Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.     

Inhibitory effect of E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on herpes simplex virus replication and DNA synthesis.

The effect of E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on herpes simplex virus (HSV) replication was examined and compared with that of E-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd). The 50% inhibitory dose against HSV type 1 (HSV-1) was 0.1 microgram/ml compared with 0.008 microgram/ml for BVdUrd; the antimetabolic 50% inhibitory dose of BVaraU ranged from 20 to 95 micrograms/ml. The addition of 50 micrograms of BVaraU per ml to HSV-1-infected Vero cells decreased the synthesis of viral and cellular DNA by 37 and 28%, respectively. The 5'-triphosphate (BVaraUTP) competed with dTTP in DNA synthesis by the herpes-viral and cellular DNA polymerases; the apparent Ki values of HSV-1 DNA polymerase, DNA polymerase alpha, and DNA polymerase beta were 0.14, 0.32, and 5 microM, respectively. Thus, BVaraU was a less effective antiherpesvirus agent than BVdUrd; unlike BVdUrd, it did not appear to be internally incorporated into replicating DNA in virus-infected cells.     

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.     

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.     

Metabolism of the carbocyclic analogue of (E)-5-(2-iodovinyl)-2'-deoxyuridine in herpes simplex virus-infected cells. Incorporation of C-IVDU into DNA

The carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), in which the sugar moiety is replaced by a cyclopentane ring and which have been designated as C-BVDU and C-IVDU, respectively, are, like their parent compounds BVDU and IVDU, potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) and, to a lesser extent, herpes simplex virus type 2 (HSV-2) replication. We have now synthesized the radiolabeled C-IVDU analogue, C-[125I]IVDU, and determined its metabolism by HSV-infected and mock-infected Vero cells. C-[125I]IVDU was effectively phosphorylated by HSV-1-infected cells and, to a lesser extent, HSV-2-infected cells. C-[125I]IVDU was not phosphorylated to an appreciable extent by either mock-infected cells or cells that had been infected with a thymidine kinase-deficient mutant of HSV-1. Furthermore, C-[125I]IVDU was incorporated into both viral and cellular DNA of HSV-1-infected Vero cells. This finding represents the first demonstration of the incorporation of a cyclopentylpyrimidine into DNA.     

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.     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

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.     

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.     

Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature-sensitive repressor

The structural gene for herpes simplex virus (type 1) thymidine kinase was cloned downstream from the lambda phage high efficiency leftward promotor in a plasmid (pHETK2) also containing the gene for the lambda cI857 temperature-sensitive repressor. Thymidine kinase is synthesized as a run-on product containing the NH2 terminus of the lambda N protein. Heat inactivation of the lambda repressor by growth at 42 degrees C results in the accumulation of thymidine kinase as approximately 4% of the total soluble cellular protein. Thymidine kinase has been purified to greater than 95% homogeneity by high speed centrifugation, ammonium sulfate fractionation, and Sephadex G-100 and hydroxylapatite column chromatography. Thymidine kinase has a subunit Mr = 42,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a dimer during Sephadex G-100 chromatography and glycerol gradient centrifugation. Thymidine kinase is enzymatically active from pH 6 to 10 with maximum activity at pH 8.5. The enzyme is protected from heat inactivation by thymidine and has a half-life at 40 degrees C of 30 min in the presence of thymidine and 3 min in its absence. Thymidine kinase displays Michaelis-Menten kinetics with apparent Michaelis constants of 0.6 and 118 microM for thymidine and ATP, respectively. Iododeoxycytidine is a competitive inhibitor of thymidine with an apparent Ki of 14 microM. The anti-herpes drug acyclovir (9-[(2-hydroxyethoxy)methyl]guanine) also appears to be a competitive inhibitor of thymidine (Ki of approximately 300 microM) but requires 3,000-fold higher concentrations than thymidine to give 50% inhibition. Other nucleoside triphosphates can substitute for ATP in the kinase reaction with the exception of dTTP which appears to inhibit thymidine kinase activity by about 50% when present in concentrations equal to that of thymidine.     

The relationship between incorporation of E-5-(2-Bromovinyl)-2'-deoxyuridine into herpes simplex virus type 1 DNA with virus infectivity and DNA integrity

E-5-(2-Bromovinyl)-2'-deoxyuridine (BrvdUrd) produced a dose-dependent shift in the density of herpes simplex virus type 1 (HSV-1) DNA at concentrations which yielded potent inhibition of virus replication in cultured Vero cells. Although the density of cellular DNA was not altered by these concentrations of BrvdUrd, incorporation of this analogue into cellular DNA of HSV-1-infected cells has been previously observed in this laboratory. The degree of inhibition correlated with the amount of BrvdUrd substituted for thymidine in HSV-1 DNA. BrvdUrd-substituted DNA was more labile as determined by a dose-dependent increase in single strand breaks when examined by centrifugation in alkaline sucrose gradients. Thus, the potent antiviral action of BrvdUrd observed in cell culture correlates not only with its incorporation into HSV-1 DNA but also with an altered stability of this DNA.     

Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Murine mammary carcinoma (FM3A TK-/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ng/ml, respectively; i.e., a concentration 5000 to 20 000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action of BVDU and related compounds.     

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.     

Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.     

