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Acta Pharmacologica Sinica Jul 2004To elucidate the potentiality of emergence of drug-resistance to carbocyclic oxetanocin G (C.OXT-G), a new effective antiviral drug for herpetic keratitis during...
AIM
To elucidate the potentiality of emergence of drug-resistance to carbocyclic oxetanocin G (C.OXT-G), a new effective antiviral drug for herpetic keratitis during treatment and the mechanism of this drug resistance.
METHODS
A C.OXT-G resistant strain (C.OXT-Gr) was established by serially propagating the herpes simplex virus (HSV) -1 in African green monkey kidney (VERO) cells in the presence of C.OXT-G. After the drug sensitivity assay and the thymidine kinase (TK) activity assay, the molecular basis for the drug resistance was studied using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and PCR direct sequencing technology.
RESULTS
After the 10th passage in 10 microm C.OXT-G, the ED50 of the C.OXT-Gr was 17.08-fold greater than that of the original strain on the average and the TK activities of these resistant strains were extremely reduced. PCR-SSCP analysis on TK gene of the wild HSV-1 and the C.OXT-Gr showed altered migration patterns in part 3 and part 4, while PCR-SSCP analysis on DNA polymerase gene showed no difference among the viruses. Sequence analysis revealed a deletion of G at position of 430 that caused frameshift, resulting in premature termination in the TK gene.
CONCLUSION
The drug resistance to C.OXT-G may appear during the treatment due to the deficiency of TK activity caused by a single mutation in the TK gene of HSV-1.
Topics: Animals; Antiviral Agents; Chlorocebus aethiops; DNA, Viral; Drug Resistance, Viral; Gene Deletion; Guanine; Herpesvirus 1, Human; Mutation; Nucleic Acid Synthesis Inhibitors; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Thymidine Kinase; Vero Cells
PubMed: 15210066
DOI: No ID Found -
Journal of Virology May 2001Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon...
Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutations on the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitro HBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by determination of inhibition constants (K(i)). Recognition of the natural substrate, dCTP, was assessed by determination of Km values. The results from the in vitro studies were as follows: (i) dCTP substrate binding was largely unaffected by the mutations, with Km changing moderately, only in a range of 0.6 to 2.6-fold; (ii) K(i)s for 3TCTP and FTCTP against Met552Ile/Val mutant HBV polymerases were increased 8- to 30-fold; and (iii) the Leu528Met mutation had a modest effect on direct binding of these beta-L-oxathiolane ring-containing nucleotide analogs. A three-dimensional homology model of the catalytic core of HBV polymerase was constructed via extrapolation from retroviral reverse transcriptase structures. Molecular modeling studies using the HBV polymerase homology model suggested that steric hindrance between the mutant amino acid side chain and lamivudine or emtricitabine could account for the resistance phenotype. Specifically, steric conflict between the Cgamma2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring (common to both beta-L-nucleoside analogs lamivudine and emtricitabine) is proposed to account for the resistance observed upon Met552Ile/Val mutation. The effects of the Leu528Met mutation, which also occurs near the HBV polymerase active site, appeared to be less direct, potentially involving rearrangement of the deoxynucleoside triphosphate-binding pocket residues. These modeling results suggest that nucleotide analogs that are beta-D-enantiomers, that have the sulfur replaced by a smaller atom, or that have modified or acyclic ring systems may retain activity against lamivudine-resistant mutants, consistent with the observed susceptibility of these mutants to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.
Topics: Acyclovir; Adenine; Amino Acid Sequence; Antiviral Agents; Cytidine Triphosphate; Deoxycytidine; Dideoxynucleotides; Drug Resistance, Microbial; Emtricitabine; Guanine; Hepatitis B virus; Humans; Lamivudine; Models, Molecular; Molecular Sequence Data; Organophosphonates; Protein Conformation; RNA-Directed DNA Polymerase; Reverse Transcriptase Inhibitors; Sequence Homology, Amino Acid
PubMed: 11312349
DOI: 10.1128/JVI.75.10.4771-4779.2001 -
British Journal of Clinical Pharmacology Dec 2000Lamivudine (3TC, 2'-deoxy-3'-thiacytidine) requires intracellular metabolism to its active 5'-triphosphate, 3TC-5'-triphosphate (3TCTP), to inhibit the replication of...
