Heart mitochondrial toxicity of antiviral nucleosides

描述（由申请人提供）： 一段时间以来，用于高效抗逆转录病毒治疗（HAART）的抗逆转录病毒核苷类似物与心血管以及其他组织毒性相关，这些毒性似乎与线粒体生物合成有关。特别是，研究表明，线粒体DNA在毒性过程中耗尽，表明DNA复制受阻。由于这些核苷类似物的三磷酸形式已被证明在不同程度上直接抑制线粒体DNA聚合酶，因此通常将其作为这种毒性的靶点。然而，正如本提案中所讨论的，我们已经表明，HAART的组成部分之一，AZT（齐多夫定）是缓慢地转化为AZT-MP，并没有进一步，在两个分离的心脏线粒体和分离的灌注大鼠心脏的条件下，标记的胸苷很容易转化为标记的TTP在这两个模型。因此，AZT在心脏中的毒性似乎不太可能是由AZT-TP抑制聚合酶介导的。相反，我们已经表明，AZT的存在下，可能通过AZT-MP，是一个有效的抑制剂，在两个分离的心脏线粒体和在分离的灌注心脏胸苷磷酸化。这些数据提出了另一种假设，即AZT和其他类似物的毒性是由于它们干扰内源性脱氧核苷的代谢，破坏线粒体DNA复制的dNTPs底物供应。这笔赠款有三个主要目标。第一个目标是记录和定量每种内源性脱氧核苷和HAART治疗中使用的每种核苷类似物在分离的心脏线粒体和分离的灌注大鼠心脏中向其三磷酸形式的摄取和转化。从这些数据中，我们可以确定线粒体在转化过程中的相对作用，我们可以估计各种类似物中间体的水平，并确定是否有足够的三磷酸合成来抑制线粒体聚合酶。该建议的第二个目标是评估这些相同的核苷类似物对分离的心脏线粒体和分离的灌注心脏中内源性脱氧核苷的摄取和磷酸化的影响。第三个目标是评估联合药物治疗对类似物和内源性脱氧核苷磷酸化的影响。从这项工作中，我们希望确定核苷类似物的潜在毒性形式以及其潜在的酶靶。

DESCRIPTION (provided by applicant): The antiretroviral nucleoside analogs used in highly active antiretroviral therapy (HAART) have been associated for some time with cardiovascular as well as other tissue toxicities that appear to be directed at mitochondrial biogenesis. In particular, studies have demonstrated that mitochondrial DNA is depleted during toxicity, suggesting a block in DNA replication. Since the triphospho-forms of these nucleoside analogs have been shown to varying degrees to directly inhibit mitochondrial DNA polymerase, this has generally been promoted as the target of this toxicity. However, as discussed in this proposal, we have shown that one of the HAART components, AZT (Zidovudine) is converted slowly only to AZT-MP and no further, in both isolated heart mitochondria and in the isolated perfused rat heart under conditions in which labeled thymidine is readily converted to labeled TTP in both models. Thus it seems unlikely that the toxicity of AZT in the heart is mediated by AZT-TP inhibition of polymerase. Rather, we have shown that the presence of AZT, perhaps via AZT-MP, is a potent inhibitor of thymidine phosphorylation in both isolated heart mitochondria and in the isolated perfused heart. These data suggest an alternative hypothesis that the toxicity of AZT and perhaps other analogs, is caused by their interference with the metabolism of endogenous deoxynucleosides disrupting the substrate supply of dNTPs for mitochondrial DNA replication. This grant has three major goals. The first goal is to document and quantitate the uptake and conversion of each of the endogenous deoxynucleosides, and each of the nucleoside analogs used in HAART therapy, to their tri-phospho-forms in isolated heart mitochondria and in the isolated perfused rat heart. From these data we can determine the relative role of the mitochondria in the conversion process and we can estimate the levels of the various analog intermediates and determine if there is sufficient synthesis of the triphosphate to inhibit the mitochondrial polymerase. The second goal of this proposal is to evaluate the effect of these same nucleoside analogs on the uptake and phosphorylation of the endogenous deoxynucleosides in isolated heart mitochondria and in the isolated perfused heart. The third goal is to evaluate the effect of combination drug therapy on analog and endogenous deoxynucleoside phosphorylation. From this work we hope to identify both the potential toxic form of the nucleoside analog as well as its potential enzymatic target.