James A. Fyfe

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.

Enzymatic phosphorylation of acyclic nucleoside analogs and correlations with antiherpetic activities

Abstract The inhibitor and substrate specificities of deoxythymidine (dThd) kinase purified from herpes simplex virus (HSV Type 1) were studied. A number of nucleosides and nucleoside analogs were phosphorylated by the virus coded enzyme. These included several compounds structurally related to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), a potent inhibitor of HSV replication. Some contained guanine with 9-substituents differing from that of acyclovir by methylene additions, methylene and thioether substitutions for the ether oxygen, and branching on the distal side of the ether oxygen. Others were various 2-substituted 6-hydroxypurines with the 9-(2-hydroxyethoxymethyl) substituent. A limitation of the specificity of the enzyme with guanine derivatives was the lack of phosphorylation of any derivative with an acyclic moiety branched on the proximal side of the ether oxygen. Many of the compounds that were phosphorylated were subsequently found to inhibit HSV replication. Such compounds apparently inhibited HSV replication via the same route of activation previously described for acyclovir [G. B. Elion, P. A. Furman, J. A. Fyfe, P. de Miranda, L. Beauchamp and H. J. Schaeffer, Proc. natn. Acad. Sci. U.S.A. 74 , 5716 (1977)]. Moreover, several compounds not phosphorylated by the enzyme did not inhibit replication. However, some other acyclic nucleoside analogs that were phosphorylated were not good antivirals, indicating that phosphorylation catalyzed by the HSV dThd kinase was not sufficient for inhibition of viral replication to occur. These results emphasize the importance of the specificity of cellular kinases and the HSV DNA polymerase to the mechanism of antiviral activity. The dThd kinase from Vero cells was also purified. With this host cell enzyme, kinetic constants of known antiviral compounds were determined and compared to those of dThd (relative V ′ max ; k ′ m ): dThd (100; 1.3 μM), 5-iodo-2′-deoxyuridine (87; 1.8 μM), 5-trifluoromethyl-2′-deoxyuridine (91; 1.2 μM), 5-bromo-2′-deoxycytidine (5; 580 μM), and 9-β- d -arabinofuransoylthymine (23; 2300 μM). None of the purine acyclic nucleoside analogs tested (at 1000 μM) was detectably phosphorylated by the Vero cell enzyme, and all had apparent K i values >300 μM. The phosphorylation catalyzed by host cell dThd kinase correlated with the toxicity of some pyrimidine nucleoside analogs.

Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with Herpesvirus genetic information

Abstract Thymidine kinase-deficient LM cells (LMTK−-) biochemically transformed to the TK+ phenotype with herpes simplex virus genetic information showed an increased uptake of and ability to phosphorylate the acyclic nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine, acyclo-Guo). In growth inhibition studies the TK+ transformants were much more sensitive to inhibition with acyclovir than the untransformed cells (13- to 90-fold more sensitive). The synthesis of DNA in the transformed cells was significantly reduced by acyclovir treatment, whereas acyclovir had little effect on the DNA synthesis of the untransformed cells. Alkaline sucrose gradient sedimentation analysis of cellular DNA synthesized in the presence of acyclovir showed that, in contrast to untreated untransformed cells, the DNA newly synthesized by transformed cells was considerably smaller in size. In pulse-chase experiments the small fragments of DNA synthesized in the presence of acyclo-Guo were not chased to high molecular weight DNA. Finally, acyclo-Guo was shown to be incorporated terminally at 3′-ends of growing DNA chains in replicating cells.

6-Methoxypurine arabinoside as a selective and potent inhibitor of varicella-zoster virus.

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.

Selection and preliminary characterization of acyclovir-resistant mutants of varicella zoster virus

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.

Activation and antiviral effect of acyclovir in cells infected with a varicella-like simian virus

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.

GMP synthetase from Escherichia coli B-96: Interactions with substrate analogs

Abstract GMP synthetase was studied with respect to its substrate and inhibitor specificity towards nine xanthosine 5′-phosphate (XMP) and GMP analogs. 8-AzaXMP and 6-thioXMP were found to be substrates, but were considerably less efficient than XMP. 1-Ribosyloxipurinol 5′phosphate was aminated with an efficiency of less than 1 18 000 that of XMP. The aminated products of the reactions were competitive inhibitors, with respect to XMP.

Xanthine phosphoribosyltransferase from Streptococcus faecalis: Properties and specificity

Streptococcus faecalis (ATCC 8043) was shown to have a purine phosphoribosyltransferase specific for xanthine. This enzyme was separated from interfering activities by heat treatment, ammonium sulfate fractionation, hydroxylapatite chromatography, and affinity chromatography. The xanthine phosphoribosyltransfer activity of this preparation was stable between pH 5.6 and 10, had a pH optimum between pH 7.4 and 8.8, and had a particle weight of 42,000 as determined by G-100 Sephadex chromatography. An initial velocity analysis when plotted in double-reciprocal form resulted in a family of parallel lines which when extrapolated to infinite concentration gave Km values for xanthine and PP-ribose-P of 20 and 53 μm, respectively. Inhibition studies with 42 purine and purine analogs indicated that oxo groups at positions 2 and 6 of the purine ring were required for optimal binding. The substitution of thio for oxo reduced binding to the enzyme ca. 20-fold. In contrast to its rigid specificity with respect to the 2,6-dioxo substituents, the enzyme bound a variety of 4,5-condensed pyrimidine systems containing a nitrogen at the position corresponding to the N-7 of xanthine. At concentrations of 1 mm, hypoxanthine, adenine, and 4,6-dihydroxypyrazolo[3,4-d]pyrimidine were converted to their corresponding ribonucleotides at rates approximately 0.1% of the rate for xanthine. Guanine was not detected as a substrate (rate <0.007% that of xanthine). The enzyme was inhibited by the ribonucleoside mono-, di-, and triphosphates of xanthine and guanine but not by those of adenine.

DNA Cleaving Activity of Purified Human Immunodeficiency Virus Integration Protein

An essential step in the life cycle of retroviruses is insertion of a double-stranded DNA copy of the viral RNA genome into the host cell DNA, to form the provirus. The insertion event depends on at least one viral protein, the integration protein (IN), which is a product of the viral pol gene. Mutations in the IN coding region of pol result in integration-negative retroviruses that will no longer replicate.1–3 The proviral DNA is identical to the precursor viral DNA except for the loss of two base pairs at each end, at the points of attachment to cellular DNA. One proposed function for IN is the removal of these two bases from the 3′-termini of both strands of the viral DNA, in preparation for integration.