Mitochondrial Toxicity of Antiviral Nucleosides in AIDS Therapy

艾滋病治疗中抗病毒核苷的线粒体毒性

• 批准号: 7421121
• 负责人: Edward E. McKee
• 金额: $ 40.41万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7421121
• 关键词: AIDS therapy; Acquired Immunodeficiency Syndrome; Acute; Address; Adipocytes; Adverse effects; Affect; Animals; Anti-Retroviral Agents; Antiviral Agents; Biological Assay; Biological Models; Brain; Cardiomyopathies; Cardiotoxicity; Cell Culture Techniques; Cell Cycle; Cell Line; Cell division; Cells; Combined Modality Therapy; Control Animal; Cultured Cells; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Deaminase; Deoxycytidine; Deoxycytidine Kinase; Deoxyguanosine kinase; Development; Dilated Cardiomyopathy; Drug usage; Echocardiography; Enzymes; Grant; Growth; Heart; Heart Mitochondria; Highly Active Antiretroviral Therapy; Human; Laboratories; Lamivudine; Lead; Liver; Long-Term Effects; Longitudinal Studies; Lysine; Measurement; Measures; Messenger RNA; Mitochondria; Mitochondrial DNA; Mitotic; Modeling; Mus; Natural regeneration; Neurites; Nuclear; Nucleosides; PC12 Cells; Pathway interactions; Peripheral Nervous System Diseases; Pharmaceutical Preparations; Phase; Phase III Clinical Trials; Phosphorylation; Polymerase; Prodrugs; Publishing; Pyrimidine; Pyrimidines; Radiolabeled; Rattus; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Ribonucleotide Reductase; Role; Sampling; Serum; Supplementation; Suspension Culture; System; TK1 gene; TK2 gene; Techniques; Testing; Thymidine; Thymidylate Synthase; Time; Tissues; Toxic effect; Ultrasonography; Uridine; Work; Zidovudine; analog; base; design; falls; human TK2 protein; improved; in vivo; inhibitor/antagonist; nucleoside analog; programs; radiotracer; research study; thymidine kinase 1; thymidylate kinase; tripolyphosphate

Antiretroviral nucleoside analogs used in highly active antiretroviral therapy (HAART) are associated with a variety of tissue toxicities associated with mitochondrial DNA depletion, suggesting a block in mt-DNA replication. Since 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. However, AZT (Zidovudine), a widely used drug in AIDS therapy, does not fit this hypothesis very well. AZT is a weak inhibitor of the mitochondrial polymerase and it is not readily converted to the triphosphate form. In recent work form our laboratory we have shown that AZT is a potent inhibitor of thymidine phosphorylation in isolated heart, liver, and brain mitochondria, and in the isolated perfused heart. We have recently demonstrated that AZT significantly decreases the TTP pool in the perfused heart. From this work we have proposed an alternative hypothesis in which the pro-drug AZT inhibits thymidine phosphorylation, reducing the TTP pool leading to imbalances in the deoxynucleotide pool that inhibit mitochondrial DNA replication and lead to mitochondrial DNA depletion. We propose to continue to test this hypothesis in several models of AZT toxicity. In the first we propose to use a previously published model of AZT toxicity in 3T3-F224A cells that are being programmed to differentiate into adipocytes. As these models develop AZT toxicity, we will measure the rates of thymidine and AZT phosphorylation, determine the effects of AZT on thymidine phosphorylation, document changes in the deoxynucleotide pools, and in levels of mitochondrial DNA relative to nuclear DNA and measure mRNA levels of select deoxynucleotide salvage and de novo enzymes. Since AZT is a competitive inhibitor of thymidine kinase 2, we will also test the effects of thymidine and uridine on reversing AZT toxicity, and on reversing changes in the deoxynucleotide pools. Additionally, we propose to use a previously published rat model of AZT toxicity in which liver and heart toxicities and mitochondrial DNA depletion have been observed. Here we will measure the same parameters as described above in the cell culture model including the effect of uridine/thymidine supplementation on AZT toxicity. We also propose to test the hypothesis that toxicity of other thymidine analogs, such as d4T is related to the ratio of analog triphosphate to the naturally occurring dNTP, particularly as cells differentiate and become post-mitotic. Nucleoside analogs represent an important component of HAART. The aims of this grant are to obtain a better understanding of the toxic effects of the nucleoside analog drugs used world-wide in the treatment of AIDS. This information is very important in developing strategies for treating these toxicities and in the rational design of new drugs with less toxic side-effects.

用于高活性抗逆转录病毒治疗（HAART）的抗逆转录病毒核苷类似物与线粒体DNA耗竭相关的多种组织毒性相关，表明其可阻断mt-DNA复制。由于这些类似物的三磷酸盐形式会不同程度地抑制mt-DNA聚合酶，因此该酶已被认为是HAART相关毒性的主要靶点。然而，在艾滋病治疗中广泛使用的药物AZT（齐多夫定）并不符合这一假设。AZT是线粒体聚合酶的弱抑制剂，不易转化为三磷酸形式。在我们实验室最近的工作中，我们已经证明AZT在离体心脏、肝脏和脑线粒体以及离体灌注心脏中是一种有效的胸苷磷酸化抑制剂。我们最近证明AZT显著降低灌注心脏的TTP池。从这项工作中，我们提出了另一种假设，即前药AZT抑制胸腺嘧啶磷酸化，减少TTP库，导致脱氧核苷酸库失衡，从而抑制线粒体DNA复制并导致线粒体DNA耗竭。我们建议在几个AZT毒性模型中继续验证这一假设。在第一项研究中，我们建议在3T3-F224A细胞中使用先前发表的AZT毒性模型，这些细胞正在被编程分化为脂肪细胞。随着这些模型产生AZT毒性，我们将测量胸腺嘧啶和AZT磷酸化的速率，确定AZT对胸腺嘧啶磷酸化的影响，记录脱氧核苷酸池的变化，以及线粒体DNA相对于核DNA的水平，并测量选择的脱氧核苷酸挽救酶和新生酶的mRNA水平。由于AZT是胸腺嘧啶激酶2的竞争性抑制剂，我们还将测试胸腺嘧啶和尿嘧啶对逆转AZT毒性的作用，以及对逆转脱氧核苷酸池变化的作用。此外，我们建议使用先前发表的AZT毒性大鼠模型，其中观察到肝脏和心脏毒性以及线粒体DNA耗竭。在这里，我们将测量与上述细胞培养模型中相同的参数，包括尿苷/胸苷补充对AZT毒性的影响。我们还建议验证其他胸腺嘧啶类似物（如d4T）的毒性与类似物三磷酸与自然发生的dNTP的比例有关的假设，特别是在细胞分化和有丝分裂后。核苷类似物是HAART的一个重要组成部分。