Interaction of herpes simplex virus-induced DNA polymerase with 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate

The triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) competitively inhibits incorporation of dGTP into DNA catalyzed by DNA polymerases specified by both type 1 and type 2 herpes simplex virus. K1 values were estimated to be 33 nM for type 1 and 46 nM for type 2-specified DNA polymerase. DHPG acted as an alternate substrate to dGTP for the virus-specified DNA polymerase. Incorporation of DHPG into DNA resulted in the slowing down of the rate of DNA synthesis. The position of DHPG incorporation was analyzed, and it was found to enter both internal and terminal linkages. DNA which contained DHPG at termini was found to competitively inhibit utilization of activated DNA as primer. DNA polymerase alpha and DNA polymerases from several phosphonoformic acid-resistant herpes simplex virus type 1 strains were examined for sensitivity to 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. A lack of correlation between the in vivo sensitivities of the virus mutants and the K1 values of the DNA polymerases was noted.     

Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate.

The effect of the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) on herpes simplex virus type 1 DNA synthesis was examined. Acycloguanosine inhibited herpesvirus DNA synthesis in virus-infected cells. The synthesis of host cell DNA was only partially inhibited in actively growing cells at acycloguanosine concentrations several hundred-fold greater than the 50% effective dose for herpes simplex virus type 1. Studies using partially purified enzymes revealed that the triphosphate of this compound inhibited the virus-induced DNA polymerases (DNA nucleotidyltransferases) to a greater degree than the DNA polymerase of the host cell, that the inhibition was dependent upon the base composition of the template, and that the triphosphate was a better substrate for the virus-induced polymerases than for the alpha cellular DNA polymerases.     

Metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new anti-herpes virus compound, in herpes simplex virus-infected cells

The metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), one of the most promising new anti-herpes virus compounds, in HeLa cells infected with herpes simplex virus type 1 was compared with that in the uninfected HeLa cells. In the virus-infected cells, the uptake of DHPG was enhanced and the major metabolites were found to be the mono-, di-, and triphosphate derivatives. The formation of these metabolites was dependent on the extracellular concentration of DHPG (0.5 to 5.0 microM). Virus-induced thymidine kinase was capable of phosphorylating DHPG to its monophosphate which could be further phosphorylated to the di- and triphosphate derivatives by the host cellular enzymes. Incorporation of the DHPG into DNA was observed in virus-infected cells. In contrast with 9-(2-hydroxyethoxymethyl)guanine, DHPG seemed not to serve as a chain terminator, but to be incorporated internally into DNA strands.     

Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviridae, human alpha and human beta polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate

The ability of human alpha and beta DNA polymerases and herpes simplex virus type 2 (HSV-2) and human cytomegalovirus (HCMV) DNA polymerases to insert and extend several nucleotide analogs has been investigated using a variation of Sanger-Coulson DNA sequencing technology. The analogs included the triphosphates of two antiviral nucleosides with incomplete sugar rings: 9-(1,3-dihydroxy-2-propoxymethyl)guanine (dhpG) and 9-(2-hydroxyethoxymethyl)guanine (acyG or acyclovir), as well as dideoxy and arabinosyl nucleoside triphosphates. Three pairs of contrasting behaviors were found, each pair distinguishing the two human polymerases from the two viral ones: first, extension behavior with araNTPs; second, insertion/extension behavior with dhpGTP; and third, the relative preference for insertion of ddGTP versus acyGTP. The relative level of insertion of the nucleotide analogs by HCMV and HSV-2 DNA polymerases was dhpGTP greater than (acyGTP and araNTP) greater than ddGTP, whereas by human alpha polymerase it was araATP greater than ddGTP much greater than (acyGTP and dhpGTP) and by human beta polymerase it was (araATP and ddGTP) much greater than (acyGTP and dhpGTP). Evidence is presented for three mechanisms of inhibition by extendible nucleotides (of dhp and ara types) exhibiting frequent internalization: araATP acted as a simple pseudoterminator of alpha and beta polymerases, but was easily extended past singlet sites by Herpesviridae polymerases and only stalled at sites requiring two or more araATP insertions in a row. Herpesviridae polymerases stalled after adding dhpGMP and one additional nucleotide, suggesting that polymerase translocation problems may be a factor in polymerase inhibition by modified sugar nucleotide analogs. The amino acid sequence of the human alpha DNA polymerase, which is acyGTP resistant, was found to vary by one amino acid from the amino sequences of the Herpesviridae polymerases in a region of significant similarity and probable functional homology. Amino acid differences at that same site differentiate acyclovir-resistant HSV-1 mutants from the acyclovir-sensitive HSV-1 wild type.     

