Mutation of Gln125 to Asn selectively abolishes the thymidylate kinase activity of herpes simplex virus type 1 thymidine kinase

The broad substrate specificity of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) has provided the basis for selective antiherpetic therapy and, more recently, suicide gene therapy for the treatment of cancer. We have now constructed an HSV-1 TK mutant enzyme, in which an asparagine (N) residue is substituted for glutamine (Q) at position 125, and have evaluated the effect of this amino acid change on enzymatic activity. In marked contrast with wild-type HSV-1 TK, which displays both thymidine kinase and thymidylate kinase activities, the HSV-1 TK(Q125N) mutant was unable to phosphorylate pyrimidine nucleoside monophosphates but retained significant phosphorylation activity for thymidine and a series of antiherpetic pyrimidine and purine nucleoside analogs. The abrogation of HSV-1 TK-associated thymidylate kinase activity resulted in a 100-fold accumulation of the monophosphate form of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in osteosarcoma cells transfected with the HSV-1 TK(Q125N) gene compared with osteosarcoma cells expressing wild-type HSV-1 TK. BVDU monophosphate accumulation gave rise to a much greater inhibition of cellular thymidylate synthase in HSV-1 TK(Q125N) gene-transfected cells than wild-type HSV-1 TK gene-transfected osteosarcoma tumor cells without significantly changing the cytostatic potency of BVDU for the HSV-1 TK gene-transfected tumor cells. Accordingly, the presence of the Q125N mutation in HSV-1 TK gene-transfected tumor cells was found to result in a multilog decrease in the cytostatic activity of those pyrimidine nucleoside analogs that in their monophosphate form do not have marked affinity for thymidylate synthase [i.e., 1-beta-D-arabinofuranosylthymine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil].     

Specific recognition of the bicyclic pyrimidine nucleoside analogs, a new class of highly potent and selective inhibitors of varicella-zoster virus (VZV), by the VZV-encoded thymidine kinase.

Recently, an entirely new class of bicyclic nucleoside analogs (BCNAs) was found to display exquisite potency and selectivity as inhibitors of varicella-zoster virus (VZV) replication in cell culture. A striking difference in their ability to convert the BCNAs to their phosphorylated derivatives was observed between the VZV-encoded thymidine kinase (TK) and the very closely related herpes simplex virus type 1 (HSV-1) TK. Whereas VZV TK efficiently phosphorylated the BCNAs, HSV-1 TK was unable to do so. In addition, the thymidylate (dTMP) kinase activity of VZV TK further converted BCNA-5'-MP to BCNA-5'-DP. The BCNAs (or their phosphorylated derivatives) were not a substrate for cytosolic TK, mitochondrial TK, or cytosolic dTMP kinase. Human erythrocyte nucleoside diphosphate (NDP) kinase was unable to phosphorylate the BCNA 5'-diphosphates to BCNA 5'-triphosphates. Under the same experimental conditions, the anti-herpetic (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) derivative was efficiently converted to BVDU-MP and BVDU-DP by both VZV TK and HSV-1 TK and further, into BVDU-TP, by NDP kinase. Our observations may account for the unprecedented specificity of BCNAs as anti-VZV agents.     

Phosphorylation of nucleoside analogs by equine herpesvirus type 1 pyrimidine deoxyribonucleoside kinase

Replication of equine herpesvirus type 1 (EHV-1) was sensitive to 9-(1,3-dihydroxy-2-propoxymethyl)guanine(DHPG) but relatively resistant to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). Likewise, plaque formation by EHV-1 was inhibited by DHPG, but not by BVDU. Plaque formation by a thymidine kinase-negative (tk-) mutant of EHV-1 was not inhibited by DHPG. In order to investigate biochemical mechanisms determining the differential sensitivity of EHV-1 to these drugs, the EHV-1-encoded thymidine kinase enzyme activity (TK)1 was partially purified from EHV-1-infected cells and analyzed. The EHV-1-induced enzyme utilized both ATP and CTP as phosphate donors and differed in relative electrophoretic mobility from the TKs of mock-infected and HSV-1-infected cells. Phosphorylation of 3H-dThd by the EHV-1 TK was inhibited by AraT, IdUrd, BVDU, and DHPG. The EHV-1 TK phosphorylated 125I-dCyd and 3H-ACV. The results indicate that EHV-1 encodes a pyrimidine deoxyribonucleoside kinase with broad nucleoside substrate specificity. These observations suggest that the failure of BVDU to inhibit EHV-1 replication is not attributable to an inability of the EHV-1 TK to phosphorylate BVDU, but may result from the incapacity of the viral TK to convert BVDU monophosphate to the triphosphate or from lack of inhibitory effect of BVDU triphosphate on viral DNA polymerase reactions.     

