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J A Fyfe

Publications and source records attributed to J A Fyfe.

At least 37 records · Page 2Linked to original sources
PubMed1991

6-N-substituted derivatives of adenine arabinoside as selective inhibitors of varicella-zoster virus.

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.

Adenosine Deaminase Inhibitors↗
PubMed1991

Mutant varicella-zoster virus thymidine kinase: correlation of clinical resistance and enzyme impairment.

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.

Acyclovir↗
PubMed1990

Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.

The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

Base Sequence↗
PubMed1989

Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers.

The enzymatic assay for deoxyribonucleoside triphosphates has been improved by using synthetic oligonucleotides of a carefully defined sequence as template primers for DNA polymerase. High backgrounds, which limit the sensitivity of the assay when calf thymus DNA or alternating copolymers are used as template primers, were eliminated with these oligonucleotide template primers. Sensitivity was further increased by designing the template primer to incorporate multiple labeled deoxyribonucleotides per limiting unlabeled deoxyribonucleotide. Each of several DNA polymerases exhibited unique reaction characteristics with the oligonucleotide template primers, which was attributed to the differing exonuclease activities associated with these various enzymes. Assay optimization therefore included matching the polymerase with the template primer to obtain the lowest background reaction and highest sensitivity. This modified assay is particularly well suited for keeping cell sample size to a minimum in experimental protocols which generate large numbers of data points or require careful timing of sampling. With this technique, we measured the levels of all four deoxyribonucleoside triphosphates in extracts from as few as 2 x 10(4) cultured cells.

Base Sequence↗
PubMed1989

3-Deazaadenosine 5'-triphosphate: a novel metabolite of 3-deazaadenosine in mouse leukocytes.

Evidence has been obtained for the metabolic formation of small amounts (1-2% of the ATP pool) of 3-deazaadenosine 5'-triphosphate (c3ATP) from 3-deazaadenosine (c3Ado) in mouse cytolytic lymphocytes and mouse resident peritoneal macrophages. With intact leukocytes, pharmacological evidence was obtained that adenosine kinase was not the enzyme chiefly responsible for the phosphorylation of c3Ado. Moreover, in the presence of MgCl2, NaCl and IMP, purified rat liver 5'-nucleotidase catalyzed the phosphorylation of c3Ado to 3-deazaadenosine 5'-monophosphate (c3AMP). Two lines of evidence suggest that the metabolic formation of c3ATP is not involved in the inhibition of leukocyte function caused by c3Ado. First, the inhibitory action of c3Ado on antibody-dependent phagocytosis and lymphocyte-mediated cytolysis was reversed markedly upon removal of the drug from the medium. However, the intracellular content of c3ATP remained constant in lymphocytes and macrophages after removal of c3Ado. Second, in macrophages and in lymphocytes, similar intracellular amounts of c3ATP were formed from both c3Ado and 3-deazaadenine under conditions in which the former was biologically active and the latter was essentially inactive. Thus, it appears unlikely that the novel c3ATP metabolite is of relevance for the mechanism of action of c3Ado in mouse leukocytes.

5'-Nucleotidase↗
PubMed1987

Ribavirin antagonizes the effect of azidothymidine on HIV replication.

Azidothymidine and ribavirin both inhibit replication of human immunodeficiency virus in vitro and show promise of clinical utility in patients infected with this virus. In this study, the possible interactions of these drugs were examined in vitro, and a reproducible antagonism between azidothymidine and ribavirin was found to occur under a variety of experimental conditions. The mechanism responsible for this antagonism appeared to be inhibition of azidothymidine phosphorylation by ribavirin. Because similar effects may occur in vivo, clinical trials of these two drugs in combination must be performed only under carefully controlled conditions.

Cell Line↗
PubMed1987

Mapping and characterization of two mutations to antibiotic supersusceptibility in Pseudomonas aeruginosa.

Two mutations associated with antibiotic supersusceptibility in Pseudomonas aeruginosa strain Z61 were transferred separately into strain PAO222, using R68.45-mediated conjugation and phage F116L transduction. One mutation (absA) was 40% contransducible with pro-82 at 26 min on the P. aeruginosa chromosome and was associated with increased susceptibility to beta-lactams, gentamicin and hydrophobic agents. Strains carrying the absA mutation also displayed enhanced uptake of a hydrophobic fluorescent probe, 1-N-phenylnaphthylamine, and were found, by SDS-PAGE, to be altered in the pattern of lipopolysaccharide O-antigen distribution. The other mutation (absB), associated with increased susceptibility to beta-lactams and gentamicin but not to hydrophobic agents, was cotransducible with met-28 and proC at 20 min on the chromosome. The absB mutation caused a structurally undefined alteration in the physical interaction of EDTA and gentamicin with the outer membrane.

1-Naphthylamine↗
PubMed1987

Nucleotide sequence changes in thymidine kinase gene of herpes simplex virus type 2 clones from an isolate of a patient treated with acyclovir.