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.     

Human cytomegalovirus-induced DNA polymerase and its interaction with the triphosphates of 1-(2''-deoxy-2''-fluoro-beta-D-arabinofuranosyl)-5-methyluracil, -5-iodocytosine, and -5-methylcytosine.

Human cytomegalovirus-induced DNA polymerase and cellular DNA polymerase alpha were purified by successive chromatography on DEAE-cellulose, phosphocellulose, heparin agarose, and single-stranded DNA agarose columns. The purified virus-induced DNA polymerase was resolved to consist of two polypeptides corresponding to molecular weights of 140,000 and 58,000, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Virus-induced DNA polymerase and cellular alpha polymerase were examined for their sensitivities to the triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAUTP), -5-iodocytosine (FIACTP), and -5-methylcytosine (FMACTP). The inhibitive effects of these triphosphates on the DNA polymerases were competitive with regard to the natural substrates; thus FMAUTP competes with dTTP, and FIACTP and FMACTP compete with dCTP. The inhibition constants (Ki) for FMAUTP, FIACTP, and FMACTP of virus-induced DNA polymerase are 0.06, 0.30, and 0.47 microM, respectively. Cellular DNA polymerase alpha is much less sensitive to these inhibitors, and its Ki values for FMAUTP, FIACTP, and FMACTP are 0.45, 3.10, and 2.90 microM, respectively. In addition, human cytomegalovirus-induced DNA polymerase, but not cellular DNA polymerase alpha, can utilize these analog triphosphates as alternate substrates for their corresponding natural deoxyribonucleoside triphosphates in in vitro DNA synthesis.     

Interaction of 9-(1,3-dihydroxy-2-propoxymethyl)guanine with cytosol and mitochondrial deoxyguanosine kinases: possible role in anti-cytomegalovirus activity

Summary The acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) is a potent inhibitor of human cytomegalovirus in vitro and in vivo. In order to investigate the phosphorylation of DHPG to the monophosphate and identify the enzyme responsible, attempts were made to isolate DHPG kinase from calf thymus and from human cytomegalovirus-infected lung cells. From calf thymus, a mitochondrial deoxyguanosine kinase was partially purified which co-migrated with DHPG phosphorylating activity on DEAE-cellulose, and had the same mobility by electrophoresis. DHPG triphosphate and DHPG kinase were elevated in cytomegalovirus-infected cells, but not enough enzyme activity was recovered to identify the kinase. However, DHPG was found to inhibit a cytosol deoxyguanosine kinase induced in these infected cells. The role of mitochondrial and cytosol deoxyguanosine kinases is discussed relative to the anti-cytomegalovirus activity of DHPG.     

Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate. Effects on primer-template function

