Sagar V. Gupta

Comparison of the mutagenic activity of 5-hydroxymethyldeoxyuridine with 5-substituted 2′-deoxyuridine analogs in the Ames Salmonella/microsome test

4 antiviral drugs 5-hydroxymethyldeoxyuridine (HMUdR), 5-trifluorothymidine (F3TdR), 5-methoxymethyldeoxyuridine (MMUdR) and 5-ethyldeoxyuridine (EtUdR) have been evaluated for mutagenic activity in the Ames Salmonella/microsome test. The antimetabolites F3TdR and HMUdR were mutagenic in a dose-dependent manner in strain TA100. F3TdR also was mutagenic in strain TA1535. Rat-liver post-mitochondrial supernatant (S9) was not required for mutagenicity.

Anti-Herpes Virus Activity of 5-Methoxymethyl-2′-Deoxycytidine in Combination with Deaminase Inhibitors

5-Methoxymethyl-2′-deoxycytidine (MMdCyd) is an anti-metabolite with selective anti-herpes activity and low cytotoxicity. MMdCyd is dependent upon initial activation by the viral-induced deoxythymidine-deoxycytidine (dThd/dCyd) kinase for its activity against herpes simplex virus (HSV). Antiviral activity of MMdCyd is cell-dependent and is influenced by the deaminase content of the cell line used for assays. The antiviral potency against HSV-1 in this study was higher in RK-13 cells (ED50 3–5 μm) than in Vero and HEP-2 cells (ED50 14–26 μm). The potency of MMdCyd increased approximately 20-fold against HSV-1 and twofold against HSV-2 in the presence of tetrahydrodeoxyuridine (H4dUrd; which inhibits both dCyd deaminase and dCMP deaminase) in Vero cells. MdCyd in combination with H4dUrd was effective in preventing the cytopathogenic effect of HSV-1 and decreasing the production of infectious virus particles. The IC99 (concentration required to reduce the yield of infectious virus obtained 72 h after infection by 99% relative to control cultures) was 1.6 μm. In combination with tetrahydrouridine (H4Urd; an inhibitor of Cyd/dCyd deaminase) the potency of MMdCyd was only slightly enhanced (ED50 7–8 μm). Dihydrodeoxyuridine and deoxyuridine reversed the antiviral activity of MMdCyd. The minimum cytotoxic concentration for rapidly dividing cells (RK-13, HEP-2 and Vero) for MMdCyd was greater than 3 mm. H4Urd and H4dUrd were devoid of cytotoxicity and antiviral activity up to 2.12 mm (the highest concentration tested). Diacetyl-MMdCyd (pro-drug form) was approximatewly 20 times less potent than MMdCyd.

Modified tetrazolium-based colorimetric method for determining the activities of anti-HIV compounds

A modified tetrazolium-based colorimetric assay was used to determine the anti-HIV activities of ddAzThd, ddCyd, ddIno, and PFA. In this assay, poly-1-lysine-coated plates were used to attach the MT-2 cells to the bottom of the plates. A fixed amount of virus (50 TCID50) was used in each well. A modified version of the formula published by Pauwels et al. (1988) was used for calculating the percentage cell protection from virus infection. Using CC10/EC90 to calculate the selective indices, the decreasing order of selectivity against HIV-1 strain A87SF, was: ddAzThd greater than PFA greater than ddCyd greater than ddIno. Against HIV-1 strain A79SK-1 the decreasing order of selectivity was: PFA greater than ddIno greater than AzThd greater than ddCyd. The modified formula showed lack of anti-HIV activity for thymidine at non-toxic concentrations.

Mechanism of selective inhibition of herpes simplex virus replication by deoxycytidine analogues : interaction of 5-methoxymethyl-2'-deoxycytidine-5'-triphosphate with DNA polymerases

5-Methoxymethyl-2′-deoxycytidine (MMdCyd) is a selective anti-herpes agent that is dependent upon initial activation by herpes simplex virus (HSV)-induced deoxythymidine/deoxycytidine kinase. 5-Methoxymethyl-2′-deoxycytidine triphosphate (MMdCTP) was synthesized. The nature of the interaction of MMdCTP and dCTP with the DNA polymerase of Escherichia coli, HSV-1 and human DNA polymerase α was determined using specific and optimized assay conditions for each enzyme. MMdCTP was a better substrate for HSV-1 DNA polymerase compared to dCTP. At a nucleotide concentration of 10 μm MMdCTP utilization was 130% that of an equimolar concentration of dCTP. Under similar conditions, human DNA polymerase α utilized MMdCTP about as efficiently as dCTP. E. coli DNA polymerase I preferentially utilized dCTP. The IC50 values of MMdCTP were 8 × 10−7 m and 29 × 10−7 m for HSV-1 and human α DNA polymerase, respectively. MMdCTP is a competitive inhibitor of HSV-1 DNA polymerase with respect to dCTP incorporation (Ki = 3.8 × 10−7 m). Preferential utilization of MMdCTP and its eventual incorporation into HSV DNA seems to account for the antiviral action of MMdCyd.