Non-nucleoside inhibitors of mitochondrial thymidine kinase (TK-2) differentially inhibit the closely related herpes simplex virus type 1 TK and Drosophila melanogaster multifunctional deoxynucleoside kinase

5'-O-Trityl derivatives of thymidine (dThd), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and their acyclic analogs 1-[(Z)-4-triphenylmethoxy-2-butenyl]thymine (KIN-12) and (E)-5-(2-bromovinyl)-1-[(Z)-4-triphenylmethoxy-2-butenyl]uracil (KIN-52) have been synthesized and evaluated for their inhibitory activity against the amino acid sequence related mitochondrial dThd kinase (TK-2), herpes simplex virus type 1 (HSV-1) TK, and Drosophila melanogaster multifunctional 2'-deoxynucleoside kinase (Dm-dNK). Several compounds proved markedly inhibitory to these enzymes and represent a new generation of nucleoside kinase inhibitors. KIN-52 was the most potent and selective inhibitor of TK-2 (IC(50), 1.3 microM; K(i), 0.50 microM; K(i)/K(m), 0.37) but was not inhibitory against HSV-1 TK and Dm-dNK at 100 microM. As found for the alternative substrate BVDU, the tritylated compounds competitively inhibited the three enzymes with respect to dThd. However, whereas BVDU behaved as a noncompetitive inhibitor (alternative substrate) of TK-2 and HSV-1 TK with respect to ATP as the varying substrate, the novel tritylated enzyme inhibitors emerged as reversible purely uncompetitive inhibitors of these enzymes. Computer-assisted modeling studies are in agreement with these findings. The tritylated compounds do not act as alternative substrates and they showed a type of kinetics against the nucleoside kinases different from that of BVDU. KIN-12, and particularly KIN-52, are the very first non-nucleoside specific inhibitors of TK-2 reported and may be useful for studying the physiological role of the mitochondrial TK-2 enzyme.     

Selective abolishment of pyrimidine nucleoside kinase activity of herpes simplex virus type 1 thymidine kinase by mutation of alanine-167 to tyrosine

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 approximately 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.     

Differential metabolism of (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) by equine herpesvirus type 1- and herpes simplex virus-infected cells

Thymidine kinase (TK) enzymes encoded by herpes simplex viruses types 1 and 2 (HSV-1, HSV-2), and equine herpesvirus type 1 (EHV-1) catalyze the phosphorylation of thymidine (dThd) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). The replication of HSV-1 is sensitive to BVDU, but the replication of HSV-2 and EHV-1 is not. To investigate the differential sensitivity of the viruses to halogenated vinyldeoxyuridine drugs, the phosphorylation of 125I-labeled (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) was studied. Cytosol enzymes from cells infected by HSV-2 and EHV-1 phosphorylated [125I]IVDU to the monophosphate, IVDUMP, but did not convert IVDUMP to higher di- plus triphosphates (IVDUDP plus IVDUTP) forms. In contrast, enzymes from HSV-1-infected cells converted [125I]IVDU to radioactive IVDUMP and IVDUDP plus IVDUTP. Experiments with mixtures of EHV-1- and HSV-1-induced enzymes showed that the EHV-1 enzyme did not inhibit formation of the IVDUDP plus IVDUTP by the HSV-1 enzyme. With [125I]IVDU as substrate, the Km values for the EHV-1 and HSV-1 TKs were 1.82 and 0.34 microM, respectively, and the Ki (dThd) value for the EHV-1 TK was 0.35 microM. In vivo experiments showed that HSV-1-infected cells converted IVDU to the mono- and the di- plus triphosphate forms. In contrast, EHV-1-infected cells converted IVDU to the monophosphate to a lesser extent than did HSV-1-infected cells, and did not produce the di- plus triphosphates. Thus, inefficient phosphorylation of the monophosphates probably contributes to the insensitivity of EHV-1 replication to IVDU, as it does to the insensitivity of HSV-2 replication to this drug.     

