Inhibition of varicella-zoster virus-induced DNA polymerase by a new guanosine analog, 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine triphosphate.

The triphosphates of the antiherpesvirus acyclic guanosine analogs 9-[4-hydroxy-2(hydroxymethyl)butyl] guanine (2HM-HBG), 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]), and 9-(3,4-dihydroxybutyl)guanine (buciclovir) were examined for their effects on partially purified varicella-zoster virus (VZV) DNA polymerase as well as cellular DNA polymerase alpha. The triphosphate of 2HM-HBG competitively inhibited the incorporation of dGMP into DNA catalyzed by the VZV DNA polymerase. 2HM-HBG-triphosphate (2HM-HBG-TP) had a higher affinity for the dGTP-binding site on the VZV DNA polymerase than did dGTP; apparent Km and Ki values of dGTP and 2HM-HBG-TP were 0.64 and 0.034 microM, respectively. ACV-triphosphate (ACV-TP) was found to be the most potent inhibitor of VZV DNA polymerase. ACV-TP had a 14 and 464 times better direct inhibitory effect than 2HM-HBG-TP and buciclovir-triphosphate, respectively. The cellular (human embryonic lung fibroblast) DNA polymerase alpha inhibition was related to viral polymerase inhibition as efficacy ratios: 2HM-HBG-TP had a ratio of more than 1,000, which appeared to be similar to that of ACV-TP.     

Improvement of the absorption of oral (R,S)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine, an anti-varicella-zoster virus drug, in rats and monkeys.

In an effort to improve the gastrointestinal absorption of (R,S)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine [(+/-)2HM-HBG], various salts and esters of the compound were synthesized and pharmacokinetic experiments were performed in rats and monkeys. The sodium or hydrochloride salts and short-chain esters of (+/-)2HM-HBG showed bioavailability characteristics that were equally as poor as those of (+/-)2HM-HBG. However, the esters given as salts tended to be better absorbed than the parent compound. The 6-deoxy and 6-deoxydiacetate analogs were extensively oxidized in vivo and represent prodrugs with considerable potential in improving the absorption of oral (+/-)2HM-HBG.     

Pharmacokinetics and antiviral activity in simian varicella virus-infected monkeys of (R,S)-9-[4-hydroxy-2-(hydroxymethyl) butyl]guanine, an anti-varicella-zoster virus drug.

The acyclic guanosine analog (R,S)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine, (+/-)2HM-HBG, is an effective inhibitor of herpes simplex virus and varicella-zoster virus infections in vitro. This report is concerned with the pharmacokinetic evaluation of the drug in rats and monkeys and its antiviral activity in African green monkeys infected with simian varicella virus (SVV), a virus closely related to varicella-zoster virus that is also susceptible to inhibition by (+/-)2HM-HBG. Elimination half-lives in plasma following intravenous administration to monkeys (100 mumol/kg of body weight) ranged from 1.8 to 2.2 h, and total body clearance was 9.0 +/- 0.4 ml/min per kg (mean +/- standard error). After oral administration, levels in plasma were low, with a maximum concentration of the drug of only 3.1 +/- 0.8 microM, a time to reach maximum concentration of drug of 2.7 +/- 0.4 h, and an oral bioavailability of 10.6 +/- 1.4%. Because of the low oral bioavailability, SVV-infected monkeys were treated intramuscularly with (+/-)2HM-HBG. (+/-)2HM-HBG at a dosage of 10 mg/kg of body weight per day allowed moderate viremia, whereas a dosage of 30 mg/kg of body weight per day strongly suppressed viremia with minimal numbers of virus plaques from blood specimens collected at days 3, 5, and 7 postinfection and complete clearance at day 9 postinfection. Titers of antibody to SVV were also low. Treatment three times daily was somewhat more efficacious than treatment twice daily. Thus, (+/-)2HM-HBG is an effective inhibitor of SVV replication in vivo, despite the fact that leves of (+/-)2HM-HBG in plasma were low at extended periods of time and below the concentration of drug giving 50% inhibition of plaque formation obtained in vitro.     

