17 results on '"Fyfe JA"'
Search Results
2. Mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine against herpesviruses.
- Author
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Lowe DM, Alderton WK, Ellis MR, Parmar V, Miller WH, Roberts GB, Fyfe JA, Gaillard R, Ertl P, and Snowden W
- Subjects
- Antiviral Agents metabolism, Base Sequence, Cell Survival drug effects, Cells, Cultured, Chromatography, High Pressure Liquid, DNA, Viral analysis, Guanine metabolism, Guanine pharmacology, Herpesviridae enzymology, Herpesvirus 1, Human drug effects, Herpesvirus 1, Human enzymology, Herpesvirus 2, Human drug effects, Herpesvirus 2, Human enzymology, Herpesvirus 3, Human drug effects, Herpesvirus 3, Human enzymology, Humans, Kinetics, Molecular Sequence Data, Nucleic Acid Synthesis Inhibitors, Phosphorylation, Thymidine Kinase metabolism, Antiviral Agents pharmacology, Guanine analogs & derivatives, Herpesviridae drug effects
- Abstract
The activity, metabolism, and mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) were studied. Compared to acyclovir (ACV), H2G has superior activity against VZV (50% inhibitory concentration of 2.3 microM) and Epstein-Barr virus (50% inhibitory concentration of 0.9 microM), comparable activity against HSV-1, and weaker activity against HSV-2. The antiviral effect on HSV-1 showed persistence after removal of compound. H2G was metabolized to its mono-, di- and triphosphate derivatives in virus-infected cells, with H2G-triphosphate being the predominant product. Only small amounts of H2G-triphosphate were detected in uninfected cells (1 to 10 pmol/10(6) cells), whereas the level in HSV-1-infected cells reached 1,900 pmol/10(6) cells. H2G was a substrate for all three viral thymidine kinases and could also be phosphorylated by mitochondrial deoxyguanosine kinase. The intracellular half-life of H2G-triphosphate varied in uninfected (2.5 h) and infected (HSV-1, 14 h; VZV, 3.7 h) cells but was always longer than the half-life of ACV-triphosphate (1 to 2 h). H2G-triphosphate inhibited HSV-1, HSV-2, and VZV DNA polymerases competitively with dGTP (Ki of 2.8, 2.2, and 0.3 microM, respectively) but could not replace dGTP as a substrate in a polymerase assay. H2G was not an obligate chain terminator but would only support limited DNA chain extension. Only very small amounts of radioactivity, which were too low to be identified by high-performance liquid chromatography analysis of the digested DNA, could be detected in purified DNA from uninfected cells incubated with [3H]H2G. Thus, H2G acts as an anti-herpesvirus agent, particularly potent against VZV, by formation of high concentrations of relatively stable H2G-triphosphate, which is a potent inhibitor of the viral DNA polymerases.
- Published
- 1995
- Full Text
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3. The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.
- Author
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Furman PA, Davis M, Liotta DC, Paff M, Frick LW, Nelson DJ, Dornsife RE, Wurster JA, Wilson LJ, and Fyfe JA
- Subjects
- Animals, Antiviral Agents toxicity, Cell Survival drug effects, Cytidine Deaminase metabolism, DNA, Viral biosynthesis, DNA, Viral drug effects, Emtricitabine analogs & derivatives, Growth Inhibitors toxicity, Hepatitis B virus genetics, Humans, Macaca fascicularis, Phosphorylation drug effects, Stereoisomerism, Substrate Specificity, Zalcitabine pharmacology, Zalcitabine toxicity, Antiviral Agents pharmacology, Hepatitis B virus drug effects, Zalcitabine analogs & derivatives
- Abstract
The anti-hepatitis B (anti-HBV) activities of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2'-deoxy-3'-thia-5-fluorocytosine [FTC]) were studied by using an HBV-transfected cell line (HepG2 derivative 2.2.15, subclone P5A). The (-) isomer was found to be a potent inhibitor of viral replication, with an apparent 50% inhibitory concentration of 10 nM, while the (+) isomer was found to be considerably less active. Both isomers showed minimal toxicity to HepG2 cells (50% inhibitory concentration, > 200 microM) and showed minimal toxicity in the human bone marrow progenitor cell assay. In accord with the cellular antiviral activity data, the 5'-triphosphate of (-)-FTC inhibited viral DNA synthesis in an endogenous HBV DNA polymerase assay, while the 5'-triphosphate of the (+) isomer was inactive. Unphosphorylated (-)-FTC did not inhibit product formation in the endogenous assay, suggesting that the antiviral activity of the compound is dependent on anabolism to the 5'-triphosphate. Both (-)- and (+)-FTC were anabolized to the corresponding 5'-triphosphates in chronically HBV-infected HepG2 cells. The rate of accumulation and the steady-state concentration of the 5'-triphosphate of (-)-FTC were greater. Also, (-)-FTC was not a substrate for cytidine deaminase and, therefore, is not subject to deamination and conversion to an inactive uridine analog. The (+) isomer is, however, a good substrate for cytidine deaminase.