Highly selective cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives for murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 thymidine kinase gene

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and various structurally related analogues thereof, i.e., (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC), and the carbocyclic analogues of BVDU, IVDU, and BVDC, were evaluated for their inhibitory effects on the growth of murine mammary carcinoma FM3A cells, deficient in thymidine kinase (TK) activity but transformed with the herpes simplex virus type 1 (HSV-1) TK gene (designated FM3A/TK-/HSV-1 TK+). BVDU and its congeners were much more inhibitory to the growth of FM3A/TK-/HSV-1 TK+ than to the growth of the wild type (FM3A/0) cells. For BVDU, for example, the 50% inhibitory dose for the FM3A/TK-/HSV-1 TK+ cells was 0.5 ng/ml, as compared to 11 micrograms/ml for the FM3A/0 cells. Evidently, BVDU and its congeners required phosphorylation by the HSV-1 TK to exert their cytostatic action. In attempts to evaluate further the mechanism of this cytostatic action, BVDU, IVDU, and their carbocyclic analogues were evaluated for their inhibitory effects on thymidylate synthetase (TS) and their incorporation into DNA. TS was identified as one, but not the sole, target in the cytostatic activity of BVDU and its derivatives. With [125I]IVDU and its carbocyclic analogue C-[125I]IVDU, clear evidence was obtained for the incorporation of these radiolabeled analogues into DNA of the FM3A/TK-/HSV-1 TK+ cell line and a TS-deficient mutant thereof, FM3A/TK-/HSV-1 TK+/TS-. No incorporation was detected with [125I]IVDU or C-[125I]IVDU into DNA of FM3A/0 and FM3A/TS- cells. To what extent the incorporation of [125I]IVDU and C-[125I]IVDU contributed to their cytostatic action against FM3A/TK-/HSV-1 TK+ cells remains the subject of further study.     

Differential binding affinities of sugar-modified derivatives of (E)-5-(2-bromovinyl)-2'-deoxyuridine for herpes simplex virus-induced and human cellular deoxythymidine kinases

The affinity of a large number of sugar-modified derivatives of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was determined towards deoxythymidine (dThd) kinases (TK) of various origin, i.e. human cytosol and mitochondrial TK, as well as herpes simplex virus (HSV) type 1 and type 2 TK. Substitution at the 3'- and 5'-position had differential effects on the interaction of BVDU with TK from different sources. The binding affinity of the nucleoside analogs for these different TKs was also influenced by the nature of the 5-substituent (2-bromovinyl vs 2- chlorovinyl ). The 5'-azido and 5'-amino derivatives of BVDU showed affinity for HSV-1 TK only and may, therefore, be useful to differentiate HSV-1 TK from all other TKs . There was no stringent correlation between the antiviral effects of the compounds and their binding constants for viral TK, suggesting that phosphorylation by viral TK is an essential but not sufficient factor in determining the antiviral activity of these analogs.     

Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 or type 2 thymidine kinase gene

Murine mammary carcinoma FM3A cells, deficient in cytosol thymidine (dThd) kinase (TK) activity and transformed by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) TK gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+, respectively) proved extremely sensitive to the cytostatic action of the potent antiherpetic drugs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU). The fact that FM3A TK-/HSV-2 TK+ cells were 5-fold more sensitive to the cytostatic action of BVDU and IVDU but incorporated [125I]IVDU to a 20-fold lower extent into their DNA than did FM3A TK-/HSV-1 TK+ cells led us to conclude that incorporation of these compounds into DNA of HSV TK gene-transformed cell lines is not directly related to their cytostatic action. In attempts to unravel the mechanism of the cytostatic effects of BVDU and IVDU on HSV TK gene-transformed FM3A cells, both compounds were submitted to an intensive biochemical study. Thymidylate synthase was identified as the principal target enzyme for the cytostatic action of BVDU and IVDU since (i) both compounds were far more inhibitory to 2(1)-deoxyuridine (dUrd) than to dThd incorporation into HSV TK gene-transformed FM3A cell DNA, (ii) the cytostatic action of BVDU and IVDU was more readily reversed by dThd than by dUrd, (iii) both compounds strongly inhibited the metabolic pathway leading to the incorporation of 2'-deoxycytidine (dCyd) into DNA thymidylate, (iv) BVDU and IVDU strongly inhibited tritium release from [5-3H]dCyd and [5-3H]dUrd in intact HSV TK gene-transformed FM3A cells, and (v) [125I]IVDU accumulated intracellularly as its 5'-monophosphate to concentration levels considerably higher than those required to inhibit partially purified thymidylate synthase. The inhibitory effects mentioned under (i) to (iv) were not observed with the parental FM3A/0 and FM3A/TK- cells; they were more pronounced for FM3A TK-/HSV-2 TK+ cells than for FM3A TK-/HSV-1 TK+ cells, which correlates with the differential cytostatic effects of BVDU and IVDU on these cells.     

