Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity.
Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity.
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DOI：
10.1385/ct:4:2:155
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发表时间：
2004-01-01
影响因子：
3.2
通讯作者：
Susan-Resiga, Delia
中科院分区：
文献类型：
--
作者：
McKee, Edward E;Bentley, Alice T;Susan-Resiga, Delia
用于高效抗逆转录病毒疗法（HAART）的抗逆转录病毒核苷类似物与心血管和其他组织毒性相关，所述心血管和其他组织毒性与线粒体DNA耗竭相关，表明线粒体（mt）-DNA复制的阻断。由于这些类似物的三磷酸盐形式可抑制线粒体DNA聚合酶，因此该酶已被提升为与HAART相关的毒性的主要靶标。我们已经使用了从大鼠心脏分离的线粒体，研究线粒体运输和磷酸化的胸苷和AZT（叠氮胸苷，或齐多夫定），在HAART中使用的一个组成部分。我们证明，分离的线粒体容易运输胸苷和磷酸化它的胸苷5 '-三磷酸（TTP）内的矩阵。在相同条件下，AZT仅磷酸化为AZT-5 '-单磷酸（AZT-MP）。胸苷和AZT的动力学表明底物与酶相互作用的负协同性，与其他人对线粒体胸苷激酶2的工作一致。结果表明，TMP和AZT-MP不跨内膜转运，表明AZT-MP可能随时间在基质中积累。由于缺乏AZT-5 '-三磷酸（AZT-TP），AZT在心脏中的毒性似乎不太可能是由AZT-TP抑制DNA聚合酶γ介导的。相反，我们的工作表明，AZT是心脏线粒体中胸苷磷酸化的有效抑制剂，抑制浓度（IC）（50）为7.0 +/- 0.9 μ M。因此，AZT在某些组织中的毒性可能是通过破坏TTP对mtDNA复制的底物供应来介导的。
Antiretroviral nucleoside analogs used in highly active antiretroviral therapy (HAART) are associated with cardiovascular and other tissue toxicity associated with mitochondrial DNA depletion, suggesting a block in mitochondrial (mt)-DNA replication. Because the triphosphate forms of these analogs variably inhibit mt-DNA polymerase, this enzyme has been promoted as the major target of toxicity associated with HAART. We have used isolated mitochondria from rat heart to study the mitochondrial transport and phosphorylation of thymidine and AZT (azidothymidine, or zidovudine), a component used in HAART. We demonstrate that isolated mitochondria readily transport thymidine and phosphorylate it to thymidine 5'-triphosphate (TTP) within the matrix. Under identical conditions, AZT is phosphorylated only to AZT-5'-monophosphate (AZT-MP). The kinetics of thymidine and AZT suggest negative cooperativity of substrate interaction with the enzyme, consistent with work by others on mitochondrial thymidine kinase 2. Results show that TMP and AZT-MP are not transported across the inner membrane, suggesting that AZT-MP may accumulate with time in the matrix. Given the lack of AZT-5'-triphosphate (AZT-TP), it seems unlikely that the toxicity of AZT in the heart is mediated by AZT-TP inhibition of DNA polymerase gamma. Rather, our work shows that AZT is a potent inhibitor of thymidine phosphorylation in heart mitochondria, having an inhibitory concentration (IC)(50) of 7.0 +/- 0.9 microM. Thus, the toxicity of AZT in some tissues may be mediated by disrupting the substrate supply of TTP for mt-DNA replication.