- Published
- 1992
- Full Text
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4. Mutant varicella-zoster virus thymidine kinase: correlation of clinical resistance and enzyme impairment.
- Author
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Roberts GB, Fyfe JA, Gaillard RK, and Short SA
- Subjects
- Acyclovir pharmacology, Amino Acid Sequence, Antibodies isolation & purification, Antiviral Agents pharmacology, Cloning, Molecular, Codon genetics, Drug Resistance, Microbial physiology, Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Genes, Viral, Herpesvirus 3, Human drug effects, Herpesvirus 3, Human genetics, Kinetics, Molecular Weight, Mutagenesis, Site-Directed, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Thymidine Kinase isolation & purification, Thymidine Kinase metabolism, Viral Structural Proteins genetics, Acyclovir metabolism, Antiviral Agents metabolism, Herpesvirus 3, Human enzymology, Thymidine Kinase genetics
- Abstract
Varicella-zoster virus (VZV) encodes a thymidine kinase (EC 2.7.2.21) which phosphorylates several antiviral nucleoside analogs, including acyclovir (ACV). A mutation in the VZV thymidine kinase coding sequence, resulting in an arginine-to-glutamine substitution at amino acid residue 130 (R130Q), is associated with clinical resistance to ACV. We have expressed the wild-type and the mutant enzymes in bacteria and have studied the kinetic characteristics of the purified enzymes. The arginine-to-glutamine substitution resulted in decreased catalytic activity and altered substrate specificity. The most striking effect was a decrease in the rates of nucleoside phosphorylation to less than 2% of the rates with the wild-type enzyme. This was accompanied by increased apparent Km values for thymidine and deoxycytidine. ACV was not detectably phosphorylated by the R130Q enzyme but still competed with thymidine for the enzyme. The inability of the R130Q enzyme to catalyze the phosphorylation of ACV correlates with resistance to ACV noted with a clinical isolate of VZV.
- Published
- 1991
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5. 6-N-substituted derivatives of adenine arabinoside as selective inhibitors of varicella-zoster virus.
- Author
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Koszalka GW, Averett DR, Fyfe JA, Roberts GB, Spector T, Biron K, and Krenitsky TA
- Subjects
- Adenosine Deaminase Inhibitors, Animals, Antiviral Agents pharmacology, Chemical Phenomena, Chemistry, Physical, L Cells drug effects, Magnetic Resonance Spectroscopy, Mice, Phosphotransferases antagonists & inhibitors, Vidarabine chemical synthesis, Vidarabine pharmacology, Antiviral Agents chemical synthesis, Herpesvirus 3, Human drug effects, Vidarabine analogs & derivatives
- Abstract
A series of 6-alkylaminopurine arabinosides were synthesized and found to inhibit varicella-zoster virus (VZV). The antiviral activities of these nucleosides were limited to VZV. None of the other viruses tested in the herpesvirus family were affected. The in vitro antiviral potencies of the 18 arabinosides correlated with their efficiencies as substrates of the VZV-encoded thymidine kinase in all but one case. The arabinosides of 6-methylaminopurine and 6-dimethylaminopurine were the most potent analogs, with 50% inhibitory concentrations against VZV of 3 and 1 microM, respectively. They were not cytotoxic to uninfected MRC-5 cells, human Detroit 98 cells, or mouse L cells (50% inhibitory concentration, greater than 100 microM). Neither 6-methylaminopurine arabinoside nor 6-dimethylaminopurine arabinoside was detectably phosphorylated by either adenosine kinase or 2'-deoxycytidine kinase. These two alkylaminopurine arabinosides were also resistant to deamination catalyzed by adenosine deaminase. The VZV-dependent phosphorylation of these nucleosides offers the possibility of a potent and highly selective therapy for VZV infection.
