for mitochondrial DNA replication. In this paper, we discuss the molecular mechanism for the stereochemical selectivity and differential toxicity.
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PMID:The molecular basis of inhibition and toxicity of modified cytosine analogues targetting HIV-1 reverse transcriptase. 1159 79

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.
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PMID:Mutations in the conserved woodchuck hepatitis virus polymerase FLLA and YMDD regions conferring resistance to lamivudine. 1207 58

Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) replication in vitro and in vivo that is currently in clinical trials for the treatment of chronic HBV infections. A major limitation of the current HBV antiviral therapy, lamivudine (3TC), is the emergence of drug-resistant HBV in a majority of treated patients due to specific mutations in the nucleotide binding site of HBV DNA polymerase (HBV Pol). To determine the effects of 3TC resistance mutations on inhibition by ETV triphosphate (ETV-TP), a series of in vitro studies were performed. The inhibition of wild-type and 3TC-resistant HBV Pol by ETV-TP was measured using recombinant HBV nucleocapsids, and compared to that of 3TC-TP. These enzyme inhibition studies demonstrated that ETV-TP is a highly potent inhibitor of wild-type HBV Pol and is 100- to 300-fold more potent than 3TC-TP against 3TC-resistant HBV Pol. Cell culture assays were used to gauge the potential for antiviral cross-resistance of 3TC-resistant mutants to ETV. Results demonstrated that ETV inhibited the replication of 3TC-resistant HBV, but 20- to 30-fold higher concentrations were required. To gain further perspective regarding the potential therapeutic use of ETV, its phosphorylation was examined in hepatoma cells treated with extracellular concentrations representative of drug levels in plasma in ETV-treated patients. At these concentrations, intracellular ETV-TP accumulated to levels expected to inhibit the enzyme activity of both wild-type and 3TC-resistant HBV Pol. These findings are predictive of potent antiviral activity of ETV against both wild-type and 3TC-resistant HBV.
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PMID:Efficacies of entecavir against lamivudine-resistant hepatitis B virus replication and recombinant polymerases in vitro. 1212 28

Lamivudine, the negative enantiomer of 2'-deoxy-3'-thiacytidine, is a dideoxynucleoside analog that prevents hepatitis B virus (HBV) DNA synthesis by competitively inhibiting the viral reverse transcriptase and DNA polymerase stages of HBV replication and by terminating proviral DNA chain extension. A dose-ranging study established that once daily oral lamivudine 3 mg/kg up to a maximum of 100 mg/day has the optimum efficacy and tolerability profile for achieving a maximal reduction in serum HBV DNA levels in children aged 2 to 12 years and adolescents aged 13 to 17 years with chronic HBV infection and active viral replication (chronic hepatitis B). Significantly more children and adolescents with chronic hepatitis B receiving lamivudine demonstrated a virologic response (undetectable serum hepatitis Be antigen and undetectable HBV DNA level) [23 vs 13%; p = 0.04] and/or biochemical response (55 vs 12%; p < 0.001) compared with placebo in a large, randomized, double-blind, 52-week phase III study. Despite the emergence of YMDD-variant HBV in 19% of lamivudine-treated children and adolescents, serum alanine aminotransferase and HBV DNA levels remained below baseline in these patients. Oral lamivudine is generally well tolerated by children and adolescents with chronic hepatitis B, with a similar tolerability profile to placebo at the recommended once daily dosage of 3 mg/kg up to a maximum of 100 mg/day.
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PMID:Lamivudine: in children and adolescents with chronic hepatitis B virus infection. 1226 43

Lamivudine (3TC) is a nucleoside analogue which inhibits replication of HIV and HBV and which is used in the treatment of chronic hepatitis B-infected patients with safety and efficacy. The activity of lamivudine was evaluated by the measurement of DNA-HBV concentration in plasma using a very sensitive assay (1,000 copies/mL) (Amplicor VHB Monitor. Roche). Ten patients chronically infected with hepatitis B (group A) and 24 patients with HIV-1 co-infection (group B) were enrolled. In 9 patients of group A, HBVDNA load was undetectable a median of 3.5 months after the beginning of treatment and remained negative for 2 years with hepatitis Be antigen disappearing and normal alanine aminotransferase concentration. In the last immunodeficient patient, the virus which had been resistant to three interferon treatments, was also resistant to lamivudine. In five patients of group B, HBV DNA load remained undetectable after 18 months with HBe antigen disappearing and baseline concentration of alanine aminotransferase. In the remaining 19 patients after a transient decrease of HBV DNA concentration for one year, HBV DNA load increased again without disappearing of HBe antigen and without decrease of alanine aminotransferase concentration showing lamivudine resistant hepatitis B virus. Mutations in the YMDD motif of the DNA polymerase gene were identified in 11 patients (3 with M550V/I mutation; 7 with M550V/I and L256M mutations; 1 with M550V/I, L526M and V519L mutations). In 6 of these patients, was found a M184V mutation in the VIH polymerase. No correlation could be observed between the mutations detected in the two viruses. Using a sensitive HBV-DNA assay, efficacy of lamivudine for a long time in HBV infected patients was proved. However, the prevalence of lamivudine resistance is related to duration of treatment and it may be necessary to use a multitherapy.
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PMID:[Evaluation of a quantitative HBV-DNA PCR assay in lamivudine treated hepatitis B-infected patients]. 1236 44