Influence of acyclovir and bucyclovir on nucleotide pools in cells infected with herpes simplex virus type 1.

The effects of the acyclic guanosine analogs acyclovir (ACV) and (R)-9-(3,4-dihydroxybutyl)guanine (bucyclovir, BCV) on the deoxyribonucleoside triphosphate (dNTP) pools of herpes simplex type 1 (HSV-1)-infected African green monkey kidney (GMK) and human embryonic lung fibroblast (HL) cells were investigated. HSV-1 infection increased the dNTP pools in both cell types compared with those in uninfected cells. Mock-infected GMK cells showed a 10-fold-higher dTTP concentration than comparable HL cells. ACV or BCV treatment of HSV-1-infected cells yielded further increases of the dNTP pools. ACV- or BCV-treated, HSV-1-infected HL cells showed 20- to 50-fold-higher concentrations of ACV triphosphate and BCV triphosphate, respectively, than similarly treated GMK cells. This is in accord with previous results, which showed that ACV and BCV are less active in GMK cells than in HL cells. This difference in activity is attributed to the substantial deoxythymidine pools previously found in GMK cells. The results are discussed in relation to known metabolic and kinetic parameters.     

Antiherpes effects and pharmacokinetic properties of 9-(4-hydroxybutyl) guanine and the (R) and (S) enantiomers of 9-(3,4-dihydroxybutyl)guanine.

Three acyclic guanosine analogs with similar structures, the (R) and (S) forms of 9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine, were compared for antiherpes activity in vivo and in vitro. The three guanosine analogs were viral thymidine kinase-dependent inhibitors of virus multiplication. In cell cultures, (S)-9-(3,4-dihydroxybutyl)guanine was the least active of these three drugs against a variety of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) strains. This was also the case for a certain HSV-1 or HSV-2 strain in different cell lines. In cell cultures, (R)-9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine had similar antiherpes activities. However, in vivo in cutaneous HSV-1 infections in guinea pigs treated topically and in systemic HSV-2 infections in mice treated orally or intraperitoneally, only (R)-9-(3,4-dihydroxybutyl)guanine had a therapeutic effect. The extremely short half-life in plasma and the high clearance of 9-(4-hydroxybutyl)guanine as compared with those of (R)-9-(3,4-dihydroxybutyl)guanine probably made 9-(4-hydroxybutyl)guanine inefficacious when given intraperitoneally or orally to mice infected with herpesvirus. On the other hand, no kinetic differences between (R)-9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine were observed in penetration through guinea pig skin ex vivo, and no preferential metabolism of 9-(4-hydroxybutyl)guanine in skin was noted. We deduced that high thymidine levels in guinea pig skin preferentially antagonize the antiviral effect of 9-(4-hydroxybutyl) guanine in cutaneous HSV-1 infections.     

Inhibition of human herpesvirus 6 replication by 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) and other antiviral compounds.

The in vitro susceptibilities of human herpesvirus 6 to foscarnet; the guanosine analogs acyclovir, ganciclovir, and two isomers of 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine; and the thymidine analogs 3'-azido-3'-deoxythymidine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil were investigated. All compounds except 3'-azido-3'-deoxythymidine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil inhibited human herpesvirus 6 replication. The highest in vitro selectivity was obtained for 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine.     

Acyclic guanosine analogs as substrates for varicella-zoster virus thymidine kinase.

This study was performed to obtain information on the enzymatic background to the antiviral activity of acyclic guanosine analogs. Five acyclic guanosine analogs, the (R)- and (S)-enantiomers of 9-(3,4-dihydroxybutyl)guanine, 9-(4-hydroxybutyl)guanine, 9-[(2-hydroxyethoxy)methyl]guanine, and 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine, were compared in enzyme kinetic experiments using purified varicella-zoster virus and human placenta mitochondrial thymidine kinase (TK). All analogs showed competitive patterns of inhibition in the phosphorylation of thymidine by varicella-zoster virus TK, but only low affinities and phosphorylation rates were observed. No affinity for the mitochondrial TK was observed for any of the analogs.     

