PHOSPHORYLATION OF 3'-AZIDO-3'-DEOXYTHYMIDINE AND SELECTIVE INTERACTION OF THE 5'-TRIPHOSPHATE WITH HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE-TRANSCRIPTASE
PHOSPHORYLATION OF 3'-AZIDO-3'-DEOXYTHYMIDINE AND SELECTIVE INTERACTION OF THE 5'-TRIPHOSPHATE WITH HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE-TRANSCRIPTASE
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DOI：
10.1073/pnas.83.21.8333
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发表时间：
1986-11-01
影响因子：
11.1
通讯作者：
BARRY, DW
中科院分区：
文献类型：
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作者：
FURMAN, PA;FYFE, JA;BARRY, DW
胸苷类似物3“-叠氮基-3”-脱氧胸苷（BW A509 U，叠氮胸苷）可以在50-500 nM范围内有效抑制人免疫缺陷病毒（HIV）复制[Mitsuya，H.，Weinhold，K. J.，弗曼，宾夕法尼亚州，圣克莱尔，M。H. Nusinoff-Lehrman，S.，加洛河C.的方法，Bolognesi，D.，巴里，D. W.和Broder，S.等人（1985）Proc. Acad. Sci. USA 82，7096-7100]。相比之下，未感染的人成纤维细胞和淋巴细胞的生长抑制已被观察到只有在浓度高于1 mM。这种选择性的性质进行了研究。叠氮胸苷对5“-单-、二-和-三磷酸衍生物的拮抗作用在未感染和HIV感染的细胞中是相似的。叠氮胸苷一磷酸的水平高，而二磷酸和三磷酸的水平低（≤ 0.001）。5 μ M和≤ 5 μ M。2 μ M）。胞质胸苷激酶（EC 2.7.1.21）负责叠氮胸苷磷酸化成其单磷酸。纯化的胸苷激酶催化胸苷和叠氮胸苷的磷酸化，表观Km值为2.9 μ M和3.0 μ M。叠氮胸苷磷酸化的最大速率等于胸苷速率的60%。叠氮胸苷一磷酸磷酸化为二磷酸似乎也被宿主细胞酶胸苷酸激酶催化（EC 2.7.4.9）。叠氮胸苷一磷酸的表观Km值比dTMP的值大2倍（8.6 μ M对4.1 μ M），但最大磷酸化速率仅为dTMP速率的0.3%。这些动力学常数与实验结果一致，表明叠氮胸苷一磷酸是胸苷酸激酶的替代底物抑制剂。这一结论反映在观察到叠氮胸苷孵育的细胞具有降低的细胞内dTTP水平。用HIV逆转录酶和用永生化人淋巴细胞（H9细胞）DNA聚合酶α测定叠氮胸苷三磷酸的IC 50（抑制酶活性50%的抑制剂浓度）值。叠氮胸苷三磷酸对HIV逆转录酶的竞争比对细胞DNA聚合酶α的竞争好约100倍。这里报告的结果表明，叠氮胸苷是非选择性磷酸化，但三磷酸衍生物有效地和选择性地结合到HIV逆转录酶。叠氮胸苷酸掺入到生长的DNA链中应终止DNA延伸，从而抑制DNA合成。
The thymidine analog 3''-azido-3''-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H. Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. and Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentration above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5''-mono-, di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di- and triphosphate were low (.ltoreq. 5 .mu.M and .ltoreq. 2 .mu.M, respectively). Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent Km values of 2.9 .mu.M and 3.0 .mu.M. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 .mu.M vs. 4.1 .mu.M), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. IC50 (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase .alpha.. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase .alpha.. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.