- Published
- 1991
- Full Text
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6. 6-Methoxypurine arabinoside as a selective and potent inhibitor of varicella-zoster virus.
- Author
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Averett DR, Koszalka GW, Fyfe JA, Roberts GB, Purifoy DJ, and Krenitsky TA
- Subjects
- Acyclovir pharmacology, Arabinonucleosides chemistry, Cell Survival drug effects, Cells, Cultured, Herpesvirus 3, Human enzymology, Humans, Kinetics, Thymidine Kinase metabolism, Vidarabine pharmacology, Antiviral Agents pharmacology, Arabinonucleosides pharmacology, Herpesvirus 3, Human drug effects
- Abstract
Seven 6-alkoxypurine arabinosides were synthesized and evaluated for in vitro activity against varicella-zoster virus (VZV). The simplest of the series, 6-methoxypurine arabinoside (ara-M), was the most potent, with 50% inhibitory concentrations ranging from 0.5 to 3 microM against eight strains of VZV. This activity was selective. The ability of ara-M to inhibit the growth of a variety of human cell lines was at least 30-fold less (50% effective concentration, greater than 100 microM) than its ability to inhibit the virus. Enzyme studies suggested the molecular basis for these results. Of the seven 6-alkoxypurine arabinosides, ara-M was the most efficient substrate for VZV-encoded thymidine kinase as well as the most potent antiviral agent. In contrast, it was not detectably phosphorylated by any of the three major mammalian nucleoside kinases. Upon direct comparison, ara-M was appreciably more potent against VZV than either acyclovir or adenine arabinoside (ara-A). However, in the presence of an adenosine deaminase inhibitor, the arabinosides of adenine and 6-methoxypurine were equipotent but not equally selective; the adenine congener had a much less favorable in vitro chemotherapeutic index. Again, this result correlated with data from enzyme studies in that ara-A, unlike ara-M, was a substrate for two mammalian nucleoside kinases. Unlike acyclovir and ara-A, ara-M had no appreciable activity against other viruses of the herpes group. The potency and selectivity of ara-M as an anti-VZV agent in vitro justify its further study.
- Published
- 1991
- Full Text
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7. Altered thymidine-thymidylate kinases from strains of herpes simplex virus with modified drug sensitivities to acyclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine.
