Deoxyribonucleoside triphosphate pools of herpes simplex virus infected cells: the influence of selective antiherpes agents and the role of the deaminase pathway.

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.

Regulatory effects of deoxyribonucleosides on the activity of 5-methoxymethyl-2'-deoxycytidine: modulation of antiherpes activity by deoxyguanosine and tetrahydrodeoxyuridine.

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.

Structure and conformation of 5-methoxymethyl-N4-methyl-2'-deoxycytidine.

C12H19N3O5, Mr = 285.25, monoclinic, P2(1), a = 7.0180 (6), b = 8.6946 (11), c = 10.7715 (10) A, beta = 91.055 (7) degrees, V = 657.15 A3, Z = 2, Dx = 1.441 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 9.63 cm-1, F(000) = 304, T = 287 K, R = 0.039 for 1424 observed reflections. The furanose ring adopts the C(1')-exo envelope conformation (E1), with the glycosyl linkage anti (chi = 193.8 degrees). The pseudo-rotational parameters are P = 130.9 degrees and tau m = 39.4 degrees. In the deoxyribose ring, the side chain on C(5') has the t conformation. In the pyrimidine ring the N4-methyl takes a cis conformation to N(3) and the methoxymethyl side chain is on the same side of the cytidine plane as O(4').

Structure and conformation of 3'-O-acetyl-2'-deoxy-5-methoxymethyluridine.

C13H18N2O7, Mr = 314.297, triclinic, P1, a = 6.0321 (4), b = 6.775 (5), c = 9.6699 (7) A, alpha = 76.917 (6), beta = 78.871 (6), gamma = 75.344 (6) degrees, V = 368.54 A3, Z = 1, Dm = 1.43, Dx = 1.416 g cm-3, Cu K alpha radiation (Ni filtered), lambda = 1.5418 A, F(000) = 166, T = 287 K, final conventional R factor = 0.034, wR = 0.044 for 1359 reflections and 268 variables. The structure was solved using the XTAL system. The conformation of the furanose ring is best described as intermediate between 2E and 2(1)T: the pseudorotational parameters are P = 148.9 degrees and tau m = 33.4 degrees. The CH2OH, C(5'), side chain has the g+ conformation, the carbonyl bond of the 3'-acetoxy group is syn to the C(3')-O(3',1) bond on the sugar ring and the glycosidic bond conformation is anti [chi = -137.6 (3) degrees]. The methoxy group of the 5-methoxymethyl substituent is on the same side of the pyrimidine plane as O(4') of the furanose ring. Comparison with 2'-deoxy-5-methoxymethyluridine shows that intermolecular attraction have little effect on the internal conformations of the molecule in the solid state.

Relationship between structure and antiviral activity of 5-methoxymethyl-2'-deoxyuridine and 5-methoxymethyl-1-(2'-deoxy-beta-D-lyxofuranosyl)uracil.

5-Methoxymethyl-1-(2'-deoxy-beta-D-lyxofuranosyl)uracil (MMdLU) was not active against the herpes simplex viruses. The relationship between molecular conformation and antiviral activity for the two epimers, 5-methoxymethyl-2'-deoxyuridine (MMdUrd) and MMdLU, is discussed. MMdUrd was phosphorylated by the virus-induced deoxythymidine kinase. In contrast, MMdLU did not serve as a substrate for the kinase. The geometry and distance between the 5'-CH2OH and 3'-OH groups of the furanose ring appear to be key factors in determining the efficiency of phosphorylation by the virus-induced deoxythymidine kinase, and hence antiviral activity.

Antiviral activity, antimetabolic activity, and cytotoxicity of 3'-substituted deoxypyrimidine nucleosides.

The antiviral activity, effect on cellular DNA and RNA synthesis, and cytotoxicity toward mammalian cells of 5-fluoro-2'-deoxyuridine, 5-methoxymethyl-2'-deoxyuridine, 2'-deoxythymidine, and their corresponding 3'-p-nitrophenylphosphate and 3'-p-aminophenylphosphate derivatives were determined. The 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine derivative was as potent as 5-methoxy-methyl-2'-deoxyuridine in inhibiting herpes simplex viruses; however, 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine was less potent than 5-fluoro-2'-deoxyuridine in inhibiting viral replication. The results suggest that the deoxypyrimidine ribonucleoside kinase has bulk tolerance for substituents at the 3-position of the ribofuranose moiety. The effect on cellular DNA and RNA synthesis and cytotoxicity toward mammalian cells were monitored by studying the incorporation of radioactive precursors. 5-Methoxymethyl-2'-deoxyuridine and 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine failed to inhibit DNA or RNA synthesis. 5-Fluoro-2'-deoxyuridine and 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine decreased incorporation of [3H]deoxyuridine by 50% at 1.0 and 40 microM, respectively. Cytotoxicity (microscopic lesions using monolayer cells) on exposure to 5-methoxymethyl-2'-deoxyuridine, 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine, 5-fluoro-2'-deoxyuridine, and 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine was observed at 3800, 1600, 1.6, and 110 microM, respectively.