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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 -
Antimicrobial Agents and Chemotherapy Dec 1997Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase...
Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.
Topics: Antiviral Agents; Cells, Cultured; Cytomegalovirus; Cytomegalovirus Infections; DNA Replication; DNA, Viral; Drug Resistance, Microbial; Enzyme Inhibitors; Fibroblasts; Ganciclovir; Guanine; Humans; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Viral Proteins
PubMed: 9420038
DOI: 10.1128/AAC.41.12.2680 -
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 -
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 -
Genitourinary Medicine Jun 1997
Review
Topics: AIDS-Related Opportunistic Infections; Antiviral Agents; Cidofovir; Clinical Trials as Topic; Cytomegalovirus Retinitis; Cytosine; Foscarnet; Ganciclovir; Guanine; Humans; Organophosphonates; Organophosphorus Compounds; Prodrugs; Prospective Studies; Recurrence
PubMed: 9306895
DOI: 10.1136/sti.73.3.169 -
Antimicrobial Agents and Chemotherapy Jun 1997Drugs commonly administered to patients infected with the human immunodeficiency virus (HIV) have been studied for their propensity to alter the intracellular...
Drugs commonly administered to patients infected with the human immunodeficiency virus (HIV) have been studied for their propensity to alter the intracellular phosphorylation of the anti-HIV nucleoside analog stavudine (2',3'-dideoxy-2',3'-didehydrothymidine; d4T) in peripheral blood mononuclear cells (PBMCs) and U937 cells in vitro. PBMCs isolated from the blood of healthy volunteers were stimulated by the mitogen phytohemagglutinin (10 microg/ml) for 72 h. Stimulated PBMCs (3 x 10(6) cells/plate) were then incubated with [3H]d4T (0.65 microCi; 3 microM) and either acyclovir, dapsone, ddC, ddI, fluconazole, foscarnet, ganciclovir, itraconazole, lobucavir, ranitidine, ribavirin, rifampin, sorivudine, sulfamethoxazole, trimethoprim, lamivudine (3TC), zidovudine, or thymidine (30 and 300 microM) for 24 h. Doxorubicin and drugs showing some evidence of inhibition were also studied at 0.3 and 3 microM. Cells were extracted overnight with 60% methanol prior to analysis by radiometric high-performance liquid chromatography. Additional data for nine of the drugs were obtained by incubation with [3H]d4T in U937 cells for 24 h. The effect of d4T (0.2 to 20 microM) on zidovudine (0.65 microCi; 0.018 microCi) phosphorylation was also studied. Zidovudine significantly reduced d4T total phosphates in PBMCs and U937 cells (in PBMCs to 33% [P < 0.001] and 17% [P < 0.001] of that in control cells at 3 and 30 microM, respectively). A small reduction in zidovudine phosphorylation was seen with d4T but only at d4T:zidovudine ratios of 100 and 1,000. Of the other compounds screened, only thymidine, ribavirin, and doxorubicin produced inhibition of d4T phosphorylation in both PBMCs and U937 cells. However, doxorubicin was cytotoxic at 3 microM. The decrease in d4T phosphorylation in the presence of ribavirin is consistent with previous findings with zidovudine. Although ddC significantly inhibited the phosphorylation of d4T in PBMCs, this was not seen in U937 cells, and it is probable that the findings in PBMCs are related to mitochondrial toxicity [based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cytotoxicity assay]. The only drugs screened which may interfere with d4T phosphorylation at clinically relevant concentrations were zidovudine, ribavirin, and doxorubicin.
Topics: Anti-HIV Agents; Cells, Cultured; Drug Interactions; Humans; Leukemia, Monocytic, Acute; Leukocytes, Mononuclear; Phosphorylation; Phytohemagglutinins; Stavudine; Stimulation, Chemical; Thymidine; Zidovudine
PubMed: 9174176
DOI: 10.1128/AAC.41.6.1231 -
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 -
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 Feb 1990Two newly synthesized carbocyclic oxetanocin analogs, (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine (cyclobut-A) and (+/-)-9-[(1 beta,2...
Two newly synthesized carbocyclic oxetanocin analogs, (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine (cyclobut-A) and (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine (cyclobut-G) were tested for activity against the infectivity of human immunodeficiency virus (HIV) in vitro. A number of other carbocyclic oxetanocin analogs failed to exert good antiretroviral effects. Both cyclobut-A and cyclobut-G protected CD4+ ATH8 cells against the infectivity and cytopathic effect of HIV type 1 (HIV-1) and suppressed proviral DNA synthesis in ATH8 cells exposed to HIV-1 in vitro at concentrations of 50 to 100 microM. These compounds also inhibited the in vitro infectivity of another human pathogenic retrovirus, HIV-2. Furthermore, both compounds completely suppressed the replication of a monocytotropic strain of HIV-1 in monocytes and macrophages at concentrations as low as 0.5 microM, as assessed by inhibition of HIV-1 p24 gag protein production. We also found that 2'-deoxyguanosine readily reversed the antiretroviral activity of cyclobut-G in our system, whereas the activity of cyclobut-A was hardly reversed by 2'-deoxyadenosine or 2'-deoxycytidine. We noted, however, that these compounds inhibited the proliferation of peripheral blood mononuclear cells at concentrations of greater than or equal to 100 microM in vitro. Although both cyclobut-A and cyclobut-G appear to have a certain level of in vitro toxicity, our observations may have theoretical and clinical implications in understanding the structure-activity relationships of antiretroviral agents active against HIV.
Topics: Adenine; Antiviral Agents; Blotting, Southern; Cell Line; Cytopathogenic Effect, Viral; Guanine; HIV-1; HIV-2; Humans; Macrophage Activation; Macrophages; Mitogens; Monocytes; Nucleosides; T-Lymphocytes; Virus Replication
PubMed: 2327778
DOI: 10.1128/AAC.34.2.287 -
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