- Author
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Fyfe JA, McKee SA, and Keller PM
- Subjects
- Bromodeoxyuridine pharmacology, Drug Resistance, Microbial, Genetic Code, Phosphorylation, Simplexvirus drug effects, Substrate Specificity, Acyclovir pharmacology, Antiviral Agents pharmacology, Bromodeoxyuridine analogs & derivatives, Nucleoside-Phosphate Kinase isolation & purification, Phosphotransferases isolation & purification, Simplexvirus enzymology, Thymidine Kinase isolation & purification
- Abstract
Virus-coded thymidine (dThd) kinases were purified by affinity chromatography from a parental strain (SC16) and two strains (SC16 B3 and SC16 S1) of herpes simplex virus, Type 1, with altered drug sensitivities. These latter two strains were less sensitive, respectively, to E-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd) and to both BrVdUrd and 9-(2-hydroxyethoxymethyl)guanine (acyclovir). The enzymes were characterized with respect to physical and catalytic properties. The enzyme from SC16 B3 was very similar to the parental enzyme except in its substrate specificity and kinetic constants. It catalyzed the phosphorylation of BrVdUrd at a relative rate that was 110% of the rate with dThd versus a relative rate of 140% with the parental enzyme. The apparent Km value for BrVdUrd was 6 microM versus 0.1 microM for the parental enzyme. The reaction kinetics with acyclovir were similar for the two enzymes. The SC16 B3 enzyme catalyzed the phosphorylation of dTMP, but at only 2% the efficiency of the parental enzyme; phosphorylation of the monophosphate of BrVdUrd (BrVdUMP) was not detected with the SC16 B3 enzyme. The enzyme from the SC16 S1 variant had a much narrower phosphate acceptor specificity than the enzyme from the parental virus. BrVdUrd was a substrate but with a relative rate of 30% and an apparent Km value of 4 microM; acyclovir was neither detectably phosphorylated nor a good inhibitor. BrVdUMP was not detectably phosphorylated. The relative efficiencies of the two variant enzymes for acyclovir phosphorylation correlated well with the sensitivities of the viruses to this compound. In contrast, the relative efficiencies of the second phosphorylation step (BrVdUMP to BrVdUDP) were most consistent with the sensitivities of the viruses to BrVdUrd.
- Published
- 1983
8. Activation and antiviral effect of acyclovir in cells infected with a varicella-like simian virus.
- Author
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Fyfe JA, Biron KK, McKee SA, Kelly CM, Elion GB, and Soike KF
- Subjects
- Acyclovir, Animals, Cell Line, Chlorocebus aethiops, Guanine metabolism, Guanine pharmacology, Herpesviridae enzymology, Herpesvirus 3, Human drug effects, Kinetics, Phosphorylation, Thymidine Kinase metabolism, Antiviral Agents pharmacology, Guanine analogs & derivatives, Herpesviridae drug effects
- Abstract
Acyclovir inhibited the replication of a varicella-like simian virus (DHV-1) in cell culture (Vero cells) with an ED50 of 38 +/- 2 microM. The activation of acyclovir in this cell culture system was compared with that in the cell system with human varicella zoster virus (VZV). Extracts of cells infected with DHV-1 catalyzed the phosphorylation of acyclovir. The phosphorylation was inhibited by dThd, suggesting the catalyst was a dThd kinase. Electrophoresis of cytosol fractions on polyacrylamide gels corroborated the existence of a virus-associated dThd kinase. This enzyme copurified with an acyclovir-phosphorylating activity. The enzyme catalyzed the phosphorylation of acyclovir at a greater relative rate than that with the VZV enzyme, but with a higher apparent Km value for acyclovir. The relative efficiencies for the two enzymes with acyclovir were similar. Anabolic studies with cells infected with DHV-1 and incubated with [14C]acyclovir indicated that triphosphate of acyclovir did accumulate. The results indicate that acyclovir is activated in cells infected with DHV-1 in a manner similar to that in cells infected with VZV.
- Published
- 1982
- Full Text
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9. Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine.
- Author
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Fyfe JA, Keller PM, Furman PA, Miller RL, and Elion GB
- Subjects
- Guanine pharmacology, Kinetics, Structure-Activity Relationship, Substrate Specificity, Antiviral Agents pharmacology, Guanine analogs & derivatives, Simplexvirus enzymology, Thymidine Kinase metabolism