Cyclobut-A and cyclobut-G, carbocyclic oxetanocin analogs that inhibit the replication of human immunodeficiency virus in T cells and monocytes and macrophages in vitro.

Two newly synthesized carbocyclic oxetanocin analogs, (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine (cyclobut-A) and (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine (cyclobut-G) were tested for activity against the infectivity of human immunodeficiency virus (HIV) in vitro. A number of other carbocyclic oxetanocin analogs failed to exert good antiretroviral effects. Both cyclobut-A and cyclobut-G protected CD4+ ATH8 cells against the infectivity and cytopathic effect of HIV type 1 (HIV-1) and suppressed proviral DNA synthesis in ATH8 cells exposed to HIV-1 in vitro at concentrations of 50 to 100 microM. These compounds also inhibited the in vitro infectivity of another human pathogenic retrovirus, HIV-2. Furthermore, both compounds completely suppressed the replication of a monocytotropic strain of HIV-1 in monocytes and macrophages at concentrations as low as 0.5 microM, as assessed by inhibition of HIV-1 p24 gag protein production. We also found that 2'-deoxyguanosine readily reversed the antiretroviral activity of cyclobut-G in our system, whereas the activity of cyclobut-A was hardly reversed by 2'-deoxyadenosine or 2'-deoxycytidine. We noted, however, that these compounds inhibited the proliferation of peripheral blood mononuclear cells at concentrations of greater than or equal to 100 microM in vitro. Although both cyclobut-A and cyclobut-G appear to have a certain level of in vitro toxicity, our observations may have theoretical and clinical implications in understanding the structure-activity relationships of antiretroviral agents active against HIV.     

Inhibiting effect of (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine on varicella-zoster virus replication in cell culture.

The activity and mode of action of the new nucleoside analog (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) against varicella-zoster virus (VZV) were determined. In cell culture, replication of different strains of VZV was inhibited to 50% by 0.4 to 0.7 microM 2HM-HBG, while 685 microM was required to inhibit 50% of the DNA synthesis in uninfected human lung fibroblasts. A thymidine kinase-negative VZV strain was not inhibited by 100 microM 2HM-HBG. Inhibition of VZV replication was not reversible after 7 to 14 days of incubation, depending on the multiplicity of VZV. 2HM-HBG was shown to be selectively phosphorylated by purified VZV thymidine kinase, with an inhibition constant of 32.5 microM. The antiviral activity of 2HM-HBG in cell culture was decreased by the addition of deoxythymidine and deoxycytidine but not by other ribo- or deoxyribonucleosides.     

In vitro activities of penciclovir and acyclovir against herpes simplex virus types 1 and 2.

Penciclovir (PCV) and acyclovir are acyclic guanine analogs which inhibit herpes simplex virus (HSV) DNA polymerase. Their 50% infective doses were 0.5 to 0.8 microgram/ml for clinical isolates of HSV-1 and 1.3 to 2.2 micrograms/ml for HSV-2. Furthermore, HSV-infected cultures receiving 2-h pulses of PCV had 2- to 50-fold less HSV than acyclovir-treated cultures, consistent with the prolonged intracellular half-life of PCV triphosphate.     

Effects of acyclovir, oxetanocin-G, and carbocyclic oxetanocin-G in combinations on the replications of herpes simplex virus type 1 and type 2 in Vero cells.