Interaction of 5-methoxymethyl-2'-deoxyuridine triphosphate with DNA polymerases : effects of the 5- substituent and comparison with the deosycytidine derivative

5-Methoxymethyl-2′-deoxyuridine (MMdUrd) is a selective anti-herpes agent that is dependent upon initial phosphorylation by Herpes simplex virus-induced deoxythymidine kinase. In order to determine its mechanism of action, MMdUrd was converted to the 5′-triphosphate (MMdUTP) and the nature of interaction of MMdUTP and dTTP with DNA polymerase of E. coli, HSV-1, and human α was investigated. The order of utilization of deoxyuridine analogues by bacterial and HSV-1 DNA polymerases for DNA synthesis was: dTTP > MMdUTP. In contrast, 5-methoxymethyl-2′-deoxycytidine-5′-triphosphate (MMdCTP) was a better substrate for HSV DNA polymerase compared to dCTP. MMdUTP is a competitive inhibitor of HSV-1 DNA polymerase with respect to dTTP incorporation (Ki = 2.9 × 10−6M). The IC50 values of MMdUTP for both HSV and human αDNA polymerases were 4.5 × 10 −6M. These data suggest that the selective activity of MMdUrd is due to its preferential phosphorylation by viral thymidine kinase and not at the DNA polymerase level. These results may also account for the difference in anti-HSV activity between MMdUrd and its deoxycytidine analogue.

Acute and delayed toxicity studies on the antiherpesvirus agents 5-methoxymethyl-2'-deoxycytidine and 5-methoxymethyl-2 -deoxyuridine

5-Methoxymethyl-2′-deoxycytidine (MMdCyd) and the corresponding deoxyuridine analogue, 5-methoxymethyl-2′-deoxyuridine (MMdUrd) are selective antiherpesvirus agents. MMdCyd (ED50 1.5 μM) is a more potent inhibitor of herpes simplex virus replication than MMdUrd (ED50 30 μM) when maintained in the deoxycytidine form (deamination prevented). The 5′-triphos-phates, MMdCTP and MMdUTP, were synthesized, and incorporation into DNA by mitochondrial DNA polymerase γ was investigated. MMdCTP and MMdUTP were incorporated into DNA in place of dCTP and dTTP, respectively. The effect of MMdCyd and MMdUrd on cell growth (acute toxicity) and prolonged exposure (delayed cytotoxicity) in CEM cells was investigated. The two analogues did not exhibit acute or delayed toxicity (2 weeks exposure) up to 1000 μM. In contrast, at a concentration as low as 0.125 μM of 2′,3′-dideoxycytidine (ddC; control drug), the doubling time of the cells increased after 10 days. At higher concentrations, a very marked increase in doubling time was observed from 6 days onward with ddC treatment. The data suggest that in uninfected cells neither MMdUrd nor MMdCyd are anabolized to the triphosphate form in significant amounts. As a result, little or no MMdCTP or MMdUTP builds up in the mitochondria and thus delayed toxicity is not observed.

RELATIONSHIP BETWEEN CONFORMATION AND ANTIVIRAL ACTIVITY. III: 3'-AZIDOTHYMIDINE (AZT) AND 3'-AZIDO-2',3'-DIDEOXY-5-HYDROXYMETHYLURIDINE

Abstract 3′-Azido-2′,3′-dideoxy-5-hydroxymethyluridine (AZHMddUrd) was synthesized to improve the potency of 5-hydroxymethyl-2′-deoxyuridine (HMdUrd) against human immunodeficiency virus (HIV). AZHMddUrd was a very poor inhibitor of HIV replication (ED50 >200 μM) and was also nontoxic up to 400 μM (highest concentration tested) to HT4-6C (HeLa CD4) cells. AZT was phosphorylated by human cellular thymidine kinase. In contrast, AZHMddUrd and HMdUrd were poor substrates for the kinase. The relationship between molecular conformation and antiretroviral activity for 3′-azidothymidine (AZT), HMdUrd and AZHMddUrd is discussed.