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.     

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.     

Substrate specificity of feline and canine herpesvirus thymidine kinase

The thymidine kinases from feline herpesvirus (FHV TK) and canine herpesvirus (CHV TK) were cloned and characterized. The two proteins are closely sequence-related to each other and also to the herpes simplex virus type 1 thymidine kinase (HSV-1 TK). Although FHV TK and CHV TK have a level of identity of 31 and 35%, respectively, with HSV-1 TK, and a general amino acid similarity of approximately 54% with HSV-1 TK, they do not recognize the same broad range of substrates as HSV-1 TK does. Instead the substrate recognition is restricted to dThd and pyrimidine analogs such as 1-beta-d-arabinofuranosylthymine (araT), 3'-azido-2',3'-dideoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). FHV TK and CHV TK differ in substrate recognition from mammalian cytosolic thymidine kinase 1 (TK1) in that TK1 does not phosphorylate BVDU and they also differ from mammalian mitochondrial thymidine kinase 2 (TK2), which, in addition to thymidine and thymidine analogs also phosphorylates dCyd. Although the nucleoside analog BVDU was a good substrate for FHV and CHV TK, the compound was poorly inhibitory to virus-induced cytopathic effect in FHV- and CHV-infected cells. The reason is likely the poor, if any, thymidylate kinase activity of FHV and CHV TK, which in HSV-1 TK-expressing cells convert BVDU-MP to its 5'-diphosphate derivative.     

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.     

2'-O-Acyl/alkyl-substituted arabinosyl nucleosides as inhibitors of human mitochondrial thymidine kinase

Introduction of a bulky lipophilic acyl entity at the 2'-OH position of both 1-beta-D-arabinofuranosylthymine (araT) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), consistently resulted in a marked ( approximately 10-fold) increase in the inhibitory activity of these new arabinosyl nucleoside analogues for the mitochondrial thymidine kinase (TK-2)-catalysed conversion of 2 microM [methyl-(3)H]dThd to [methyl-(3)H]dTMP. The most potent derivatives were inhibitory to [methyl-(3)H]dThd phosphorylation by TK-2 within the lower micromolar concentration range. Substitution of the arabinosyl nucleoside derivatives with the acyl groups also dramatically increased the selectivity of these compounds. The inhibitory activity of araT and BVaraU to dThd phosphorylation by other related nucleoside kinases, including herpes simplex virus type 1 TK, varicella-zoster virus TK, and cytosolic TK-1, was completely annihilated upon 2'-O-acyl substitution (IC(50) > or = 1000 microM). Kinetic analysis revealed purely competitive inhibition of 2'-O-acyl-BVaraU against TK-2-catalysed thymidine phosphorylation (K(i)/K(m): 2.3). However, 2'-O-acyl-BVaraU was extremely poorly converted to the corresponding arabinosyl nucleoside 5'-monophosphate by TK-2 as revealed by [gamma-(32)P]phosphate transfer studies from [gamma-(32)P]ATP. Thus, the 2'-O-acyl derivatives of BVaraU did not behave as substrates, but rather as potent and highly selective inhibitors of TK-2. This is the first report on such a highly selective arabinosyl nucleoside inhibitor of mitochondrial TK-2, and opens perspectives for the rational design of selective mitochondrial TK-2 inhibitors.     

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.     

Acyclic nucleoside analogues as novel inhibitors of human mitochondrial thymidine kinase

A series of acyclic nucleoside analogues of 5'-O-tritylthymidine have been synthesized and evaluated as potential human mitochondrial thymidine kinase (TK-2) inhibitors. In this series, the sugar moiety of the parent 5'-O-tritylthymidine has been replaced by aliphatic chains including (E)- and (Z)-butenol, butynol, or butanol. Among them the (Z)-butenyl derivative (10) showed an IC(50) against TK-2 of 1.5 microM, being 1 order of magnitude more potent than the parent 5'-O-tritylthymidine. This lead compound has been further modified by replacing the thymine base by other pyrimidine bases such as 5-iodouracil, 5-ethyluracil, 5-methylcytosine, 3-N-methylthymine, or 5,6-dihydrothymine, as well as by the purine base guanine. The trityl group has also been replaced by different aliphatic and aromatic acyl moieties including tert-butylacetyl, hexanoyl, decanoyl, and diphenylacetyl moieties. The evaluation of the compounds against TK-2 and the phylogenetically close HSV-1 TK has shown that the base moiety plays a crucial role in their interaction against these pyrimidine nucleoside kinases. Also, the presence of a lipophilic substituent, preferentially an aromatic moiety such as diphenylmethyl or triphenylmethyl, is required for efficient TK-2 inhibition. Whereas some compounds showed marked specificity for either TK-2 (i.e, the 5,6-dihydrothymine derivative, 26) or HSV-1 TK (i.e., the butynyl derivative, 11), some others, including the (Z)-and (E)-butenyl derivatives 10 and 12, showed significant inhibition against both enzymes. They also proved to be inhibitory against HSV-1 TK in intact human osteosarcoma cells that were transduced with the HSV-1 TK gene.     

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.     

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].     

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 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.     

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.     

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.     

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.     

