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.     

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.     

Synthesis and antiviral activity of the carbocyclic analogues of (E)-5-(2-halovinyl)-2'-deoxyuridines and (E)-5-(2-halovinyl)-2'-deoxycytidines

The carbocyclic analogues of the potent and selective antiherpes agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) were synthesized by conventional methods with use of carbocyclic 2'-deoxyuridine as starting material. C-BVDU, C-IVDU, and C-BVDC were equally selective, albeit slightly less potent, in their antiherpes action than BVDU, IVDU, and BVDC. Although resistant to degradation by pyrimidine nucleoside phosphorylases, C-BVDU did not prove more effective than BVDU in the systemic (oral, intraperitoneal) or topical treatment of HSV-1 infections in mice.     

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.     

Incorporation of (E)-5-(2-iodovinyl)-2'-deoxyuridine into deoxyribonucleic acids of varicella-zoster virus (TK+ and TK- strains)-infected cells

The incorporation of (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) into DNA of varicella-zoster virus (VZV)-infected human embryo fibroblasts was studied, using thymidine kinase-positive (TK+) and thymidine kinase-negative (TK-) VZV strains. [125I]IVDU was taken up by cells infected with TK+ VZV-, but not by TK- VZV- or mock-infected cells. [125I]IVDU was incorporated into both VZV DNA and cellular DNA of TK+ VZV-infected cells. When the cells were exposed to 0.3 microM IVDU, a more marked shift was noted in the buoyant density of viral DNA than of host DNA. In contrast, the DNAs isolated from TK- VZV- or mock-infected cells did not exhibit a detectable incorporation of [125I]IVDU. [125I] IVDU-labeled VZV DNA was purified from the viral nucleocapsids of TK+ VZV-infected cells. Substitution of no more than 0.1-1% of the thymidine residues in the VZV DNA by IVDU seemed to suffice to inhibit the replication of VZV.     

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.     

Role of the incorporation of (E)-5-(2-iodovinyl)-2'-deoxyuridine and its carbocyclic analogue into DNA of herpes simplex virus type 1-infected cells in the antiviral effects of these compounds

The carbocyclic analogue of (E)-5-(2-iodovinyl)-2'-deoxyuridine (C-IVDU) is, like its parent compound (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1). There is a close correlation between the inhibition of viral DNA synthesis and the antiviral activity of both IVDU and C-IVDU. IVDU and C-IVDU inhibit viral DNA synthesis at 0.2 and 0.5 microM, respectively, and interfere with cellular DNA synthesis at concentrations that are 10- to 40-fold in excess of their antivirally effective doses. At concentrations affording a similar antiviral effect, C-[125I]IVDU is incorporated into viral and cellular DNA of HSV-1-infected Vero cells to a 7- to 10-fold lesser extent than IVDU. [125I]IVDU but not C-[125I]IVDU leads to breakage of both DNA strands when incorporated into HSV-1 DNA.     

Synthesis and antiviral activity of (E)-5-(2-bromovinyl)uracil and (E)-5-(2-bromovinyl)uridine

(E)-5-(2-Bromovinyl)uracil (BVU) and (E)-5-(2-bromovinyl)uridine (BVRU) were synthesized starting from 5-formyluracil via (E)-5-(2-carboxyvinyl)uracil or starting from 5-iodouridine via (E)-5-(2-carbomethoxyvinyl)uridine and (E)-5-(2-carboxyvinyl)uridine, respectively. Depending on the choice of the cell system, BVU and BVRU exhibited a marked activity against herpes simplex virus type 1 (HSV-1) in vitro. Although BVU and BVRU were less potent than the reference compound (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), their antiviral activity spectrum was remarkably similar to that of BVDU. The latter findings suggest that BVU and BVRU are metabolically converted to BVDU or a phosphorylated product thereof. In vivo, BVU protected mice against a lethal disseminated HSV-1 infection.     

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.     

(E)-5-(2-bromovinyl)uridine requires phosphorylation by the herpes simplex virus (type 1)-induced thymidine kinase to express its antiviral activity

(E)-5-(2-Bromovinyl)uridine (BVUrd), the riboside counterpart of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd), effected a dose-dependent inhibition of viral progeny formation and viral DNA synthesis in herpes simplex virus type 1 (HSV-1, strain KOS)-infected human (E6SM) diploid fibroblast cells. BVUrd was directly phosphorylated in HSV-1-infected cells, presumably by the virus-encoded thymidine kinase (TK), since (i) BVUrd was not phosphorylated by extracts of cells infected with a HSV-1 strain deficient in TK expression and (ii) the phosphorylation was inhibited by a polyclonal anti-HSV-1 antibody. Within the HSV-1-infected cells, BVUrd was incorporated into the viral DNA as BVdUMP (BVdUrd 5'-monophosphate). This incorporation may account for the antiviral action of BVUrd, and implies that, following its initial phosphorylation by the viral TK, BVUrd is converted to its 2'-deoxy counterpart, most likely at the 5'-diphosphate level (BVUDP----BVdUDP).     