The intracellular activation of lamivudine (3TC) and determination of 2'-deoxycytidine-5'-triphosphate (dCTP) pools in the presence and absence of various drugs in HepG2 cells.
AIMS
Lamivudine (3TC, 2'-deoxy-3'-thiacytidine) requires intracellular metabolism to its active 5'-triphosphate, 3TC-5'-triphosphate (3TCTP), to inhibit the replication of hepatitis B virus (HBV). We have investigated the activation of 3TC, in the presence and absence of a range of compounds, in HepG2 cells. The intracellular levels of the endogenous competitor of 3TCTP, 2'-deoxycytidine-5'-triphosphate (dCTP), were also determined and 3TCTP/dCTP ratios calculated.
METHODS
The effects of a number of compounds on 3TC (3H; 1 microM) phosphorylation were investigated by radiometric h.p.l.c. dCTP levels were determined using a template primer extension assay. 3TCTP/dCTP ratios were calculated from these results.
RESULTS
The phosphorylation of 3TC was significantly increased in the presence of either hydroxyurea (HU), methotrexate (MTX), or fludarabine (FLU). For example, at 100 microM HU, control 3TCTP levels were increased to 361% of control, whereas at 100 microM FLU, control 3TCTP levels were increased to 155%. dCTP pools were significantly reduced in the presence of HU and FLU, at 100 microM concentrations only. However, for all the above three compounds investigated, the ratio of 3TCTP/dCTP was favourably enhanced (e.g. at 1 microM MTX, 255% of control). Neither ganciclovir (GCV), lobucavir (LCV), penciclovir (PCV), adefovir dipivoxil (ADV), nor foscarnet (FOS) had any significant effects on 3TC phosphorylation or dCTP pools.
CONCLUSIONS
These results suggest that the activity of 3TC may be potentiated when combined with one of the modulators studied. The lack of an interaction between 3TC and the other anti-HBV agents is reassuring. These in vitro studies can be used as an initial screen to examine potential interactions at the phosphorylation level.
Topics: Anti-HIV Agents; Cell Survival; Cytidine Triphosphate; Deoxycytosine Nucleotides; Dideoxynucleotides; Drug Interactions; Humans; Hydroxyurea; Lamivudine; Methotrexate; Phosphorylation; Tumor Cells, Cultured; Vidarabine
PubMed: 11136299
DOI: 10.1046/j.1365-2125.2000.00302.x -
Journal of Viral Hepatitis May 1999Significant advances have been made, during the last 5 years, in the treatment of chronic hepatitis B. Several new antiviral agents: lamivudine, famciclovir, lobucavir... (Review)
Review
Significant advances have been made, during the last 5 years, in the treatment of chronic hepatitis B. Several new antiviral agents: lamivudine, famciclovir, lobucavir and adefovir, have been shown to be safe and effective in inhibiting hepatitis B virus (HBV) replication. These compounds can be administered orally and are well tolerated. However, virus clearance is uncommon after short courses (<6 months) of therapy. Lamivudine and famciclovir have been evaluated in Phase III clinical trials in patients with chronic hepatitis B as well as in liver transplant recipients. Unfortunately, drug-resistant mutants involving the HBV polymerase gene, leading to breakthrough infection, have been reported in some patients who have received long courses (>/= 12 months) of treatment. The incidence, clinical outcome and biological significance of these mutants will be reviewed.