The inhibition of highly purified herpes simplex virus (HSV)-induced and host cell DNA polymerases by the triphosphate form of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; acycloguanosine) was examined. Acyclovir triphosphate (acyclo-GTP) competitively inhibited the incorporation of dGMP into DNA, catalyzed by HSV DNA polymerase; apparent Km and Ki values of dGTP and acyclo-GTP were 0.15 microM and 0.003 microM, respectively. HeLa DNA polymerase alpha was also competitively inhibited; Km and Ki values of dGTP and acyclo-GTP were 1.2 microM and 0.18 microM, respectively. In contrast, HeLa DNA polymerase beta was insensitive to the analogue. The "limited" DNA synthesis observed when dGTP was omitted from HSV or alpha DNA polymerase reactions was inhibited by acyclo-GTP in a concentration-dependent manner. Prior incubation of activated DNA, acyclo-GTP, and DNA polymerase (alpha or HSV resulted in a marked decrease in the utilization of the primer-template in subsequent DNA polymerase reactions. This decreased ability of preincubated primer-templates to support DNA synthesis was dependent on acyclo-GTP, enzyme concentration, and the time of prior incubation. Acyclo-GMP-terminated DNA was found to inhibit HSV DNA polymerase-catalyzed DNA synthesis. Kinetic experiments with variable concentrations of activated DNA and fixed concentrations of acyclo-GMP-terminated DNA revealed a noncompetitive inhibition of HSV-1 DNA polymerase. The apparent Km of 3'-hydroxyl termini was 1.1 X 10(-7) M, the Kii and Kis of acyclo-GMP termini in activated DNA were 8.8 X 10(-8) M and 2.1 X 10(-9) M, respectively. Finally, 14C-labeled acyclo-GMP residues incorporated into activated DNA by HSV-1 DNA polymerase could not be excised by the polymerase-associated 3',5'-exonuclease activity.     

Phosphorylation of the antiviral precursor 9-(1,3-dihydroxy-2-propoxymethyl)guanine monophosphate by guanylate kinase isozymes

Guanylate kinase was purified from human erythrocytes by affinity chromatography using GMP-agarose, and the four isozymes which are present were separated by chromatofocusing. The kinetic properties of each isozyme were analyzed with respect to the natural substrates GMP and dGMP, and the 5'-monophosphate derivatives of the antiviral nucleoside analogs 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and 9-(2-hydroxyethoxymethyl)guanine (ACV, Acyclovir). The analysis of substrate kinetics yielded Km values for DHPG 5'-monophosphate which were similar with all isozymes (42-54 microM), and about 3-fold higher than the Km values obtained for GMP. Km values obtained with ACV 5'-monophosphate were 10-20-fold higher than the GMP values and varied nearly 4-fold among isozymes (209-753 microM). GMP produced the highest enzyme velocities with all isozymes, followed by dGMP, DHPG 5'-monophosphate, and ACV 5'-monophosphate, in that order. Differences in maximal velocities among isozymes were generally small. DHPG 5'-monophosphate inhibited the isozymes by a simple competitive mechanism with respect to GMP. In contrast, ACV 5'-monophosphate acted as an apparent hyperbolic mixed-type inhibitor. Similar patterns of inhibition were obtained with all isozymes. It is probable that differences is the reactivity of DHPG 5'-monophosphate and ACV 5'-monophosphate with individual guanylate kinase isozymes do not contribute significantly to differences in their antiviral effects.     

Herpes simplex virus type 1 and human DNA polymerase interactions with 2'-deoxyguanosine 5'-triphosphate analogues. Kinetics of incorporation into DNA and induction of inhibition

The ability of herpes simplex virus type 1 (HSV-1) DNA polymerase, HeLa polymerase alpha, and HeLa polymerase beta to utilize several dGTP analogues has been investigated using a defined synthetic template primer. The relative efficiencies of the triphosphates of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir triphosphate, ACVTP), 9-[(1,3-dihydroxy-2-propoxy)methyl] guanine (ganciclovir triphosphate, DHPGTP), and 2',3'-dideoxyguanosine (ddGTP) as substrates for the three polymerases were: HSV-1 polymerase, dGTP greater than ACVTP approximately equal to DHPGTP greater than ddGTP; polymerase alpha, dGTP greater than ACVTP approximately equal to DHPGTP much greater than ddGTP; polymerase beta, ddGTP greater than dGTP much greater than ACVTP approximately equal to DHPGTP. The potent inhibition of HSV-1 polymerase by ACVTP has been shown previously to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3' end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism was shown here to be a general mechanism for inhibition of polymerases by the obligate chain terminators, ACVTP and ddGTP. The ACVTP-induced inhibition was 30-fold more potent with HSV-1 polymerase than with polymerase alpha. This difference may contribute to the antiviral selectivity of this nucleotide analogue. The effect of ganciclovir monophosphate incorporation (a nonobligate chain terminator) on subsequent primer extension was also evaluated. With HSV-1 polymerase and polymerase alpha, although there was a considerable reduction in the efficiency of utilization of the 3'-DHPGMP-terminal primer, contrasting kinetic behavior was observed. With HSV-1 polymerase, insertion of DHPGTP resulted in a significant reduction in Vmax for subsequent nucleotide incorporations. In contrast, with polymerase alpha, a relatively small decrease in Vmax was accompanied by increased Km values for subsequent nucleotide incorporations.     

