ABSTRACT
The effect of 5-methoxymethyl-2'-deoxycytidine (MMdCyd), in combination with tetrahydrodeoxyuridine (H4dUrd) and 5-methoxymethyl-2'-deoxyuridine (MMdUrd) on deoxyribonucleoside triphosphate pools was assessed. The dNTP pool content was almost 5 times as high in herpes simplex virus (HSV) infected VERO cells compared with mock-infected cells. Significant differences in dNTP pool sizes were observed with the different treatments. Treatment of HSV-infected cells with MMdCyd and MMdUrd resulted in a massive expansion of the dTTP pool, whereas pools of dCTP and dGTP were not affected substantially. MMdUrd and MMdCyd produced dATP pools that were 4 and 2.5 times that of the controls, respectively. Treatment with H4dUrd resulted in the dCTP pool increasing 12 times and barely detectable levels of dTTP. MMdCyd in combination with H4dUrd resulted in a marked reduction of the total deoxyribonucleoside triphosphate level. These results indicate that during viral replication the bulk of the thymidine nucleotides are derived from the dCyd/dCMP deaminase de novo pathway.
Subject(s)
Deoxycytidine/analogs & derivatives , Deoxyribonucleotides/metabolism , Deoxyuridine/analogs & derivatives , Simplexvirus/physiology , Tetrahydrouridine/analogs & derivatives , Animals , Binding Sites , Cytidine Deaminase , Deamination , Deoxycytidine/pharmacology , Deoxycytosine Nucleotides/metabolism , Deoxyuridine/pharmacology , Drug Interactions , Nucleoside Deaminases/metabolism , Simplexvirus/drug effects , Tetrahydrouridine/pharmacology , Thymine Nucleotides/metabolism , Vero CellsABSTRACT
The effect of purine and pyrimidine deoxyribonucleosides on the activity of 5-methoxymethyl-2'-deoxycytidine (MMdCyd) against herpes simplex virus type 1 (HSV-1) was investigated. The antiviral activity of MMdCyd was decreased by deoxythymidine, deoxyuridine and deoxycytidine. Deoxyadenosine had no effect at concentrations up to 500 microM. In contrast, deoxyguanosine (dGuo) potentiated MMdCyd activity. The mean ED50 (1.5 microM) for the combination (MMdCyd plus 100 microM dGuo) was approximately 20-fold lower than that of MMdCyd (ED50 26 microM). When tetrahydrodeoxyuridine (H4dUrd, 540 microM) was added along with MMdCyd and dGuo, anti-HSV-1 activity of MMdCyd was further potentiated by 25-fold (ED50 0.06 microM). The inhibition of virus replication, as determined by the plaque reduction assay, was further confirmed by virus yield studies and by parallel observations on virus-induced cytopathogenicity. The order of decreasing effectiveness for reducing the production of infectious virus particles (virus yield) by different treatments was: MMdCyd + dGuo + H4dUrd greater than MMdCyd + DGuo greater than MMdCyd + H4dUrd greater than MMdCyd greater than dGuo + H4dUrd greater than dGuo greater than H4dUrd. The effect of dGuo and dGuo in combination with H4dUrd on deoxyribonucleoside triphosphate (dNTP) pools was determined in Vero cells infected with multiplicity of infection of 5 PFU/cell. In the presence of 100 microM dGuo, there was approximately a 3-fold, 2-fold and 12-fold increase in dCTP, dTTP and dGTP pool sizes respectively, as compared to control (untreated) cells. Treatment with H4dUrd (1.06 mM) in combination with dGuo (100 microM), resulted in an increase of the dCTP pool and a marked fall in the dTTP and dGTP pool. The possible mechanisms for potentiation of MMdCyd activity by dGuo and H4dUrd are discussed.
Subject(s)
Antiviral Agents/pharmacology , DCMP Deaminase/antagonists & inhibitors , Deoxycytidine/analogs & derivatives , Deoxyguanosine/pharmacology , Deoxyribonucleosides/pharmacology , Simplexvirus/drug effects , Tetrahydrouridine/analogs & derivatives , Animals , Antiviral Agents/toxicity , Deoxycytidine/metabolism , Deoxyguanosine/toxicity , Simplexvirus/growth & development , Simplexvirus/metabolism , Tetrahydrouridine/pharmacology , Vero CellsABSTRACT
The antiviral activity and cytotoxicity of (E)-5-(2-bromovinyl)-2'-deoxycytidine (BrVdCyd) against herpes simplex virus type 1 (HSV-1), singly and in combination with deaminase inhibitors was determined using rabbit kidney (RK-13), HEP-2, BHK-21 and VERO cells. BrVdCyd was a potent inhibitor of HSV-1 replication with ED50 values of 0.30 to 1.20 microM depending on the cell line used. In the presence of tetrahydrouridine or tetrahydrodeoxyuridine (H4dUrd), potency of BrVdCyd increased approximately two fold (ED50: 0.54 microM) in HSV-infected VERO cells. The combination of BrVdCyd and H4dUrd was also effective in decreasing virus yield. Dihydrodeoxyuridine (H2dUrd) reversed the activity of BrVdCyd (ED50: 6 to 7 microM). The effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd), BrVdCyd and BrVdCyd in combination with H4dUrd on deoxyribonucleoside triphosphate (dNTP) pools was assessed in VERO cells infected with a high multiplicity of infection (10 PFU/cell). Significant differences in dNTP poll sizes (pmol/10(6) cell) were observed with different treatments. BrVdUrd and BrVdCyd treatment resulted in marked expansion of the dTTP pool (greater than 1200 pmol) compared to HSV-infected VERO cells (303 pmol). Exposure to H4dUrd resulted in a 12-fold expansion of the dCTP pool (326 pmol) and barely detectable levels of dTTP (less than 1.0 pmol). BrVdCyd plus H4dUrd treatment resulted in a slight expansion of the dTTP pool (515 pmol). These results indicate: (i) H4dUrd inhibits de novo dCyd/dCMP deaminase pathway and (ii) exposure to BrVdCyd plus H4dUrd puts a strain on viral DNA synthesis to such an extent that even though dTTP is being formed from alternative pathways, its eventual utilization as a substrate is reduced and hence it builds up.
