Herpesviruses are a commonvirus infecting man. Twodrugs have shown clinical efficacyin the treatment of herpesvirus infections, acyclovir and ganciclovir. Acyclovir has excellent antiviral activity against herpes simplex types 1 and 21}; ganciclovir is potent against human cytomegalovirus2). Both drugs are nucleoside analogs and are metabolized to their respective triphosphates which then block viral DNA replication by inhibiting the viral DNA polymerase3'4).

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This chapter discusses the polynucleotide polymerization and prostate proliferation. Many enzymes in animal tissues are involved in DNA replication and in the related processes of DNA repair and genetic recombination. The monomeric building blocks are invariably the deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP. Several characteristics of the replicative DNA polymerase reactions that are greatly increased during the hyperplastic phase of rat ventral prostate growth. The characteristics of reactions as catalyzed by prostate extracts were examined in considerable detail so as to establish optimal assay conditions. It is found that when various adult rat organs were homogenized in dilute thiol-containing buffers and then ultracentrifuged, high D-DNA polymerase activities were observed only with soluble extracts of tissues, such as spleen and testis that contained large numbers of dividing cells. The remarkable difference between the two deoxyribonucleotide polymerizations catalyzed by soluble prostate extracts is that the R-DNA polymerase reaction was much more inhibited by the direct addition of low concentrations of ethidium bromide than was the D-DNA polymerase. It is found that the rat prostate soluble D-DNA and R-DNA polymerase reactions exhibit very different patterns of changes in response to alterations in the androgenic status of the animals.

The syntheses of the enantiomeric cyclobutyl guanine nucleoside analogues [1R-1 alpha, 2 beta, 3 alpha]- and [1S-1 alpha, 2 beta, 3 alpha]-2- amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one (7 and 8, respectively) and the enantiomeric cyclobutyl adenine analogues [1R-1 alpha, 2 beta, 3 alpha]- and [1S-1 alpha, 2 beta, 3 alpha]-6-amino-9-[2,3-bis(hydroxymethyl) cyclobutyl]purine (9 and 10, respectively) are described. trans-3,3-Diethoxy-1,2-cyclobutanedicarboxylic acid (14) was coupled with R-(-)-2-phenylglycinol to provide a mixture of diastereomeric bis-amides, 15a and 15b, which was readily separated by crystallization. Conversion of each bis-amide to the corresponding diol enantiomer, 16a and 16b, respectively, was effected by a facile three-step sequence in high overall yield. Homochiral diol 16a was converted in a straightforward manner to 7 and 9, and homochiral diol 16b was similarly converted to the corresponding optical isomers 8 and 10. Compounds 7 and 9, which mimic the absolute configuration of natural nucleosides, are highly active against a range of herpesviruses in vitro while the isomers of opposite configuration, 8 and 10, are devoid of antiherpes activity. The corresponding triphosphates of 7 and 8 (7-TP and 8-TP) were prepared enzymatically. Compound 7-TP selectively inhibits HSV-1 DNA polymerase, compared to human (HeLa) DNA polymerase, while 8-TP is much less inhibitory than 7-TP against both types of enzymes. Compounds 7 and 9 are efficacious in a mouse cytomegalovirus model infection.

(+/-)-(1 alpha,2 beta,3 alpha)-9-[2,3-Bis(hydroxymethyl)cyclobutyl] guanine [(+/-)-BHCG] is a nucleoside analog with potent in vitro activity against herpesviruses [Tetrahedron Lett. 30:6453-6456 (1989)]. The two enantiomers have been synthesized, and their biochemical characterization is reported here. [1S(1 alpha,2 beta,3 alpha)]-9-[2,3-Bis(hydroxymethyl)cyclobutyl]guanine [(S)-BHCG] was phosphorylated by herpes simplex virus type 1 (HSV-1) thymidine kinase (Vmax = 8 nmol/hr/micrograms of enzyme), whereas [1R(1 alpha,2 beta,3 alpha)]-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine [(R)-BHCG] was a poor substrate for the viral thymidine kinase under these conditions. The triphosphate of each enantiomer was enzymatically synthesized, and both enantiomers competitively inhibited HSV-1 DNA polymerase with respect to dGTP. However, the potency of (R)-BHCG-TP was 4 orders of magnitude greater than that of (S)-BHCG-TP. (R)-BHCG-TP inhibited HeLa DNA polymerase alpha, but the inhibition constant was 30-fold higher than that for the viral DNA polymerase. In comparison, (S)-BHCG-TP was a very poor inhibitor of DNA polymerase alpha. (R)-[3H]BHCG-TP could be incorporated into a synthetic DNA template by HSV-1 DNA polymerase at 80% the extent of dGTP under the assay conditions used and, therefore, could act as an alternative substrate. Incorporation of (R)-BHCG-TP was similar to that observed for acyclovir triphosphate and ganciclovir triphosphate, based on maximal velocities. In contrast, HSV-1 DNA polymerase did not incorporate (S)-BHCG-TP into DNA. Compared with dGTP, only limited extension (10%) of the DNA primer by HSV-1 DNA polymerase occurred after incorporation of (R)-BHCG-TP and, therefore, (R)-BHCG-TP acts as a nonobligate chain terminator.