Topics: Amino Acid Sequence; Antiviral Agents; DNA-Directed DNA Polymerase; Drug Resistance, Microbial; Hepatitis B; Hepatitis B virus; Humans; Molecular Sequence Data; Mutation; RNA-Directed DNA Polymerase; Ribonuclease H
PubMed: 10607230
DOI: 10.1046/j.1365-2893.1999.00160.x -
Antimicrobial Agents and Chemotherapy Dec 1999Hydroxyurea has been shown to potentiate the anti-human immunodeficiency virus activities of 2',3'-dideoxynucleoside analogs such as didanosine. We have now evaluated in...
Hydroxyurea has been shown to potentiate the anti-human immunodeficiency virus activities of 2',3'-dideoxynucleoside analogs such as didanosine. We have now evaluated in vitro the effect of hydroxyurea on the antiherpesvirus activities of several nucleoside analogs (acyclovir [ACV], ganciclovir [GCV], penciclovir [PCV], lobucavir [LBV], (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine [H2G], and brivudin and nucleoside phosphonate analogs (cidofovir [CDV] and adefovir [ADV]). When evaluated in cytopathic effect (CPE) reduction assays, hydroxyurea by itself had little effect on CPE progression and potentiated in a subsynergistic (herpes simplex virus type 1 [HSV-1]) to synergistic (HSV-2) fashion the antiviral activities of ACV, GCV, PCV, LBV, H2G, ADV, and CDV. Hydroxyurea also caused marked increases in the activities of ACV, GCV, PCV, LBV, and H2G (compounds that depend for their activation on a virus-encoded thymidine kinase [TK]) against TK-deficient (TK(-)) HSV-1. In fact, in combination with hydroxyurea the 50% effective concentrations of these compounds for inhibition of TK(-) HSV-1-induced CPE decreased from values of 20 to > or = 100 microg/ml (in the absence of hydroxyurea) to values of 1 to 5 microg/ml (in the presence of hydroxyurea at 25 to 100 microg/ml). When evaluated in a single-cycle virus yield reduction assay, hydroxyurea at a concentration of 100 microg/ml inhibited progeny virus production by 60 to 90% but had little effect on virus yield at a concentration of 25 microg/ml. Under these assay conditions hydroxyurea still elicited a marked potentiating effect on the antiherpesvirus activities of GCV and CDV, but this effect was less pronounced than that in the CPE reduction assay. It is conceivable that the potentiating effect of hydroxyurea stems from a depletion of the intracellular deoxynucleoside triphosphate pools, thus favoring the triphosphates of the nucleoside analogues (or the diphosphates of the nucleoside phosphonate analogues) in their competition with the natural nucleotides at the viral DNA polymerase level. The possible clinical implications of these findings are discussed.
Topics: Animals; Antiviral Agents; Cell Division; Cell Survival; Chlorocebus aethiops; Drug Synergism; HIV Infections; Herpesvirus 1, Human; Humans; Hydroxyurea; Nucleic Acid Synthesis Inhibitors; Nucleosides; Organophosphonates; Purine Nucleosides; Pyrimidine Nucleosides; Vero Cells; Virus Replication
PubMed: 10582877
DOI: 10.1128/AAC.43.12.2885 -
BioTechniques Oct 1999We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the...
We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) into inhibitors of cell growth. The method is based on the use of budding yeast (Saccharomyces cerevisiae) transformed with the HSV-1 TK gene on a multicopy plasmid. Eight nucleoside analogs (acyclovir, ganciclovir, penciclovir, lobucavir, brivudin, sorivudine, IVDU and ara-T), for which the cytostatic action against mammalian cells expressing the HSV-1 TK gene has been well documented, were studied for their inhibitory effect on the growth of yeast expressing the viral TK. These nucleoside analogs had little or no inhibitory effect on the growth of yeasts transformed with the empty vector, but inhibited to a significant extent the growth of yeast expressing the viral TK. Use of HSV-1 TK-expressing yeast allows quick screening in multi-well plate format for compounds with potential use in HSV-1 TK suicide gene therapy. The method may also be used as a tool to selectively suppress or arrest the growth of one population of yeast out of mixed yeast cell cultures.