9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV) and novel nucleosides, 9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanocyl)guanine (oxetanocin-G, OXT-G) and (+)-9-[(1R, 2R, 3S)-2, 3-bis(hydroxymethyl)cyclobutyl]guanine (carbocyclic oxetanocin-G, carbocyclic OXT-G) possessed substantial antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). ACV inhibited only viral thymidine kinase positive (TK+) herpes viruses, although the latter two compounds inhibited the replications of the TK deficient (TK-) mutants of HSV-1 and HSV-2 as well as the TK+ parent strains in vitro. The TK- mutants of HSV-1 and HSV-2 (HSV-1 TK- and HSV-2 TK-) were as susceptible to OXT-G as the TK parent strains. However, the TK- mutants were less susceptible to carbocyclic OXT-G than the TK+ parent strains. We demonstrated synergistic inhibition of the replications of HSV-1 and HSV-2 by ACV and OXT-G in combination, additive inhibition of HSV-1 and HSV-2 by ACV and carbocyclic OXT-G in combination, synergistic inhibition of HSV-1 by OXT-G and carbocyclic OXT-G in combination, and additive inhibition of HSV-2 by these two compounds. We investigated the metabolism of ACV and OXT-G in HSV-1 TK(+)-, HSV-1 TK(-)- and mock-infected Vero cells by thin layer chromatography. ACV-triphosphate increased more in HSV-1 TK(+)-infected Vero cells than in HSV-1 TK(-)- and mock-infected Vero cells. The metabolism of OXT-G had almost the same pattern in HSV-1 TK(+)-, HSV-1 TK(-)- and mock-infected Vero cells. These results suggest that ACV is phosphorylated by virus-induced TK, and OXT-G is phosphorylated by cellular nucleoside and nucleotide kinases.     

Phosphorylation of four acyclic guanosine analogs by herpes simplex virus type 2 thymidine kinase.

Four acyclic guanosine analogs, the (R) and (S) enantiomers of 9-(3,4-dihydroxybutyl)guanine, 9-(4-hydroxybutyl)guanine, and acyclovir were compared in enzyme kinetic experiments, using purified herpes simplex virus type 2 thymidine kinase. All four analogs showed competitive patterns of inhibition in the phosphorylation of thymidine by the viral thymidine kinase, but different affinities and relative rates of phosphorylation were observed.     

Inhibition of cellular alpha DNA polymerase and herpes simplex virus-induced DNA polymerases by the triphosphate of BW759U.

The triphosphate form of the acyclovir analog BW759U (9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine) inhibited the DNA polymerases (EC 2.7.7.7) from several strains of herpes simplex virus type 1. Two acyclovir triphosphate-resistant DNA polymerases were as sensitive to BW759U-triphosphate as were the DNA polymerases induced by wild-type viruses (Ki = 0.05 to 0.1 microM). The Ki value for cellular alpha DNA polymerase was 35- to 50-fold greater than those for the DNA polymerases induced by the various herpes simplex virus strains investigated. Incubation of Vero cells infected by the KOS strain of herpes simplex virus type 1 with the acyclovir analog resulted in the formation of substantial quantities of (9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine) triphosphate.     

Mutation within the herpes simplex virus DNA polymerase gene conferring resistance to (R)-9-(3,4-dihydroxybutyl)guanine.

Five herpes simplex virus mutants known or presumed to contain mutations in their DNA polymerase genes conferring resistance to acyclovir and arabinosyladenine also proved to exhibit some degree of resistance to (R)-9-(3,4-dihydroxybutyl)guanine (buciclovir). For one mutant, a buciclovir resistance mutation was mapped to a region of the viral DNA polymerase gene proposed to encode the deoxynucleoside 5'-triphosphate binding domain. These data implicate the viral polymerase as a target of buciclovir action that contributes to its antiviral selectivity.     

Analysis of phosphorylation pathways of antiherpesvirus nucleosides by varicella-zoster virus-specific enzymes.