Carbocyclic 5-iodo-2'-deoxyuridine (C-IDU) and carbocyclic (E)-5-(2-bromovinyl)-2'-deoxyuridine (C-BVDU) as unique examples of chiral molecules where the two enantiomeric forms are biologically active: interaction of the (+)- and (-)-enantiomers of C-IDU and C-BVDU with the thymidine kinase of herpes simplex virus type 1

The (+)- and (-)-enantiomers of the carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (C-BVDU) and 5-iodo-2'-deoxyuridine (C-IDU) were synthesized by separate routes. Both the (+)- and (-)-enantiomers of C-BVDU and C-IDU were markedly inhibitory to herpes simplex virus type 1 (HSV-1) replication. (+)-C-BVDU and (+)-C-IDU were as inhibitory to HSV-1 as the racemic (+/-)-C-BVDU and (+/-)-C-IDU, respectively, whereas the (-)-enantiomers were only 10-fold less active. Also, the (+)- and (-)-enantiomers of C-BVDU were equally inhibitory to the growth of murine mammary carcinoma cells transformed by the HSV-1 or HSV-2 thymidine kinase (TK) gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+). The (+)- and (-)-enantiomers of C-BVDU and the (+)- and (-)-enantiomers of C-IDU had a remarkably similar affinity for HSV-1 TK [Ki, 0.09 and 0.19 microM for (+)-C-BVDU and (+)-C-IDU and 0.16 and 0.19 microM for (-)-C-BVDU and (-)-C-IDU, respectively]. The inhibition of HSV-1 TK by BVDU, IDU, (+)-C-BVDU, and (+)-C-IDU was purely competitive with regard to the natural substrate (thymidine), whereas (-)-C-BVDU, (-)-C-IDU, (+/-)-C-BVDU, and (+/-)C-IDU showed a linear mixed-type inhibition of HSV-1 TK. C-BVDU and C-IDU are examples of chiral molecules of which both isomeric forms are markedly active at both the cellular and enzymatic level.     

Distinct thymidine kinases encoded by cowpox virus and herpes simplex virus contribute significantly to the differential antiviral activity of nucleoside analogs

Orthopoxviruses and herpesviruses are both large enveloped DNA viruses, yet these virus families exhibit very different susceptibilities to antiviral drugs. We investigated the activation of nucleoside analogs by the types I and II thymidine kinase (TK) homologs expressed by herpes simplex virus type 1 (HSV-1) and cowpox virus (CV). Antiviral activity against TK(-) and TK(+) strains of HSV-1 and CV was determined, and the ratio of the EC(50) values was used as a measurement of TK dependence. As to HSV-1, most of the selected compounds were markedly less effective against the TK(-) strains, suggesting that this enzyme was required for the activation of these nucleoside analogs. This differs from the results for CV where only idoxuridine and bromodeoxyuridine appeared to be activated, putatively by the type II TK expressed by this virus. These data confirm that the type II TK encoded by CV exhibits a more limited substrate specificity than the type I TK encoded by HSV-1. These data suggest that the inefficient activation of nucleoside analogs by the orthopoxvirus TK significantly limits their activity. Additional screening against orthopoxviruses will be required to identify nucleoside analogs that are efficiently activated by their type II TK.     

Antiviral activity of cyclosaligenyl prodrugs of the nucleoside analogue bromovinyldeoxyuridine against herpes viruses

A series of 42 lipophilic bromovinyldeoxyuridine monophosphates (BVDUMPs) are presented as potential prodrugs of the antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). The 5'-cycloSal-masking group technique has been applied to this cyclic nucleoside analogue to achieve delivery of the monophosphate of BVDU inside the target cells. The new substances have been tested for their antiviral activity against herpes simplex virus types 1 and 2 (HSV-1 and -2), thymidine kinase-deficient (TK(-)) HSV-1, varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). The XTT-based tetrazolium reduction assay EZ4U (for HSV), the plaque inhibition test (for VZV and HCMV) and a DNA hybridisation assay (for EBV) were used to assess antiviral activity. The results indicate that cycloSal-BVDUMP triesters proved to be potent and selective inhibitors of HSV-1 comparable with aciclovir. VZV replication was inhibited by very low concentrations, and two substances had a slightly better anti-VZV activity than the parent compound BVDU. No antiviral effect could be demonstrated against TK(-)-HSV-1, HSV-2 and HCMV, most likely owing to the lack of phosphorylation to BVDU diphosphate. Most remarkably, several cycloSal-BVDUMP triesters yielded promising anti-EBV activity whereas the parent compound BVDU was entirely inactive.     