Prolonged inhibitory effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine against replication of Epstein-Barr virus.

The effects of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), a new antiviral drug, and acyclovir (ACV) [9-(2-hydroxyethoxymethyl)guanine] on the replication of Epstein-Barr virus (EBV) were compared. Both drugs inhibited EBV DNA replication in P3HR-1 cells and superinfected Raji cells, but neither inhibited replication of the plasmid form of the EBV genome in latently infected Raji cells. However, DHPG had a more prolonged inhibitory effect than ACV. Although the effect of the drugs is prompt, the kinetics of inhibition of EBV replication indicated that a drug exposure of 14 days was needed to reduce the EBV genome copy number to the residual plasmid level (30 copies per cell). The inhibitory effect of ACV was readily reversed within 11 days after removal of the drug, in contrast to the more prolonged effect exerted by DHPG, which persisted for more than 21 days. The 50% inhibitory doses for cell growth of ACV and DHPG were estimated to be 250 and 200 microM, respectively. The viral 50% and 90% effective doses of inhibition were, respectively, 0.3 and 9 microM for ACV and 0.05 and 3 microM for DHPG. The therapeutic indices (50% inhibitory dose/50% effective dose) for ACV and DHPG were 833 and 4,000, respectively. Synthesis of EBV-associated polypeptides was also affected. In superinfected Raji cells, ACV (100 microM) and DHPG (30 microM) inhibited synthesis of polypeptides with molecular weights of 145,000 and 140,000; in addition, synthesis of polypeptides with molecular weights of 110,000 and 85,000 was markedly reduced by DHPG but not by ACV. However, after drug removal, the inhibitory effect of ACV on polypeptide synthesis was abolished in contrast to the more persistent effect of DHPG.     

(Difluoromethylene)phosphates of guanine nucleosides as probes of DNA polymerases and G proteins

5'-Polyphosphates of N2-(p-n-butylphenyl)-2'-deoxyguanosine and -guanosine which contain a difluoromethylene group in place of a phosphoanhydride oxygen have been synthesized. 5'-[beta,gamma-(Difluoromethylene)triphosphates], including that of 2'-deoxyguanosine, were prepared by reaction of the corresponding 5'-phosphates, activated by 1,1'-carbonyldiimidazole, with difluoromethanediphosphonate. The 5'-[(difluoromethylene)diphosphate] of N2-(p-n-butylphenyl)guanosine was prepared by treatment of a protected 5'-tosyl nucleoside with difluoromethanediphosphonate, followed by deprotection. Condensation of this nucleotide, activated with 1,1'-carbonyldiimidazole, with orthophosphate gave N2-(p-n-butylphenyl)guanosine 5'-[(alpha,beta-difluoromethylene)triphosphate]. Products were characterized by 31P and 19F NMR spectroscopy. The phosphonates were tested for their ability to displace [3H]GDP from the GTP binding proteins cellular (EC) and oncogenic (Leu-61) Ha-ras p21, and for their ability to inhibit DNA polymerase alpha from Chinese hamster ovary cells. The p21s bound weakly to a triphosphonate when the CF2 group was in the beta,gamma position, but not when it was in the alpha,beta position, and they did not bind to the corresponding (difluoromethylene)diphosphate. In contrast, the CF2 group had no effect on inhibition of DNA polymerase alpha by N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphospate]. 2'-Deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphosphate] was found to be a bona fide substrate for several DNA polymerases and had a lower apparent Km than dGTP with Bacillus subtilis DNA polymerase III.     

Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.     

Phosphorylation of acyclovir [9-(2-hydroxyethoxymethyl)guanine] in Epstein-Barr virus-infected lymphoblastoid cell lines.

The extent of phosphorylation of 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]) in fresh peripheral leukocytes, in Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines, and in herpes simplex virus type 1-infected lymphoblastoid (P3HR-1) and monkey kidney (Vero) cells was determined by high-pressure liquid chromatography, Mono-, di-, and triphosphorylated derivatives of [8-14C]ACV were detected at low levels at various times after superinfection of Raji cells with EBV. The extent of phosphorylation appeared to be related to the concentration of ACV in the medium. Small amounts of ACV mono-, di-, and triphosphates were formed in fresh peripheral leukocyte preparations from EBV- seropositive and -seronegative donors. Comparable ACV monophosphate levels were detected in EBV-negative BJAB and the EBV-positive BJAB/GC cell lines; however, no di- or triphosphate derivatives were detected. Comparable ACV-monophosphate levels were detected in both P3HR-1 and HSV-infected P3HR-1 cell lines; however, larger amounts of ACV di- and triphosphorylated derivatives were detected in the HSV-infected P3HR-1 cells. ACV was converted to the triphosphate to a greater extent in HSV-infected Vero cells than in mock-infected Vero cells or in HSV-infected P3HR-1 cells. ACV or its phosphorylated derivatives were converted to guanine nucleotides to a greater extent in lymphoblastoid cells than in fibroblasts (Vero). In conclusion, neither the productive replication of EBV nor the presence of latent viral DNA is required for ACV monophosphate formation in B lymphoblastoid cells. ACV triphosphate, however, was detected only in cells infected productively with EBV.     

Comparative effects of the 5'-triphosphates of 9-beta-(2'-azido-2'-deoxy-D-arabinofuranosyl)adenine and 9-beta-D-arabinofuranosyladenine on DNA polymerases from L1210 leukemia cells.

9-beta-(2'-Azido-2'-deoxy-D-arabiofuranosyl)adenine (arazide) causes greater and significantly more persistent inhibition of [3H]-thymidine incorporation into the DNA of neoplastic cells than the related agent 9-beta-D-arabinofuranosyladenine (araA). To elucidate the mechanism(s) responsible, we compared the effects of arazide and araA 5'-triphosphates on DNA polymerases alpha and beta of L1210 leukemia cells. Both nucleoside triphosphate analogs inhibited DNA polymerase alpha activity by competing with dATP; only araATP was inhibitory to DNA polymerase beta. Arazide triphosphate was at least four times more active than araATP as an inhibitor of DNA polymerase alpha. Preincubation of DNA polymerase alpha with either agent did not result in enzyme inactivation. The results suggest that interference with DNA polymerase alpha activity by arazide triphosphate may be in part responsible for the inhibition of DNA synthesis produced by arazide in neoplastic cells.     

Differential effects of acyclovir and 9-(1,3-dihydroxy-2-propoxymethyl)guanine on herpes simplex virus and Epstein-Barr virus in a dually infected human lymphoblastoid cell line.

We investigated the effects of acyclovir and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) on a lymphoblastoid cell line dually infected with Epstein-Barr virus and herpes simplex virus (HSV) type 1. The numbers of Epstein-Barr virus genomes were reduced during 70 days of treatment with either drug. Both drugs suppressed HSV replication in a dose-related manner. In the continued presence of the drugs, HSV developed resistance, rapidly to acyclovir and much more slowly to 30 microM DHPG. Analysis of HSV glycoprotein C production and viral DNA showed that treatment with 100 microM DHPG eliminated HSV production, curing the cell line of HSV persistent infection.     