- Published
- 1978
10. Selection and preliminary characterization of acyclovir-resistant mutants of varicella zoster virus.
- Author
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Biron KK, Fyfe JA, Noblin JE, and Elion GB
- Subjects
- Acyclovir, DNA-Directed DNA Polymerase metabolism, Drug Resistance, Microbial, Guanine metabolism, Guanine pharmacology, Herpesvirus 3, Human enzymology, Herpesvirus 3, Human genetics, Mutation, Phosphorylation, Thymidine Kinase metabolism, Antiviral Agents pharmacology, Guanine analogs & derivatives, Herpesvirus 3, Human drug effects
- Abstract
A series of acyclovir-resistant mutants of varicella zoster virus (VZV) were selected in vitro by serial passage of VZV-infected human fibroblasts in increasing drug concentrations, or by continuous exposure of cultures infected at high multiplicity to 100 microM acyclovir. The in vitro susceptibility of these mutants to several antiherpetic agents was measured by the plaque-reduction assay. The capacity of extracts of cells infected with these mutants to phosphorylate acyclovir was examined and compared with that of their acyclovir-sensitive parent strains. Based on these studies, VZV could be shown to acquire resistance to acyclovir through diminished acyclovir phosphorylation. This was presumable due to loss of viral specific thymidine kinase (TK) function. Two acyclovir-resistant mutants remained TK competent but demonstrated phenotypic changes in sensitivity to antiviral agents known to act at the herpes simplex virus (HSV)-specific DNA polymerase level. These results suggest that the resistance of VZV to acyclovir results from qualitative or quantitative alterations in the virus-specified TK or DNA polymerase.
- Published
- 1982
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11. Modifications on the heterocyclic base of acyclovir: syntheses and antiviral properties.
- Author
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Beauchamp LM, Dolmatch BL, Schaeffer HJ, Collins P, Bauer DJ, Keller PM, and Fyfe JA
- Subjects
- Acyclovir chemical synthesis, Acyclovir pharmacology, Chemical Phenomena, Chemistry, Simplexvirus drug effects, Simplexvirus enzymology, Thymidine Kinase antagonists & inhibitors, Acyclovir analogs & derivatives, Antiviral Agents chemical synthesis
- Abstract
A group of compounds was prepared in which variations of the ring portion of the acyclovir (ACV) structure were made. These modifications included monocyclic (isocytosine, triazole, imidazole), bicyclic (8-azapurine, pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine) and tricyclic (linear benzoguanine) congeners. The derivatives were evaluated against herpes simplex virus type 1 (HSV-1) by the plaque-inhibition and plaque-reduction methods with only the 8-azapurine analogue 28 showing some activity. In a test measuring the ability of these compounds to inhibit the HSV-1 thymidine kinase, 28 and the tricyclic derivative 38 exhibited competition with ACV for binding to the enzyme. The inability of the group to exert significant antiherpetic action is attributed to their lack of phosphorylation to the requisite triphosphate stage.
- Published
- 1985
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12. Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase.
- Author
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Furman PA, Fyfe JA, St Clair MH, Weinhold K, Rideout JL, Freeman GA, Lehrman SN, Bolognesi DP, Broder S, and Mitsuya H
- Subjects
- Antiviral Agents metabolism, Cell Survival drug effects, HIV enzymology, Humans, Kinetics, Nucleic Acid Synthesis Inhibitors, Phosphorylation, Thymidine metabolism, Thymidine pharmacology, Thymidine Monophosphate metabolism, Thymine Nucleotides analysis, Virus Replication drug effects, Zidovudine, Antiviral Agents pharmacology, HIV drug effects, Reverse Transcriptase Inhibitors, Thymidine analogs & derivatives
- Abstract
The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di- and triphosphate were low (less than or equal to 5 microM and less than or equal to 2 microM, respectively). Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent Km values of 2.9 microM and 3.0 microM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 microM vs. 4.1 microM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. IC50 (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase alpha. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase alpha. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.
- Published
- 1986
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13. Enzymatic phosphorylation of acyclic nucleoside analogs and correlations with antiherpetic activities.
- Author
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Keller PM, Fyfe JA, Beauchamp L, Lubbers CM, Furman PA, Schaeffer HJ, and Elion GB
- Subjects
- Animals, Chlorocebus aethiops, Kinetics, Nucleosides pharmacology, Phosphorylation, Simplexvirus drug effects, Structure-Activity Relationship, Substrate Specificity, Thymidine Kinase analysis, Virus Replication drug effects, Antiviral Agents metabolism, Nucleosides metabolism, Simplexvirus metabolism
- Published
- 1981
- Full Text
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14. Selectivity of antiviral effectiveness derived from differences of herpes simplex virus-coded thymidine kinases.