Bystander effects of cancer cell lines transduced with the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster and synergistic enhancement by hydroxyurea.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human cells with retained enzymatic activity. The cells expressing Dm-dNK exhibit increased sensitivity to several cytotoxic nucleoside analogs. In this study, we further evaluated Dm-dNK as a potential novel suicide gene in combination with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as the prodrug. We used two human cancer cell lines transduced with a retrovirus encoding the Dm-dNK cDNA and investigated whether the cells expressing the enzyme can induce cell death of untransduced cells, a phenomenon known as the "bystander effect". A bystander effect was observed in a thymidine kinase-deficient human osteosarcoma cell line but not in the MIA PaCa-2 human pancreatic adenocarcinoma cell line. The cytotoxicity of BVDU increased in both cell lines when the compound was used in combination with subtoxic concentrations of hydroxyurea. Hydroxyurea also enhanced the bystander effect in the osteosarcoma cells, but not in the MIA PaCa-2 cells, treated with BVDU. These findings indicate that BVDU phosphorylated by Dm-dNK in transduced cancer cells may also induce bystander cell death in certain cell lines.     

Mitochondrial expression of the Drosophila melanogaster multisubstrate deoxyribonucleoside kinase

The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) is studied as a candidate suicide gene for applications in combined gene/chemotherapy of cancer. We have created an engineered Dm-dNK nucleoside kinase that is targeted to the mitochondrial matrix. The enzyme was expressed in a thymidine kinase 1-deficient osteosarcoma cell line, and the sensitivity of the cells to cytotoxic nucleoside analogs was determined when the enzyme was targeted to either the nucleus or the mitochondrial matrix. Although the total deoxythymidine (dThd) phosphorylation activity was similar in cells expressing Dm-dNK in the nucleus or in the mitochondria, the cells expressing the enzyme in the mitochondria showed higher sensitivity to the antiproliferative activity of several pyrimidine nucleoside analogs, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine. Labeling studies using [3H]dThd showed that the cells expressing the mitochondrial enzyme had an increased incorporation of [3H]dThd into DNA, shown to be due to a higher [3H]dTTP specific activity of the total dTTP pool in the cells in which Dm-dNK was targeted to the mitochondria. The difference in the specific activity of the dTTP pool is a result of different contributions of the de novo and the salvage pathways for the dTTP synthesis in transduced cells. In summary, these findings suggest that mitochondrial targeting of Dm-dNK facilitates nucleoside and nucleoside analog phosphorylation and could be used as a strategy to enhance the efficacy of nucleoside analog phosphorylation and concomitantly their cytostatic potential.     

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.     

Cellular pharmacology of 5-fluorouracil in a human colon adenocarcinoma cell line selected for thymidine kinase deficiency

A human colon adenocarcinoma cell line (GC3TK-) was selected for thymidine kinase (TK) deficiency from cloned parental cells (GC3C1) by exposure to 5-bromodeoxyuridine (BrdUrd). The cellular pharmacology of 5-fluorouracil (FUra) and the influence of physiological concentrations of thymidine (dThd; 0.1 to 1 microM) on FUra cytotoxicity during brief exposure in both cell lines were examined. The uptake of FUra during a 1-hr drug exposure, its metabolism to ribo- and deoxyribonucleotides, incorporation into RNA, and inhibition of thymidylate synthase were similar in GC3C1 and GC3TK- cells as were the IC50 values for FUra (26 and 23 microM respectively). TK deficiency did not reduce the intracellular concentrations of FdUMP generated from FUra. In GC3C1, at FUra concentrations up to 100 microM, cytotoxicity was prevented by co-administration of dThd (0.1 to 20 microM). The relationship between cell survival and thymidylate synthase inhibition was close under these conditions. At higher drug concentrations, less dThd protection was observed, and none was detected in GC3TK- cells. Thus, the metabolism of FUra did not appear to be altered substantially in GC3C1 cells selected for TK deficiency. Also in these cells, at concentrations of FUra less than 100 microM, FUra cytotoxicity appeared to be mediated via the inhibition of thymidylate synthase.     

Phosphonoformate and phosphonoacetate derivatives of 5-substituted 2'-deoxyuridines: synthesis and antiviral activity

The synthesis of potential "combined prodrugs" wherein phosphonoformate or phosphonoacetate was attached to the 5'-position of 2'-deoxyuridine, 2'-deoxythymidine, 5-iodo-2'-deoxyuridine (IDU), 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), or 5-(2-bromovinyl)-2'-deoxyuridine (BVDU) or to the 3'-position of CEDU is described. The antiviral activities of these derivatives and of reference compounds were compared in Vero, HEp-2, and primary rabbit kidney cells against herpes simplex virus types 1 and 2 (HSV-1 and -2). The CEDU and BVDU analogues were also evaluated against systemic and intracutaneous HSV-1 infection in mice. The nature of the 5-substituent proved critical for antiviral activity, since only the 5-iodo-, 5-(2-bromovinyl)-, and 5-(2-chloroethyl)-substituted derivatives were inhibitory to the herpesviruses. Furthermore, the type specificity is determined by the nature of the 5-substituent: the IDU analogues were similarly inhibitory to HSV-1 and -2 whereas the CEDU and BVDU analogues inhibited HSV-2 replication only at considerably higher concentrations than HSV-1. In vivo, several derivatives were shown to possess significant antiviral activity; however, none surpassed its respective parent compound, CEDU or BVDU, in potency. It seems improbable, therefore, that a synergistic effect between PFA or PAA and the nucleoside analogue occurred. The extent of in vitro and in vivo activity of the CEDU and BVDU 5'-phosphonoformates and 5'-phosphonoacetates is most plausibly explained by the ease by which the "combined prodrugs" are hydrolyzed and the parent compound, CEDU and BVDU, respectively, is released.