2'-Nor-cGMP: a seco-cyclic nucleotide with powerful anti-DNA-viral activity

As part of our study of antiherpetic acyclonucleosides, we synthesized a cyclic GMP analog, 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide, sodium salt (2'-nor-cGMP), and discovered its potent and broad spectrum anti-DNA-viral activities. 2'-Nor-cGMP inhibits the replication of many DNA viruses, including herpes simplex virus, human cytomegalovirus, vaccinia, SV40, and adenovirus, but does not inhibit RNA viruses. In plaque reduction studies this potent antiviral agent is also approximately 10-fold more potent than 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) against varicella-zoster virus and inhibits cell transformation by bovine papilloma virus. Unlike 2'NDG, the potent activity of 2'-nor-cGMP against herpes virus is not dependent upon the action of virus-specified thymidine kinase. Intercellular metabolism of 2'-nor-cGMP produced small amounts of 2'NDG triphosphate which were insufficient to account for the antiviral activity observed, implying that this potent anti-DNA-viral agent operates by a mechanism different from that of known acyclonucleosides.     

Potent synergistic inhibition of herpes simplex virus-2 by 9-[(1, 3-dihydroxy-2-propoxy)methyl]guanine in combination with recombinant interferons

DHPG, an acyclic guanine nucleoside with the structure 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine], showed potent synergism with recombinant alpha or beta interferons and modest synergism with gamma interferon in inhibiting the replication of herpes simplex virus type 2 in vitro. The most potent direct anti-herpes viral synergism was obtained by combination of DHPG and recombinant human interferon-beta-ser17; when combined, doses of each near their separate effective dose50's resulted in almost complete elimination of production of infectious virus within a single viral replication cycle. The anti-herpes viral activity of DHPG-interferon combinations was significantly greater than that obtained with acyclovir-interferon combinations.     

Differential inhibition of various mammalian DNA polymerase activities by ammonium 21-tungsto-9-antimoniate (HPA23)

The 21-tungsto-9-antimoniate ammonium salt (HPA23), known as an antiviral agent, has been shown to be a potent inhibitor of both human and murine DNA polymerase alpha and murine DNA polymerase gamma. HPA23 inhibited the activity of DNA polymerase alpha in noncompetitive fashion with respect to either deoxynucleotide substrate or nucleic acid template.primer. The Ki of murine DNA polymerase alpha for HPA23 was determined to be 24 nM. The activity of mouse DNA polymerase gamma also was strongly inhibited by HPA23 (Ki, 20 nM), and the mode of inhibition was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive to substrate, dTTP. DNA polymerase beta and terminal deoxynucleotidyltransferase, however, were relatively resistant to inhibition by HPA23. The observed inhibitions by HPA23 seem to be closely related to the polyanionic property of this drug.     

Stereochemical considerations in the enzymatic phosphorylation and antiviral activity of acyclonucleosides. I. Phosphorylation of 2'-nor-2'-deoxyguanosine

The antiviral compound 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (2'-nor-2'-deoxyguanosine, 2'-NDG) is phosphorylated by the HSV-1-induced thymidine kinase to the monophosphate (2'-NDG-MP) and this is further phosphorylated by cellular kinases to the triphosphate (2'-NDG-TP) which is a potent inhibitor of DNA polymerases. Since phosphorylation of 2'-NDG creates a chiral center in the molecule, it was of interest to examine whether both monophosphate enantiomers were produced by the viral thymidine kinase, whether they both could be further phosphorylated by cellular kinases and, if so, whether the respective triphosphates were equally inhibitory to the DNA polymerases. The time course of the phosphorylation by GMP kinase of a chemically synthesized, racemic 2'-NDG-MP was compared to that of a 2'-NDG-MP preparation obtained by enzymatic phosphorylation of 2'-NDG with HSV-1 thymidine kinase. The results indicated that the two enantiomeric monophosphates were phosphorylated by GMP kinase with different rates and that phosphorylation of 2'-NDG by HSV-1 thymidine kinase gave only one of the isomers, whose structure was determined to be S. Both enantiomeric diphosphates were further phosphorylated to the respective triphosphates and it was shown that, in contrast to the triphosphate obtained from the 2'-NDG-MP prepared by viral thymidine kinase which was a potent inhibitor of HSV-1 DNA polymerase, the triphosphate obtained from the slow-reacting R isomer had little or no inhibitory activity against this enzyme.