The inhibitory activities of acyclovir (ACV), 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), ganciclovir (GCV), 9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanosyl)guanine (OXT-G), and (+)-9-[(1R,2R,3S)-2,3-bis(hydroxymethyl)Cyclobutyl]guanine (cOXT-G) on the replication of wild-type and thymidine kinase (TK)-negative strains of herpes simplex virus types 1 and 2 and varicella-zoster virus (VZV) and the wild-type strain of human cytomegalovirus were tested to clarity whether the phosphorylation of these compounds is catalyzed by viral TK or other enzymes. ACV and BV-araU had little effect on the replication of TK-negative virus strains. On the other hand, GCV, OXT-G, and cOXT-G inhibited the replication of TK-negative VZV at concentrations 10 times higher than those at which they inhibited wild-type VZV, indicating that a kinase other than TK phosphorylates GCV and OXT-G in VZV-infected cells. GCV phosphorylation activity was not detected in VZV-infected cell lysates; therefore, this activity was evaluated in COS 1 cells expressing viral TK and viral protein kinase (PK). The COS 1 cells expressing VZV TK were shown to be susceptible to all compounds tested. In contrast, VZV Pk-expressing COS 1 cells were susceptible to only GCV, OXT-G, and cOXT-G. These results suggest that VZV PK phosphorylates some nucleoside analogs, for example, GCV, OXT-G, and cOXT-G. This phosphorylation pathway may be important in the anti-VZV activities of some nucleoside analogs.     

Evaluation of SQ 34,514: pharmacokinetics and efficacy in experimental herpesvirus infections in mice.

The new antiviral nucleoside SQ 34,514 [(1R-1 alpha, 2 beta, 3 alpha)-2-amino-9- [2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one], the active R isomer of racemic SQ 33,054 (cyclobut-G), was evaluated for efficacy in the treatment of herpesvirus infections in mice. SQ 34,514 was orally efficacious in a herpes simplex virus type 1 (HSV-1) systemic infection, an intracerebral HSV-2 infection, a vaginally induced HSV-2 infection in ovariectomized mice, and in a systemic murine cytomegalovirus infection. SQ 34,514 compared favorably with acyclovir and ganciclovir in the treatment of these experimental infections. In mice, SQ 34,514 had an oral bioavailability of 80% based on urinary excretion. SQ 34,514 may have potential value in the therapy of HSV and cytomegalovirus infections in humans.     

Efficacy of the acyclic guanosine analog buciclovir [(R)-9-(3,4-dihydroxybutyl)guanine] in experimental genital herpes.

The efficacy of the anti-herpesvirus drug buciclovir [(R)-9-(3,4-dihydroxybutyl)guanine] was investigated in guinea pigs and mice infected intravaginally with herpes simplex virus type 2. Topical treatment initiated early after infection was efficacious, in contrast to topical treatment delayed 24 h or more. Systemic treatment of infected mice could not prevent the spread of virus to the brain and mortality. Systemically administered buciclovir had an effect in guinea pigs, even after delayed onset of treatment, but this effect required high doses of the drug. Our results suggest that buciclovir has only a limited effect against herpesvirus infections once the virus is present in the nervous systems of infected animals.     

Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxymethylbut-1-yl) guanine (BRL 39123) in animals.

The antiviral activity of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) was assessed in several animal models of herpes simplex virus (HSV) infection. BRL 39123 was as active as acyclovir (ACV) when applied topically to guinea pigs with a cutaneous HSV type 1 (HSV-1) infection and was also active topically in an HSV-2 genital infection. Before systemic administration to infected animals, BRL 39123 and ACV were administered orally and subcutaneously to mice, and the blood was assayed for each compound by high-pressure liquid chromatography. When given systemically to mice infected cutaneously with HSV-1, BRL 39123 was as active as ACV. In mice infected intranasally with HSV-1 or HSV-2, single daily subcutaneous doses of BRL 39123 were more effective than equivalent treatment with ACV, reflecting the more persistent activity seen in cell culture and a more stable triphosphate within the infected cell. When the compounds were supplied in drinking water for this infection, BRL 39123 and ACV had similar potencies against HSV-1, although ACV was more active against an HSV-2 infection than BRL 39123 was. In mice infected intraperitoneally with HSV-1, BRL 39123 was 10-fold more potent than ACV and a single dose of BRL 39123 reduced virus replication within the peritoneal cavity more effectively than 3 doses of ACV given 1, 5, and 20 h after infection. Although BRL 39123 failed to eradicate the virus from mice latently infected with HSV-1, treatment initiated 5 h after infection of the ear pinna reduced the numbers of mice that developed latent infections.     