Topics: Acyclovir; Antiviral Agents; Gene Expression; Genetic Therapy; Genetic Vectors; Herpesvirus 1, Human; Plasmids; Promoter Regions, Genetic; Saccharomyces cerevisiae; Thymidine Kinase; Transfection
PubMed: 10524320
DOI: 10.2144/99274st08 -
The Journal of Clinical Investigation Jun 1999The emergence of resistant hepatitis B virus (HBV), with mutations in the YMDD motif of the polymerase gene after treatment with lamivudine, is becoming an important...
The emergence of resistant hepatitis B virus (HBV), with mutations in the YMDD motif of the polymerase gene after treatment with lamivudine, is becoming an important clinical problem. In this study, susceptibility of wild-type and lamivudine-resistant HBV M552I, M552V, and L528M/M552V mutants to other reverse transcriptase inhibitors was investigated by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, which showed the presence of a single-stranded band (representative of the HBV replicative intermediates) in the drug-free, wild-type HBV-transfected cells. This band was diminished in the samples of wild-type HBV DNA treated with either lamivudine, adefovir, or lobucavir. The band intensities from the lamivudine-resistant mutants were not decreased by treatment with lamivudine, but were decreased by the treatments with adefovir or lobucavir. In contrast, penciclovir and nevirapine did not diminish the intensity of the single-stranded band of wild-type HBV or the lamivudine-resistant mutants. These results demonstrate that lamivudine-resistant HBV is susceptible to adefovir and lobucavir. Lamivudine-resistant HBV should be treated with adefovir or lobucavir, and combination therapy with lamivudine and adefovir/lobucavir may prevent the emergence of lamivudine-resistant HBV.
Topics: Acyclovir; Adenine; Carcinoma, Hepatocellular; Drug Resistance, Microbial; Guanine; Hepatitis B virus; Humans; Lamivudine; Mutagenesis, Site-Directed; Nevirapine; Organophosphonates; Reverse Transcriptase Inhibitors; Transfection; Tumor Cells, Cultured
PubMed: 10377169
DOI: 10.1172/JCI5882 -
Antimicrobial Agents and Chemotherapy Jan 1999BMS-200475 was recently shown to have potent antiviral activity against hepatitis B virus (50% effective concentration = 3.7 nM; 50% cytotoxic concentration = 30...
BMS-200475 was recently shown to have potent antiviral activity against hepatitis B virus (50% effective concentration = 3.7 nM; 50% cytotoxic concentration = 30 microM). In metabolic studies in both HepG2 and hepatitis B virus-transfected 2.2.15 human hepatoma cell lines, the metabolism was similar, the primary products being the di- and triphosphates. The accumulation of triphosphate was rapid and detectable down to a 5 nM concentration of added drug. When cells were labeled at 25 microM, the intracellular triphosphate concentration attained 30 pmol/10(6) cells ( approximately 30 microM). The intracellular half-life of the triphosphate was about 15 h. Compared with five other nucleoside analogs of medical interest (lamivudine, penciclovir, ganciclovir, acyclovir, and lobucavir), BMS-200475 was most efficiently phosphorylated to the triphosphate in HepG2 cells.
Topics: Antiviral Agents; Deoxyguanosine; Half-Life; Hepatitis B virus; Humans; Phosphorylation; Spectrophotometry, Ultraviolet; Transfection; Tumor Cells, Cultured
PubMed: 9869593
DOI: 10.1128/AAC.43.1.190 -
Antimicrobial Agents and Chemotherapy Dec 1998The guanosine analogs BMS-200475 and lobucavir have previously been shown to effectively suppress propagation of the human hepatitis B virus (HBV) and woodchuck...