Two herpes simplex virus mutants containing mutated residues within the conserved Exo III motif of the polymerase gene were previously shown to be defective in 3′–5′ exonuclease activity and exhibited extremely high mutation frequencies. In this study, we have shown that these mutants also exhibited higher resistance to phosphonoacetic acid and sensitivity to aphidicolin and all nucleoside analogs tested, including acyclovir and gancicolvir, compared to wild-type virus. Marker transfer experiments and sequencing analyses demonstrated that these altered phenotypes were the result of mutations within the Exo III motif. The data indicate that, aside from leading to exonuclease deficiency, mutations in the Exo III motif may also affect interaction of nucleoside triphosphates with the catalytic sites of polymerase activity.

A novel nucleoside analogue, (±)-(1α, 2β, 3α)-9-[2-hydroxy -3-(hydroxymethyl)cyclobutyl]guanine [(±)-HHCG] was synthesized and has antiviral activity against herpes simplex virus (HSV) types 1 and 2, human cytomegalovirus (HCMV) and varicella-zoster virus (VZV) in plaque reduction assays. The antiviral activity of (±)-HHCG against HSV-2 shows a 10-40-fold dependence on the presence of a virally encoded thymidine kinase. (±)-HHCG is a substrate for HSV-1 thymidine kinase with a phosphorylation rate of 28 μm h −1 compared to 15 μm h −1 for acyclovir under identical conditions. Enzymatically prepared HHCG-triphosphate is a competitive inhibitor of dGTP incorporation into DNA by HSV-1 DNA polymerase with an inhibition constant corresponding to 0.0077 μm. Hybridization studies using an HSV-1-specific DNA probe indicated that DNA synthesis is reduced in HSV-1-infected WI-38 cells treated with (±)-HHCG, with an ED 50 comparable to that of acyclovir. These results suggest that the antiviral activity of (±)-HHCG is due to preferential inhibition of viral DNA synthesis.

(±)-(1α,2β,3α)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine [(±)-BHCG or SQ 33 054] is a newly synthesized nucleoside analog with potent and selective antiviral activity against members of the herpesvirus group, including human cytomegalovirus. The activity against a thymidine kinase deficient HSV-2 mutant was 25-fold poorer than against the parent virus, suggesting that phosphorylation is an important prerequisite for antiviral activity against HSV-2. (±)-BHCG is readily phosphorylated by purified HSV-1 thymidine kinase, and BHCG triphosphate synthesized enzymatically is a selective inhibitor of HSV-1 DNA polymerase. (±)-BHCG did not inhibit host cell growth at concentrations at least 1000-fold higher than HSV-2 inhibitory concentrations. Subcutaneous administration of (±)-BHCG was protective against HSV-1 systemic infections in mice. BHCG is an exciting antiviral agent and represents a new class of nucleoside analogs.

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl] guanine [(+-)-BHCG or SQ 33,054] is a newly synthesized nucleoside analog with potent and selective antiviral activity against members of the herpesvirus group, including human cytomegalovirus. The activity against a thymidine kinase deficient HSV-2 mutant was 25-fold poorer than against the parent virus, suggesting that phosphorylation is an important prerequisite for antiviral activity against HSV-2. (+-)-BHCG is readily phosphorylated by purified HSV-1 thymidine kinase, and BHCG triphosphate synthesized enzymatically is a selective inhibitor of HSV-1 DNA polymerase. (+-)-BHCG did not inhibit host cell growth at concentrations at least 1000-fold higher than HSV-2 inhibitory concentrations. Subcutaneous administration of (+-)-BHCG was protective against HSV-1 systemic infections in mice. BHCG is an exciting antiviral agent and represents a new class of nucleoside analogs.

The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.