Mode of Action of (1′S,2′R)-9-{[1′,2′-Bis(hydroxymethyl) cycloprop-1′-yl]methyl}guanine (A-5021) against Herpes Simplex Virus Type 1 and Type 2 and Varicella-Zoster Virus

The mode of action of (1′S,2′R)-9-{[1′,2′-bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) was studied. A-5021 was monophosphorylated at the 2′ site by viral thymidine kinases (TKs). The 50% inhibitory values for thymidine phosphorylation of A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for penciclovir (PCV) and lower than those for acyclovir (ACV). Of these three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated more than ACV triphosphate but less than PCV triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5 cells had comparable levels of A-5021 and ACV triphosphates. The intracellular half-life of A-5021 triphosphate was considerably longer than that of ACV triphosphate and shorter than that of PCV triphosphate. A-5021 triphosphate competitively inhibited HSV DNA polymerases with respect to dGTP. Inhibition was strongest with ACV triphosphate, followed by A-5021 triphosphate and then (R,S)-PCV triphosphate. A DNA chain elongation experiment revealed that A-5021 triphosphate was incorporated into DNA instead of dGTP and terminated elongation, although limited chain extension was observed. Thus, the strong antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV and on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV.     

Hydroxyurea potentiates the antiherpesvirus activities of purine and pyrimidine nucleoside and nucleoside phosphonate analogs

Hydroxyurea has been shown to potentiate the anti-human immunodeficiency virus activities of 2',3'-dideoxynucleoside analogs such as didanosine. We have now evaluated in vitro the effect of hydroxyurea on the antiherpesvirus activities of several nucleoside analogs (acyclovir [ACV], ganciclovir [GCV], penciclovir [PCV], lobucavir [LBV], (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine [H2G], and brivudin and nucleoside phosphonate analogs (cidofovir [CDV] and adefovir [ADV]). When evaluated in cytopathic effect (CPE) reduction assays, hydroxyurea by itself had little effect on CPE progression and potentiated in a subsynergistic (herpes simplex virus type 1 [HSV-1]) to synergistic (HSV-2) fashion the antiviral activities of ACV, GCV, PCV, LBV, H2G, ADV, and CDV. Hydroxyurea also caused marked increases in the activities of ACV, GCV, PCV, LBV, and H2G (compounds that depend for their activation on a virus-encoded thymidine kinase [TK]) against TK-deficient (TK(-)) HSV-1. In fact, in combination with hydroxyurea the 50% effective concentrations of these compounds for inhibition of TK(-) HSV-1-induced CPE decreased from values of 20 to > or = 100 microg/ml (in the absence of hydroxyurea) to values of 1 to 5 microg/ml (in the presence of hydroxyurea at 25 to 100 microg/ml). When evaluated in a single-cycle virus yield reduction assay, hydroxyurea at a concentration of 100 microg/ml inhibited progeny virus production by 60 to 90% but had little effect on virus yield at a concentration of 25 microg/ml. Under these assay conditions hydroxyurea still elicited a marked potentiating effect on the antiherpesvirus activities of GCV and CDV, but this effect was less pronounced than that in the CPE reduction assay. It is conceivable that the potentiating effect of hydroxyurea stems from a depletion of the intracellular deoxynucleoside triphosphate pools, thus favoring the triphosphates of the nucleoside analogues (or the diphosphates of the nucleoside phosphonate analogues) in their competition with the natural nucleotides at the viral DNA polymerase level. The possible clinical implications of these findings are discussed.