Preferential inhibition of 5-trifluoromethyl-2'-deoxyuridine phosphorylation by 5'-amino-5'-deoxythymidine in uninfected versus herpes simplex virus-infected cells.
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.
D000818:Animals; D000998:Antiviral Agents / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D015224:Dideoxynucleosides; D006367:HeLa Cells; D006561:Herpes Simplex / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D010766:Phosphorylation; D013936:Thymidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors; D014271:Trifluridine / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D015224:Dideoxynucleosides; C024082:5'-amino-5'-deoxythymidine; D013937:Thymidine Kinase; D014271:Trifluridine; D013936:Thymidine