To identify the nucleotide changes that occur in drug-induced thymidine kinase (TK) mutants of herpes simplex virus type 2 (HSV-2), we compared the nucleotide sequences of the tk genes of two mutant HSV-2 clones isolated from a patient who had been treated with acyclovir [9-(2-hydroxyethoxymethyl)guanine; ACV] with the nucleotide sequence of the parental TK+ HSV-2(8703) strain isolated from the same patient. One of the mutants, TK-altered (TKA) HSV-2(9637), was ACV resistant but induced the incorporation of [14C]thymidine into the DNA of infected rabbit skin cells. The nucleotide sequence of the tk gene of mutant TKA HSV-2(9637) had a single change (G to A) at nucleotide 668, which would cause an arginine-to-histidine substitution at amino acid residue 223 of the TK polypeptide. The second ACV-resistant mutant, TK- HSV-2(8710), did not induce detectable incorporation of [14C]thymidine into the DNA of infected rabbit skin cells. This mutant exhibited a deletion of a single base at nucleotide 217 of its nucleotide sequence. This deletion would cause a frameshift mutation at amino acid residue 73 and chain termination at amino acid residue 86 of the TK polypeptide. The nucleotide sequence of TK+ HSV-2(8703) was the same as that of the laboratory strain, TK+ HSV-2(333). The nucleotide sequence of a bromodeoxyuridine-resistant TK- HSV-2(333) mutant of TK+ HSV-2(333) also exhibited a single-base deletion, but at nucleotide 439. This deletion would cause a frameshift mutation at amino acid residue 147 and chain termination at amino acid residue 182. The frameshift mutations of TK- HSV(8710) and TK- HSV-2(333), respectively, occurred in sequences in which C was repeated three times and G was repeated seven times. The results raise the possibility that TK- frameshift mutations of HSV-2 may be common.

Acyclovir↗
PubMed1987

Antibacterial activity and mechanism of action of 3'-azido-3'-deoxythymidine (BW A509U).

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U; azidothymidine [AZT]) had potent bactericidal activity against many members of the family Enterobacteriaceae, including strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri, and Enterobacter aerogenes. AZT also had bactericidal activity against Vibrio cholerae and the fish pathogen Vibrio anguillarum. AZT had no activity against Pseudomonas aeruginosa, gram-positive bacteria, anaerobic bacteria, Mycobacterium tuberculosis, nontuberculosis mycobacteria, or most fungal pathogens. Several lines of evidence indicated that AZT must be activated to the nucleotide level to inhibit cellular metabolism: AZT was a substrate for E. coli thymidine kinase; spontaneously arising AZT-resistant mutants of E. coli ML-30 and S. typhimurium were deficient in thymidine kinase; and intact E. coli ML-30 cells converted [3H]AZT to its mono-, di-, and triphosphate metabolites. Of the phosphorylated metabolites, AZT-5'-triphosphate was the most potent inhibitor of replicative DNA synthesis in toluene-permeabilized E. coli pol A mutant cells. AZT-treated E. coli cultures grown in minimal medium contained highly elongated cells consistent with the inhibition of DNA synthesis. AZT-triphosphate was a specific DNA chain terminator in the in vitro DNA polymerization reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I. Thus, DNA chain termination may explain the lethal properties of this compound against susceptible microorganisms.

Antiviral Agents↗
PubMed1987

Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity.

In vitro and in vivo studies were done on a herpes simplex virus type 2 strain recovered from a patient on acyclovir (ACV) which was ACV resistant but expressed thymidine (dThd) kinase (EC 2.7.1.21) activity. Plaque-purified clones derived from the original clinical sample were heterogeneous with respect to plaque size and drug susceptibility. The heterogeneity of this viral mixture was also evident from varied 125I-labeled 5-iodo-2'-deoxycytidine autoradiographic patterns and from varied expression of dThd kinase-associated phosphorylating activities. Four clones from this mixture were 1-beta-D-arabinofuranosylthymine (ara-T) susceptible and ACV resistant. Extracts of cells infected with these clones catalyzed the phosphorylation of ara-T but little of ACV. The virus-coded dThd kinase was purified from one of these clones to determine whether its substrate specificity was altered. The amount of virus-coded dThd phosphorylating activity with the cell extracts was estimated to be sevenfold lower with the resistant clone than with the MS strain of herpes simplex virus type 2. The dThd kinase eluted from a dThd-agarose affinity column under the same conditions with extracts from both sources and substrate saturations of both enzymes by acyclic nucleoside analog phosphate acceptors were classical hyperbolic functions. However, there were significant differences in the kinetic parameters of substrates between the two enzymes. Apparent Km (Km') values for dThd, deoxycytidine, ara-T, ACV, and the acyclic guanosine analog 9-[[2-hydroxyl-1-(hydroxymethyl)ethoxy]methyl]guaine (BW B759U) were 2- to 60-fold higher with the variant enzyme than with the enzyme from laboratory strain MS. Comparing these two enzymes, relative maximal phosphorylation rates (Vm) were eightfold lower for ACV but unchanged for BW B759U. In contrast, the relative rates for deoxycytidine and ara-T were eight- and twofold higher, respectively. The surprisingly good substrate activity with BW B759U compared with that of ACV (Vm/Km' = 0.39 versus 0.01) coincided with susceptibility of the ACV-resistant virus to BW B759U. This clinical variant retained its pathogenicity for mice and was only moderately less neurovirulent than wild-type virus. Although such mutants have the potential to induce illness less responsive to therapy, the recurrence from which the isolate was obtained was typical for this patient in severity and duration. Since this episode, the patient has been treated successfully with ACV.