in vitro metabolism of 1-β-d-arabinofuranosylcytosine and 1-β-2'-fluoro-arabino-5-iodocytosine in normal and herpes simplex type 1 virus-infected cells

Phosphorylation of 1-β-D-2'-F-arabino-5-iodocytosine (FIAC), a newly synthesized pyrimidine nucleoside with potent antiherpesvirus activity, was compared with that of its parent compound, 1-β-D-arabinofuranosylcytosine (ara-C). While ara-C was phosphorylated extensively by homogenates of normal, rapidly proliferating mouse tissues, FIAC was a poor substrate for the nucleoside kinase occurring in such normal tissues. With cell homogenates of noninfected Vero cells, thymidine (TdR) was phosphorylated about fifty and twenty times more efficiently than FIAC and ara-C, while infection of Vero cells with Herpes Simplex Virus Type 1 (HSV-1) resulted in a 23-fold increase of TdR- and a 1270-fold increase of FIAC phosphorylation. In contrast, phosphorylation of ara-C was increased only by a factor of 2.6. While the reaction products obtained with homogenates of normal mouse tissues were 5'-mono-, di- and triphosphates of ara-C and FIAC, the reaction products with noninfected and infected Vero cell homogenates were predominantly monophosphates. In contrast, TdR was efficiently phosphorylated to its 5'-mono-, di- and triphosphates by such homogenates. In intact HSV-1-infected Vero cells, FIAC was rapidly taken up and phosphorylated to FIACMP and to an as yet unidentified metabolite. In contrast, TdR was taken up and phosphorylated to 5'-mono-, di- and triphosphates and ara-C was taken up moderately but metabolized poorly to its 5'-mono-, di- and triphosphates. Thus, in normal tissues, FIAC was a poorer substrate than ara-C for nucleoside kinases, but in intact HSV-1-infected Vero cells FIAC was efficiently phosphorylated and thus behaved like a TdR analog, except that it was phosphorylated only to the 5'-monophosphate and a hitherto unidentified metabolite. The greatly increased phosphorylation of FIAC by HSV-1-infected Vero cells probably accounts, at least in part, for its great selectivity of action.     

Mechanism of action of 5-(2-chloroethyl)-2'-deoxyuridine, a selective inhibitor of herpes simplex virus replication

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1). CEDU is preferentially phosphorylated by HSV-infected (Vero) cells, as compared with mock-infected cells or cells infected with a thymidine kinase-deficient strain of HSV-1. The end product of this phosphorylation process, CEDU 5'-triphosphate, is a competitive inhibitor of HSV-1 DNA polymerase activity and, to a lesser extent, of cellular DNA polymerase alpha activity. However, in the absence of the natural substrate dTTP, CEDU 5'-triphosphate also serves as an alternative substrate for viral and cellular DNA polymerase. When exposed to HSV-1-infected cells, [2-14C]CEDU was incorporated into both viral and cellular DNA. The extent to which [2-14C]CEDU was incorporated remained approximately constant over a concentration range of 0.5 to 50 microM. Within this concentration range, CEDU effected a concentration-dependent inhibition of viral DNA synthesis that closely paralleled the inhibition of viral progeny formation. It is postulated that CEDU owes (i) its selectivity as an antiviral agent to its preferential phosphorylation by the virus-infected cell and (ii) its antiviral potency to an inhibition of viral DNA synthesis at the level of the viral DNA polymerization reaction.     

Synthesis and antiviral activity of the enantiomeric forms of carba-5-iodo-2'-deoxyuridine and carba-(E)-5-(2-bromovinyl)-2'-deoxyuridine

Both enantiomers of the carbocyclic analogues of 5-iodo-2'-deoxyuridine (14 and ent-14) and of (E)-5-(2-bromo-vinyl)-2'-deoxyuridine (16 and ent-16) were synthesized by using (+)- or (-)-endo-norborn-5-en-2-yl acetate or butyrate, respectively, as starting materials. Against herpes simplex virus type 1 (+)-C-BVDU (16) was only slightly less active than BVDU itself, whereas (-)-C-BVDU (ent-16) proved to be 10-400-fold less effective, depending on the strain investigated. Against HSV-2 both (+)- and (-)-C-BVDU as well as (+)- and (-)-C-IDU showed minor activity. All carbocyclic analogues were inactive against TK-HSV-1 strains, pointing to the prerequisite of phosphorylation (activation) by the viral thymidine kinase (TK).     

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].     

