Schneider B, Biondi R, Sarfati R, Agou F, Guerreiro C, Deville-Bonne D, Veron M
Unité de Régulation Enzymatique des Activités Cellulaires, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1773, 558, Institut Pasteur, Paris, France.
Mol Pharmacol. 2000 May;57(5):948-53.
The last step in the intracellular activation of antiviral nucleoside analogs is the addition of the third phosphate by nucleoside diphosphate (NDP) kinase resulting in the synthesis of the viral reverse transcriptase substrates. We have previously shown that dideoxynucleotide analogs and 3'-deoxy-3'-azidothymidine (AZT) as di- or triphosphate are poor substrates for NDP kinase. By use of protein fluorescence, we monitor the phosphotransfer between the enzyme and the nucleotide analog. Here, we have studied the reactivity of D4T (2',3'-dideoxy-2',3'-didehydrothymidine; stavudine) as di- (DP) or triphosphate (TP) at the pre-steady state. The catalytic efficiency of D4T-DP or -TP is increased by a factor of 10 compared with AZT-DP or -TP, respectively. We use an inactive mutant of NDP kinase to monitor the binding of a TP derivative, and show that the affinity for D4T-TP is in the same range as for the natural substrate deoxythymidine triphosphate, but is 30 times higher than for AZT-TP. Our results indicate that D4T should be efficiently phosphorylated after intracellular maturation of a prodrug into D4T-monophosphate.
抗病毒核苷类似物细胞内激活的最后一步是由核苷二磷酸（NDP）激酶添加第三个磷酸基团，从而合成病毒逆转录酶底物。我们之前已经表明，双脱氧核苷酸类似物和作为二磷酸或三磷酸的3'-脱氧-3'-叠氮胸苷（AZT）是NDP激酶的不良底物。通过蛋白质荧光法，我们监测了酶与核苷酸类似物之间的磷酸转移。在此，我们研究了双脱氧胸苷（D4T；2',3'-二脱氧-2',3'-二氢胸苷；司他夫定）作为二磷酸（DP）或三磷酸（TP）在前稳态下的反应活性。与AZT-DP或-TP相比，D4T-DP或-TP的催化效率分别提高了10倍。我们使用NDP激酶的无活性突变体来监测TP衍生物的结合，并表明对D4T-TP的亲和力与天然底物脱氧胸苷三磷酸处于相同范围，但比AZT-TP高30倍。我们的结果表明，前体药物在细胞内成熟为单磷酸D4T后，D4T应能被有效磷酸化。