Comparative inhibition of DNA polymerases from varicella zoster virus (TK+ and TK-) strains by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

The inhibitory effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) 5'-triphosphate on varicella zoster virus (VZV) DNA polymerase was studied using the parent strain (TK+-VZV) and the mutant strain (TK--VZV). The mutant strain was deficient in thymidine kinase (TK)-inducing activity and resistant to BVDU. In the absence of BVDU, TK--VZV and TK+-VZV induced an equivalent level of viral DNA polymerase activity in human embryo fibroblasts. In the presence of 5 microM BVDU, TK--VZV still induced viral DNA polymerase activity, whereas TK+-VZV failed to do so. BVDU 5'-triphosphate (BVDUTP) was considerably more inhibitory to the TK+- and TK--VZV DNA polymerases than to the cellular DNA polymerases. There were no significant differences in the affinity for dTTP as substrate and the sensitivity to BVDUTP as inhibitor between the TK+- and TK--VZV DNA polymerases. The Km value for dTTP and the Ki value for BVDUTP of the VZV DNA polymerases were 1.43 microM and 0.55 microM, respectively. The inhibitory effect of BVDUTP to VZV DNA polymerase was competitive with respect to the natural substrate.     

The multifunctional deoxynucleoside kinase of insect cells is a target for the development of new insecticides

The antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was found to be an efficient substrate for recombinant Drosophila melanogaster-deoxyribonucleoside kinase with a K(m) of 4.5 microM and a V(max) of 400 nmol/microg protein/h compared with 1.3 microM and 62.5 nmol/microg protein/h, respectively, for the natural substrate thymidine. Mammalian cytosolic thymidine kinase-1 does not recognize BVDU as a substrate. In sharp contrast to mammalian cells, the insect D. melanogaster and Spodoptera frugiperda (Sf) embryonic cells proved highly sensitive to the cytostatic action of BVDU. BVDU was efficiently metabolized to its 5'-mono-, 5'-di- and 5'-triphosphate derivatives in the insect cell cultures and abundantly incorporated into the insect cell DNA. BVDU prevented the D. melanogaster cells to initiate the S phase of their cell cycle, and exposure of S. frugiperda cells to BVDU led to a dose-dependent retardation of the insect cells in the S phase of their cell cycle. Both inhibition of nucleic acid synthesis (through the 5'-triphosphate of BVDU) and inhibition of thymidylate synthase (through the 5'-monophosphate of BVDU) would account for the cytostatic activity of BVDU against the insect cells. Because of the virtual lack of cytotoxicity of BVDU against mammalian cells, the drug should be considered highly selective in its cytostatic action against the insect cells. When added to the food of S. frugiperda larvae, BVDU caused a remarkable decrease in the weight gain of the larvae and heavily compromised the transformation of the larvae to the pupae and their subsequent adult (moth) phase. Our data indicate that insect multifunctional deoxyribonucleoside kinase should be considered an entirely novel and attractive target in the development of new nucleoside types of highly selective insecticidal drugs.     

Antiherpetic activity and mechanism of action of 9-(4-hydroxybutyl)guanine

9-(4-Hydroxybutyl)guanine was synthesized and tested for antiherpes activity. In cell cultures, different strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were inhibited by 50% at 2–14 μM of 9-(4-hydroxybutyl)guanine, while a HSV-1 mutant lacking thymidine kinase (HSV-1 TK^-) was resistant. Linear competitive inhibition of purified HSV-1-induced thymidine kinase (TK) with thymidine as a variable substrate was observed for 9-(4-hydroxybutyl)guanine with an apparent K_i value of 2.06 μM while the corresponding K_i value for the cellular TK was > 250 μM. By using high performance liquid chromatography, the formation of 9-(4-hydroxybutyl)guanine monophosphate by HSV-1 TK was measured and the rate of product formation was found to be about 10% of that found by using thymidine as a substrate. A selective inhibition of HSV-1 DNA synthesis by 9-(4-hydroxybutyl)guanine was observed in infected Vero cells. 9-(4-Hydroxybutyl)guanine had a low cellular toxicity. A weak therapeutic effect on herpes keratitis in rabbits was observed whereas cutaneous HSV-1 infection in guinea pigs and systemic HSV-2 infection in mice were not affected by this compound.     