- Author
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Fyfe JA
- Subjects
- Animals, Bromodeoxyuridine toxicity, Cell Line, Chlorocebus aethiops, Kidney, Simplexvirus drug effects, Thymidine Kinase isolation & purification, Acyclovir toxicity, Antiviral Agents toxicity, Bromodeoxyuridine analogs & derivatives, Simplexvirus genetics, Thymidine Kinase genetics
- Published
- 1984
- Full Text
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15. 3'-Azido-3'-deoxythymidine inhibits the replication of avian leukosis virus.
- Author
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Olsen JC, Furman P, Fyfe JA, and Swanstrom R
- Subjects
- Animals, Antiviral Agents metabolism, Avian Leukosis Virus enzymology, Avian Leukosis Virus physiology, Chick Embryo, DNA, Viral biosynthesis, Phosphorylation, Reverse Transcriptase Inhibitors, Thymidine metabolism, Thymidine pharmacology, Thymine Nucleotides pharmacology, Zidovudine, Antiviral Agents pharmacology, Avian Leukosis Virus drug effects, Thymidine analogs & derivatives, Virus Replication drug effects
- Abstract
We tested the ability of the thymidine analog 3'-azido-3'-deoxythymidine (BWA509U) to inhibit the replication of the retrovirus avian leukosis virus. Inhibition was measured with two different assays: inhibition of a single round of virus replication and inhibition of virus spread through a cell culture. With both assays, we detected inhibition of virus growth, although inhibition of a single round of virus replication required a 40-fold higher drug concentration than did inhibition of virus spread. We also detected variations in the concentrations of drug needed to inhibit virus replication in different cell types. Higher concentrations of drug were needed to inhibit virus replication in chicken embryo fibroblasts than in the continuous quail cell line QT6. Viral DNA synthesis in infected cells was shown to be inhibited in the presence of the drug. The triphosphate form of the analog acted as a competitive inhibitor of purified viral reverse transcriptase, with a Ki of 0.09 +/- 0.003 microM, and was incorporated as a chain terminator during reverse transcription of the natural viral RNA substrate in vitro.
- Published
- 1987
- Full Text
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16. Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine.
- Author
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Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, and Schaeffer HJ
- Subjects
- Cell Line, DNA-Directed DNA Polymerase metabolism, Guanine metabolism, Guanine Nucleotides metabolism, Kinetics, Phosphotransferases metabolism, Simplexvirus enzymology, Thymidine Kinase metabolism, Antiviral Agents, Guanine analogs & derivatives, Simplexvirus drug effects
- Abstract
A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED(50)) = 0.1 muM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virus-specified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di- and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine. The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsA1).
- Published
- 1977
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17. A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate.
- Author
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Biron KK, Fyfe JA, Stanat SC, Leslie LK, Sorrell JB, Lambe CU, and Coen DM
- Subjects
- Acyclovir metabolism, Acyclovir pharmacology, Antiviral Agents metabolism, Drug Resistance, Microbial, Ganciclovir, Humans, Mutation, Nucleosides metabolism, Phosphorylation, Simplexvirus enzymology, Thymidine Kinase genetics, Acyclovir analogs & derivatives, Antiviral Agents pharmacology, Cytomegalovirus drug effects
- Abstract
We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate approximately equal to 1/10th the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed. The induction of normal nucleoside and deoxynucleoside triphosphates and certain cellular nucleoside kinases was comparable in mutant and wild-type virus infections. These results provide strong evidence for the importance of phosphorylation in the selectivity of this antiviral compound and raise the possibility that human cytomegalovirus encodes a nucleoside kinase. The mutant may identify the existence of a cytomegalovirus function whose properties could facilitate genetic analysis of this important pathogen.
- Published
- 1986
- Full Text
- View/download PDF
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