Genotoxic Properties of (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU)

(E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) is a 5-substituted2'-deoxyuridine antiviral compound that inhibits thymidylatesynthetase. The selectivity of BVDU for virus-infected cellshas been attributed to phosphorylation of BVDU by a virus-inducedthymidine kinase. Since the closely related compounds 5-bromo-2'-deoxyuridineand 5-iodo-2'-deoxyuridine are in vitro and in vivo mutagens,BVDU was tested for genotoxic activity in bacterial and mammaliancell mutation assays as well as in assays measuring DNA damage/repairand clastogenic activity. Mutation assays with BVDU at concentrationsranging from 10 to 5000 µg/plate using Salmonella typhimuriumstrains TA1535, TA1537, TA1538, TA98, and TA100 were negative,both with and without S9 activation. BVDU was also negativein the in vitro rat hepatocyte unscheduled DNA synthesis assayat concentrations of 750 and 1000 µg/ml. In contrast,BVDU was positive in the L5178Y TK^± mouse lymphoma mutationassay without S9 activation at five concentrations ranging from500 to 2000 µg/ml. A Chinese hamster ovary cell (CHO)/hypoxanthineguanine phosphoribosyl transferase gene mutation assay conductedwithout S9 over similar concentrations was negative. However,micronucleus induction by BVDU was detected with out S9 activationat concentrations between 500 and 1750 µg/ml using bothCHO and L5178Y cells. These results indicate that BVDU is apotential human clastogen.     

Intracellular phosphorylation of broad-spectrum anti-DNA virus agent (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and inhibition of viral DNA synthesis

The acyclic nucleotide analogue (S)-9-(3-hydroxy-2-phosphonyl-methoxypropyl)-adenine [(S)-HPMPA], which contains a phosphonate-substituted aliphatic chain, is a potent and selective inhibitor of the replication of various DNA viruses, including herpes simplex virus type 1 (HSV-1). We have synthesized radiolabeled (S)-[U-14C-adenine]HPMPA and investigated its metabolism by HSV-1-infected and mock-infected cells. The drug is as such taken up by the cells and subsequently converted to its monophosphoryl [(S)-HPMPAp] and diphosphoryl [(S)-HPMPApp] derivatives by cellular enzymes. It is incorporated to a very low extent into DNA of both mock-infected and HSV-1-infected Vero cells. (S)-HPMPA inhibits HSV-1 DNA synthesis at a concentration that is several orders of magnitude lower than the concentration required for inhibition of cellular DNA synthesis. Thus the selectivity of (S)-HPMPA as an antiviral agent cannot be attributed to a differential phosphorylation by virus-infected or uninfected cells but resides in a specific inhibitory effect on viral DNA synthesis. The exact basis for the latter effect is under investigation.     

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.     

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.     

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.     

Comparative metabolism of E-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in herpes simplex virus-infected cells

The antiviral activities and metabolic fates of E-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd) and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BrVaraUra) were compared in a dThd kinase-deficient human fibroblast cell line, infected with parental strains of herpes simplex virus, and other strains expressing no viral dThd kinase activity. Metabolic experiments were performed at concentrations well above the ID50 for each compound because radiolabeled agents were not available. BrVaraUra and its nucleotides qualitatively displayed chromatographic and anabolic characteristics which closely paralleled those of BrVdUrd and its nucleotides. Monophosphorylation of both drugs was dependent upon the presence of viral dThd kinase activity except in the case of one dThd kinase-negative type 1 mutant (SC16R5C1) which retained BrVdUrd/BrVaraUra kinase activity. Intracellular uptake of either parent compound was absent during mock-infection and minimal in the cases of infection with mutants unable to phosphorylate the parent compound. Parental type 1 strains were able to induce diphosphorylation and triphosphorylation of both compounds to a similar, dose-dependent degree. Extracts of type 2-infected cells contained greater quantities of BrVdUrd and its monophosphate compared with BrVaraUra and its monophosphate, after identical drug exposure and infection conditions. As previously observed for BrVdUrd, diphosphorylated and triphosphorylated nucleotides of BrVaraUra were not detected after type 2 infection. BrVdUrd and BrVaraUra metabolic breakdown pathways differed, however, as evidenced by the formation of E-5-(2-bromovinyl)uracil (BrVUra). Unlike BrVdUrd, BrVaraUra formed no BrVUra in infected cells, suggesting that replacement of 2'-deoxyribose with arabinose makes the compound biologically more stable, presumably because of resistance to enzymatic breakdown by pyrimidine nucleoside phosphorylases. In this dThd kinase-negative cell line, BrVdUrd and BrVaraUra displayed qualitatively similar susceptibility profiles in that activities were type 1 selective and dThd kinase dependent. Antiviral activities against dThd kinase-positive type 1 strains were similar with both compounds. These data would suggest that BrVdUrd and BrVaraUra have identical type-specific dThd-dTMP kinase-dependent mechanisms of cellular uptake and phosphorylation, but that the latter is not subjected to phosphorolysis and resultant formation of an inactive metabolite. Furthermore, the absence of presence of phosphorolysis of the parent nucleoside does not apparently adversely affect in vitro antiviral activity.