Molecular Pharmacology 1983-07-01

Preferential inhibition of 5-trifluoromethyl-2'-deoxyuridine phosphorylation by 5'-amino-5'-deoxythymidine in uninfected versus herpes simplex virus-infected cells.

P H Fischer, D G Murphy, R Kawahara

Index: Mol. Pharmacol. 24(1) , 90-6, (1983)

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Abstract

The cytotoxic effects of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) were effectively antagonized by 5'-amino-5'-deoxythymidine (5'-AdThd). The antiproliferative actions of CF3dUrd were reduced in a dose-dependent manner by 5'-AdThd in both HeLa and Vero cells. In addition, the ability of CF3dUrd to kill HeLa cells (95% at 1 microM and 99% at 3 microM), as measured by cloning efficiency, was ablated entirely by 5'-AdThd (300 microM). In contrast, the inhibition of herpes simplex virus Type 2 (HSV-2) replication in HeLa cells was not antagonized by 5'-AdThd. In Vero cells, the combination of CF3dUrd and 5'-AdThd produced a greater antiviral effect than either agent alone. The reduction in CF3dUrd cytotoxicity caused by 5'-AdThd in uninfected HeLa and Vero cells was associated with decreased intracellular levels of CF3dUrd nucleotides. In contrast, in HSV-2-infected Vero cells the intracellular levels of CF3dUrd nucleotides were slightly elevated by 5'-AdThd and, in virally infected HeLa cells, a 300-fold excess of 5'-AdThd reduced CF3dUrd uptake only marginally. Since the relative abundance of these phosphorylated derivatives of CF3dUrd was not markedly changed by 5'-AdThd, preferential inhibition of the mammalian thymidine kinase (EC 2.7.1.21) was suggested. Using CF3dUrd as the substrate, the ability of 5'-AdThd to inhibit thymidine kinase activity in extracts prepared from parallel cultures of mock-infected or HSV-2 infected HeLa cells was compared. CF3dUrd was phosphorylated to a lesser extent and the reaction was more potently inhibited by 5'-AdThd in the extracts prepared from the uninfected cells. The HeLa cell and HSV-2 thymidine kinases were purified by affinity column chromatography, and kinetic analyses were then done. Using CF3dUrd as the variable substrate, the Ki values for 5'-AdThd were 2.2 microM for the HeLa enzyme and 36 microM for the viral enzyme. Km values for CF3dUrd were 2.6 microM and 4 microM for the viral and mammalian enzymes, respectively. These data account for the ability of 5'-AdThd to inhibit preferentially the phosphorylation of CF3dUrd in uninfected host cells. The presence of 5'-AdThd substantially increased the therapeutic index of CF3dUrd, indicating that this drug combination, an example of specific inhibition, warrants investigation in vivo.