Acyclovir↗
PubMed1987

3'-Azido-3'-deoxythymidine inhibits the replication of avian leukosis virus.

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.

Animals↗
PubMed1986

Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase.

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.

Antiviral Agents↗
PubMed1986

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.

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.

Acyclovir↗
PubMed1985

Cytoplasmic 5'-nucleotidase catalyzes acyclovir phosphorylation.

A cytoplasmic 5'-nucleotidase (EC 3.1.3.5) can catalyze the phosphorylation of inosine (Worku, Y., and Newby, A.C. (1982) Biochem. J. 205, 503-510). This enzyme was purified to determine whether it could catalyze the formation of trace levels of phosphorylated acyclovir (ACV), a nucleoside analog with antiherpes activity. Acyclovir phosphorylating activity from rat liver co-chromatographed with the enzyme throughout the 1200-fold purification and through size exclusion chromatography or polyacrylamide-gel electrophoresis. In addition, the pH optimum, ATP stimulation, and phosphate inhibition of the ACV phosphorylating activity paralleled those of the 5'-nucleotidase. Finally, ACV phosphorylation was competitively inhibited by inosine (Kis = 6.5 mM; K'm (inosine) = 5.0 mM). This was consistent with phosphorylation at a common catalytic site. In addition to inosine and ACV, the guanine derivatives Guo, dGuo, 9-beta-D-arabinofuranosylguanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were substrates for the enzyme. The relative phosphorylation rates were, respectively, 100, 0.7, 19, 4, 0.3, and 0.7, at 0.1 mM phosphate acceptor. Approximate K'm values were, respectively, 5, 90, 10, 10, greater than 100, and greater than 100 mM. Although the substrate activity of ACV with the 5'-nucleotidase was inefficient, it appeared to be sufficient to account for the small amounts of ACV phosphates formed in uninfected cells.

5'-Nucleotidase↗
PubMed1985

Orthophosphate determination in the presence of high concentrations of ATP.

A method to measure orthophosphate which contaminates samples of ATP was developed. Concentrations of orthophosphate as low as 0.4% of the ATP concentration were determined using a zinc-molybdate reagent [D. A. Bencini, J. R. Wild, and G. A. O'Donovan, Anal. Biochem. 132, 254-258 (1983)] and continuous spectrophotometric monitoring of chromophore formation. Since the rate of ATP hydrolysis was pseudo-first order and was slow compared to the rate of chromophore formation, the initial concentration of phosphate could be readily determined by extrapolation to zero time. The method is rapid and reproducible, and requires a single, stable reagent.

Adenosine Triphosphate↗
PubMed1985

Modifications on the heterocyclic base of acyclovir: syntheses and antiviral properties.

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.

Acyclovir↗
PubMed1985

Metabolic activation of the nucleoside analog 9-[( 2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus.

9-[( 2-Hydroxy-1-(hydroxymethyl)ethoxy]-methyl)guanine (BW B759U) is a more potent inhibitor of human cytomegalovirus (HCMV) in vitro than is the related nucleoside analog acyclovir (ACV). BW B759U was selectively activated to the 5'-triphosphate (BW B759U-triphosphate) in cells infected with HCMV to levels at least 10-fold higher than those measured for ACV-triphosphate and up to as much as 100-fold higher than the levels found in uninfected cells. BW B759U-triphosphate accumulated in HCMV-infected cells with time; the rate of this increase was dependent upon the drug dose and virus multiplicity of infection. Enzyme activities that catalyzed the phosphorylation of thymidine and 2'-deoxycytidine increased 3- to 7-fold in extracts of cells early after HCMV infection but thereafter declined. No concomitant increase in the rate of BW B759U phosphorylation was detected under these assay conditions. Maximal rate of accumulation of both BW B759U-triphosphate and ACV-triphosphate after a short exposure to drug occurred in the late phase of the infective cycle, as the titer of extracellular virus reached a peak in untreated cultures, but after the decline of stimulated host deoxypyrimidine kinase activities. Once formed, the BW B759U-triphosphate pool decreased very slowly and thus it persisted for several days in both HCMV-infected and uninfected cells.

Acyclovir↗