Conservative mutations of glutamine-125 in herpes simplex virus type 1 thymidine kinase result in a ganciclovir kinase with minimal deoxypyrimidine kinase activities

The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures. To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants and wild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic properties compared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the K(m) of thymidine increased from 0.9 microM for the parent Gln-125 enzyme to 3 microM for the Glu-125 mutant, to 6000 microM for the Asp-125 mutant, and to 20 microM for the Asn-125 mutant. In contrast, the K(m) of ganciclovir decreased from 69 microM for the parent Gln-125 enzyme to 50 microM for the Asn-125 mutant and increased to 473 microM for the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines.     

Herpes Simplex Virus Thymidine Kinase Gene-Transfected Tumor Cells; Sensitivity to Antiherpetic Drugs

Abstract

A series of antiherpetic 5-substituted 2′-deoxyuridine derivatives (i. e. BVDU) and guanine derivatives (i. e. ganciclovir) have been evaluated for their cytostatic activity against murine mammary carcinoma FM3A cell lines that are deficient in cytosol thymidine kinase, but transfected by the herpes simplex virus type 1 (HSV-1)- or type 2 (HSV-2)-specified thymidine kinase gene. Most compounds were endowed with a markedly higher cytostatic activity against the HSV TK gene-transfected tumor cells than against wild-type tumor cells. The principal target for cytostatic activity of the BVDU derivatives proved thymidylate synthase, whereas the guanine derivatives inhibited HSV TK gene-transfected tumor cell proliferation by competing with cellular DNA polymerase(s) and subsequent incorporation into the cellular genome.

     

DNA sequence of an immediate-early gene (IEmRNA-5) of herpes simplex virus type I

We describe a 2560 base pair herpes simplex virus type 1 (HSV-1) DNA sequence containing the entire immediate-early mRNA-5 (IEmRNA-5) gene. The 3' and 5' termini of IEmRNA-5 were mapped within this DNA sequence by single-strand specific endonuclease protection experiments. The IEmRNA-5 gene contains DNA sequences from both the unique (Us) and reiterated (TRs/IRs) regions of the HSV-1 DNA short component and is interrupted by a single intron mapping in TRs/IRs. A search of the transcribed DNA sequence revealed no initiator codon within TRs/IRs. The first ATG was located 6 bases into Us sequences and this reading frame (316 codons) was also observed in the 3' transcribed region. The oligonucleotide sequences adjacent to the IEmRNA-5 termini are discussed in relation to those of the HSV-1 thymidine kinase gene and other genes transcribed by RNA polymerase II.     

Effect of phosphorothioate homo-oligodeoxynucleotides on herpes simplex virus type 2-induced DNA polymerase

Effects of phosphorothioate oligodeoxynucleotides of different chain length and base composition on herpes simplex virus (HSV) type 2 (strain 333)-induced DNA polymerase have been examined in vitro. The anti-HSV-2 DNA polymerase activity was related to the base composition of the analogs, with the order of potency: deoxycytidine greater than thymidine greater than deoxyadenosine, for compounds with equal chain length. The potency was also related to oligomer chain length, since it was observed that the longer the chain length, the more potent the inhibition exerted. Among all the compounds tested, the phosphorothioate oligodeoxycytidine 28-mer (S-(dC)28) was the most potent inhibitor of HSV-2-induced DNA polymerase. This inhibition was competitive with an activated DNA template with a Ki value of 7 nM. It was also a competitive inhibitor of the DNA polymerase-associated exonuclease activity with a Ki value of 5 nM. In contrast, this compound showed less inhibition of human DNA polymerase alpha, beta, and gamma, as well as HSV-1 (strain KOS) and Epstein-Barr virus-induced DNA polymerase. The possibility that S-oligomers can serve as primers for DNA elongation was also investigated. Poly(dG).S-(dC)28 and poly(dA).S-(T)28 are poor substrates for DNA elongation catalyzed by HSV-2 DNA polymerase. In summary, phosphorothioate oligonucleotides could be anti-template inhibitors of HSV DNA polymerase. This information may lead to the development of a new class of selective anti-HSV agents.     

Synthesis and enzymatic transformations of 5-halo-6-methoxy-5,6-dihydro derivatives of 5-[1-methoxy-2-halo (or 2,2-dihalo)ethyl]-2'-deoxyuridines as potential herpes simplex virus inhibitors

The 5-halo-6-methoxy-5,6-dihydro derivatives of 5-[1-methoxy-2-halo(or 2,2-dihalo)ethyl]-2'-deoxyuridines (3-12) were synthesized and investigated as potential anti-herpes agents. These 5,6-dihydro derivatives were designed to act as potential prodrugs to 5-[1-methoxy-2-halo(or 2,2-dihalo)ethyl]-2'-deoxyuridines (2a-e), with enhanced metabolic stability, and ready conversion to the parent molecules. These 5,6-disubstituted-5,6-dihydro analogs are stable to E. coli thymidine phosphorylase, and undergo regeneration of the 5,6-olefinic bond to provide parent moieties (2a-e), upon incubation with glutathione at 37 degrees C. The compounds (3-12) themselves were found to be non-inhibitory against herpes simplex virus type-1 (HSV-1), likely due in part to their inability to undergo conversion to parent compounds in cell culture medium.     

Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.