Differential mechanism of cytostatic effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, and other antiherpetic drugs on tumor cells transfected by the thymidine kinase gene of herpes simplex virus type 1 or type 2

2-Amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) represents the first antivirally active nucleoside analog with the side chain attached to the N-7 position of the purine ring. Compound S2242 strongly inhibits the in vitro replication of both herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) (50% effective concentration [EC50], 0.1 to 0.2 microgram/ml), varicella-zoster virus (EC50, 0.01 to 0.02 microgram/ml) and thymidine kinase (TK)-deficient strains of HSV (EC50, 0.4 microgram/ml) and varicella-zoster virus (EC50, 0.2 to 0.5 microgram/ml). Potent activity was also observed against murine cytomegalovirus (EC50, 1 microgram/ml), human cytomegalovirus (HCMV) (EC50, 0.04 to 0.1 microgram/ml), and human herpesvirus 6 (EC50, 0.0005 microgram/ml). Compound S2242 (i) was not cytotoxic to confluent Vero, HeLa, or human fibroblast cells at concentrations of > 100 micrograms/ml, (ii) proved somewhat more cytostatic to Vero, HEL, HeLa, and C127I cells than ganciclovir, and (iii) was markedly more cytostatic than ganciclovir to the growth of the human lymphocytic cell lines HSB-2 and CEM degrees. In contrast to ganciclovir, (i) compound S2242 proved not to be cytocidal to murine mammary carcinoma (FM3A) cells transfected with the HSV-1 or HSV-2 TK gene, (ii) exogenously added thymidine had only a limited effect on its anti-HSV-1 activity, and (iii) the compound was not phosphorylated by HSV-1-encoded TK derived from HSV-1 TK-transfected FM3A cells, indicating that the compound is not activated by a virally encoded TK. Compound S2242 inhibited (i) the expression of late HCHV antigens at an EC50 of 0.07 microgram/ml (0.6 microgram/ml for ganciclovir) and (ii) HCMV DNA synthesis at an EC50 of 0.1 microgram/ml (0.32 microgram/ml for ganciclovir), i.e., values that are close to the EC50S for inhibition of HCMV-induced cytopathogenicity. Neither ganciclovir nor S2242 had any effect on the expression of immediate-early HCMV antigens, which occurs before viral DNA synthesis. In time-of-addition experiments, S2242 behaved like ganciclovir and acyclovir; i.e., the addition of the drugs could be delayed until the onset of viral DNA synthesis.

The N-7-substituted acyclic nucleoside analog 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine is a potent and selective inhibitor of herpesvirus replication.

Ten pyrimidine nucleoside analogues, including (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and closely related analogues, were evaluated for their cytostatic activity against human osteosarcoma cells transfected with the varicella-zoster virus (VZV) thymidine kinase (tk) (ATP:thymidine 5' phosphotransferase, EC 2,7,2,21) gene. (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), (E)-5-(2-iodovinyl)-2'-deoxy-2'fluoro-1 beta-D-arabinofuranosyluracil (IVFAU) and (E)-5-(2-bromovinyl)-2'deoxy-4'-thiouridine (S-BVDU) were among the most potent inhibitors of VZVtk gene-transfected cell proliferation. They displayed an inhibitory activity at drug concentrations that were up to four orders of magnitude lower than those required to inhibit the corresponding nontransfected tumor cells. inhibition of cellular DNA polymerase and/or incorporation of the drugs into cellular DNA may be a likely target for the cytostatic activity of the BVDU derivatives against the VZVtk gene-transfected tumor cells. These compounds were approximately 40- to 80-fold more potent cytostatic agents in VZVtk gene-transfected cells than the anti-VZV compound 6-methoxy-9-beta-D-arabinofuranosylpurine (araM), and at least five-to 50-fold more cytostatic than ganciclovir in HSV-1tk gene-transfected murine mammary carcinoma FM3A cells. In addition, the intrinsic resistance of BVaraU, IVFAU and S-BVDU to glycosidic bond cleavage by mammalian dThd phosphorylases makes them promising candidate compounds for the treatment of VZVtk gene-transfected tumors in vivo.

Varicella-zoster virus thymidine kinase gene and antiherpetic pyrimidine nucleoside analogues in a combined gene/chemotherapy treatment for cancer

Superior cytostatic activity of the ganciclovir elaidic acid ester due to the prolonged intracellular retention of ganciclovir anabolites in herpes simplex virus type 1 thymidine kinase gene-transfected tumor cells

Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2′-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2′-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced ∼2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2′-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.

Selective Abolishment of Pyrimidine Nucleoside Kinase Activity of Herpes Simplex Virus Type 1 Thymidine Kinase by Mutation of Alanine-167 to Tyrosine

To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.

Establishment of Mutant Murine Mammary Carcinoma FM3A Cell Strains Transformed with the Herpes Simplex Virus Type 2 Thymidine Kinase Gene

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Establishment of Mutant Murine Mammary Carcinoma FM3A Cell Strains Transformed with the Herpes Simplex Virus Type 2 Thymidine Kinase Gene