The guanosine analogs BMS-200475 and lobucavir have previously been shown to effectively suppress propagation of the human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) in 2.2.15 liver cells and in the woodchuck animal model system, respectively. This repression was presumed to occur via inhibition of the viral polymerase (Pol) by the triphosphate (TP) forms of BMS-200475 and lobucavir which are both produced in mammalian cells. To determine the exact mode of action, BMS-200475-TP and lobucavir-TP, along with several other guanosine analog-TPs and lamivudine-TP were tested against the HBV, WHV, and duck hepatitis B virus (DHBV) polymerases in vitro. Estimates of the 50% inhibitory concentrations revealed that BMS-200475-TP and lobucavir-TP inhibited HBV, WHV, and DHBV Pol comparably and were superior to the other nucleoside-TPs tested. More importantly, both analogs blocked the three distinct phases of hepadnaviral replication: priming, reverse transcription, and DNA-dependent DNA synthesis. These data suggest that the modest potency of lobucavir in 2.2.15 cells may be the result of poor phosphorylation in vivo. Kinetic studies revealed that BMS-200475-TP and lobucavir-TP competitively inhibit HBV Pol and WHV Pol with respect to the natural dGTP substrate and that both drugs appear to bind to Pol with very high affinities. Endogenous sequencing reactions conducted in replicative HBV nucleocapsids suggested that BMS-200475-TP and lobucavir-TP are nonobligate chain terminators that stall Pol at sites that are distinct yet characteristically two to three residues downstream from dG incorporation sites.
Topics: Antiviral Agents; DNA-Directed RNA Polymerases; Deoxyguanosine; Enzyme Inhibitors; Guanine; Hepadnaviridae; Hepatitis B Virus, Duck; Hepatitis B Virus, Woodchuck; Hepatitis B virus; Humans; Kinetics; Nucleic Acid Synthesis Inhibitors; Phosphates; RNA-Directed DNA Polymerase; Structure-Activity Relationship
PubMed: 9835515
DOI: 10.1128/AAC.42.12.3200 -
Journal of Virology Jul 1998A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to...
Characterization of drug resistance-associated mutations in the human cytomegalovirus DNA polymerase gene by using recombinant mutant viruses generated from overlapping DNA fragments.
A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to antiviral agents and consequently with clinical failure. To precisely determine the roles of UL54 mutations in HCMV drug resistance, recombinant UL54 mutant viruses were generated by using cotransfection of nine overlapping HCMV DNA fragments into permissive fibroblasts, and their drug susceptibility profiles were determined. Amino acid substitutions located in UL54 conserved region IV (N408D, F412C, and F412V), region V (A987G), and delta-region C (L501I, K513E, P522S, and L545S) conferred various levels of resistance to cidofovir and ganciclovir. Mutations in region II (T700A and V715M) and region VI (V781I) were associated with resistance to foscarnet and adefovir. The region II mutations also conferred moderate resistance to lobucavir. In contrast to mutations in other UL54 conserved regions, those residing specifically in region III (L802M, K805Q, and T821I) were associated with various drug susceptibility profiles. Mutations located outside the known UL54 conserved regions (S676G and V759M) did not confer any significant changes in HCMV drug susceptibility. Predominantly an additive effect of multiple UL54 mutations with respect to the final drug resistance phenotype was demonstrated. Finally, the influence of selected UL54 mutations on the susceptibility of viral DNA replication to antiviral drugs was characterized by using a transient-transfection-plus-infection assay. Results of this work exemplify specific roles of the UL54 conserved regions in the development of HCMV drug resistance and may help guide optimization of HCMV therapy.
Topics: Antiviral Agents; Cidofovir; Cytomegalovirus; Cytosine; DNA Replication; DNA, Viral; DNA-Directed DNA Polymerase; Drug Resistance, Microbial; Foscarnet; Ganciclovir; Genes, Viral; Humans; Mutation; Organophosphonates; Organophosphorus Compounds; Recombination, Genetic; Virus Replication
PubMed: 9621055
DOI: 10.1128/JVI.72.7.5927-5936.1998