Adefovir is a potent nucleotide analog inhibitor of hepatitis B virus (HBV) DNA polymerase. Its oral prodrug adefovir dipivoxil has been approved for the treatment of chronic HBV infection. In this study, adefovir was characterized for its in vitro effects on mitochondrial DNA (mtDNA) synthesis and compared with the nucleoside analogues lamivudine (3TC), fialuridine (FIAU), and zalcitabine (ddC). No substantial changes in mtDNA content were detected in human hepatoblastoma HepG2 cells and normal human skeletal muscle cells following a 9-day treatment with 0.3-30 microm adefovir, concentrations up to 500-fold higher than the peak serum levels in patients treated with adefovir dipivoxil. Similarly, mtDNA was unchanged in both cell types following treatment with 3TC. In contrast, 30-55% and > 90% reductions in mtDNA were observed following incubation with 30 microm FIAU and ddC, respectively. The effects of FIAU on mtDNA became more pronounced following prolonged 18-day treatment of skeletal muscle cells while the effects of other drugs remained unchanged.
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PMID:Comparative effects of adefovir and selected nucleoside inhibitors of hepatitis B virus DNA polymerase on mitochondrial DNA in liver and skeletal muscle cells. 1255 12

Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side-effects by inducing mitochondrial toxicity. We and others have demonstrated that the mitochondrial polymerase, DNA polymerase gamma (pol gamma), participates in mitochondrial toxicity by incorporating these chain-terminating antiviral nucleotide analogs into DNA. Here, we explore the role of three highly conserved amino acid residues in the active site of human pol gamma that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. Steady-state kinetic analysis of the pol gamma derivatives with the normal and antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol gamma to incorporate dideoxynucleotides and D4T-MP. Mutation of Tyr951 to Phe renders the enzyme resistant to dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal nucleotides, producing dramatic increases in K(m(dNTP)) and large decreases in k(cat). Mutation of Tyr955 in pol gamma causes the degenerative disease progressive external ophthalmoplegia in humans, and we show that this residue partially accounts for the ability of pol gamma to incorporate D4T-MP and carbovir. Alteration of Glu895 to Ala slightly increased discrimination against dideoxynucleotides and D4T-TP. The mechanisms by which pol gamma selects certain nucleotide analogs are discussed.
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PMID:Structural determinants in human DNA polymerase gamma account for mitochondrial toxicity from nucleoside analogs. 1274 17

Incorporation of nucleoside analogues by the mitochondrial DNA polymerase has been implicated as the primary cause underlying many of the toxic side effects of these drugs in HIV therapy. Recent success in reconstituting recombinant human enzyme has afforded a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. The toxic side effects of nucleoside analogues are correlated with the kinetics of incorporation by the mitochondrial DNA polymerase, varying over 6 orders of magnitude in the sequence zalcitabine (ddC) > didanosine (ddI metabolized to ddA) > stavudine (d4T) >> lamivudine (3TC) > tenofovir (PMPA) > zidovudine (AZT) > abacavir (metabolized to carbovir, CBV). In this review, we summarize our current efforts to examine the mechanistic basis for nucleotide selectivity by the mitochondrial DNA polymerase and its role in mitochondrial toxicity of nucleoside analogues used to treat AIDS and other viral infections. We will also discuss the promise and underlying challenges for the development of new analogues with lower toxicity.
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PMID:Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. 1467 45

The use of L(-)SddC [beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC)] for the treatment of Herpes B virus (HBV) infection is hindered by the emergence of drug-resistance associated with the L526M, L550V, and L526M/M550V mutations of the viral DNA polymerase (DP). The interactions of the anti-HBV compounds 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorode-oxycytidine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil triphosphate with HBV DP and its L(-)SddC-associated mutants have not been studied. The e antigen-negative variant of HBV associated with the G1896A mutation in the precore region has a high prevalence. Its effect on HBV DP is unclear. Because HBV DNA synthesis occurs in the nucleocapsid, we examined the kinetics of the reverse transcriptase activity from wild-type (wt) and mutated DPs with the wt or G1896A-mutated RNA template in the nucleocapsid. The effects of this template mutation on the activities of these L-nucleoside triphosphates were also examined. Results indicated that these DP mutations increased the Km values of deoxy-NTPs and decreased the efficiencies (Vmax/Km) of DPs. The additional L526M mutation increased the efficiency of the M550V-mutated DP but no more than that of the L526M-mutated DP. The G1896A mutation had impacts on the interactions between different DPs and deoxy-NTPs, except dCTP. It also had different impacts on the actions of the L-nucleoside triphosphates toward DPs. The L526M and M550V mutations caused a greater decrease in the Vmax using the wt RNA template compared with the G1896A-mutated template. The L526M, M550V, and L526M/M550V mutations caused varying degrees of resistance to the different M-nucleoside triphosphates.
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PMID:Reverse transcriptase activity of hepatitis B virus (HBV) DNA polymerase within core capsid: interaction with deoxynucleoside triphosphates and anti-HBV L-deoxynucleoside analog triphosphates. 1474 82


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