Inhibition of herpes simplex virus thymidine kinases by 2-phenylamino-6-oxopurines and related compounds: structure-activity relationships and antiherpetic activity in vivo

Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 microM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.     

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)-cyclobutyl] guanine [(+-)-BHCG or SQ 33,054]: a potent and selective inhibitor of herpesviruses

(+-)-(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.     

Effects of phosphonylmethoxyalkyl-purine and -pyrimidine derivatives on TK+ and TK- HSV-1 keratitis in rabbits.

The phosphonylmethoxyalkyl derivatives HPMPA [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine], HPMPC [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] and PMEA [9-(2-phosphonylmethoxyethyl)adenine] were evaluated as 0.2% eyedrops for their efficacy in the treatment of experimental herpes simplex virus type 1 (HSV-1) keratitis in the rabbit model. BVDU 0.2% eyedrops were used as the reference treatment. HPMPA, HPMPC, PMEA and BVDU eyedrops showed a rapid and highly significant healing effect (P less than 0.005) on keratitis caused by TK+ HSV-1 (McIntyre strain) when compared with placebo eyedrops, whereas BVDU treatment did not affect the course of TK- HSV-1 (VMW-1837) keratitis. HPMPA and HPMPC treatment again caused a highly significant healing (P less than 0.005, compared with placebo eyedrops). Although PMEA eyedrops were less effective than HPMPA or HPMPC eyedrops, the effect of PMEA eyedrops was significantly (P less than 0.05) different from the effect of either BVDU or placebo eyedrops.     

Acetylenic nucleosides. 4. 1-beta-D-arabinofuranosyl-5-ethynylcytosine. Improved synthesis and evaluation of biochemical and antiviral properties

5-Ethynyl-1-beta-D-arabinofuranosylcytosine (EAC) was prepared from 1-(2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)cytosine by iodination followed by coupling with (trimethylsilyl)acetylene and deblocking. At 50 microM, EAC was found to inhibit the in vitro replication of herpes simplex virus type 1 and type 2 by greater than 99%. EAC also showed activity against a strain of HSV-1 resistant to (E)-5-(2-bromovinyl)-2'-deoxyuridine which has an alteration of the virus-induced thymidine kinase (TK). At 100 microM, EAC did not inhibit the in vitro growth of leukemia L1210 and HeLa cells. EAC was resistant to the action of dCR-CR deaminase, its rate of deamination being approximately 2% that of dCR. The compound was a poor substrate for dCR kinase, but it was phosphorylated by HSV-1- and HSV-2-induced TKs at 50% and 30%, respectively, the rate of thymidine.     

N1-substituted thymine derivatives as mitochondrial thymidine kinase (TK-2) inhibitors

Novel N1-substituted thymine derivatives related to 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine have been synthesized and evaluated against thymidine kinase-2 (TK-2) and related nucleoside kinases [i.e., Drosophila melanogaster deoxynucleoside kinase (Dm-dNK) and herpes simplex virus type 1 thymidine kinase (HSV-1 TK)]. The thymine base has been tethered to a distal triphenylmethoxy moiety through a polymethylene chain (n = 3-8) or through a (2-ethoxy)ethyl spacer. Moreover, substitutions at position 4 of one of the phenyl rings of the triphenylmethoxy moiety have been performed. Compounds with a hexamethylene spacer (18, 26b, 31) displayed the highest inhibitory values against TK-2 (IC50 = 0.3-0.5 microM). Compound 26b competitively inhibited TK-2 with respect to thymidine and uncompetitively with respect to ATP. A rationale for the biological data was provided by docking some representative inhibitors into a homology-based model of human TK-2. Moreover, two of the most potent TK-2 inhibitors (18 and 26b) that also inhibit HSV-1 TK were able to reverse the cytostatic activity of 1-(beta-D-arabinofuranosyl)thymine (Ara-T) and ganciclovir in HSV-1 TK-expressing OST-TK-/HSV-1 TK+ cell cultures.