Mitochondrial Toxicity of Antiviral Nucleosides

抗病毒核苷的线粒体毒性

• 批准号: 7851315
• 负责人: Edward E. McKee
• 金额: $ 39.99万
• 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/7851315
• 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; 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.

高活性抗逆转录病毒疗法 (HAART) 中使用的抗逆转录病毒核苷类似物与多种与线粒体 DNA 耗竭相关的组织毒性有关，表明线粒体 DNA 复制受阻。由于这些类似物的三磷酸盐形式不同程度地抑制 mt-DNA 聚合酶，因此该酶已被推广为与 HAART 相关的毒性的主要目标。然而，广泛用于艾滋病治疗的药物 AZT（齐多夫定）却不太符合这一假设。 AZT 是线粒体聚合酶的弱抑制剂，并且不易转化为三磷酸形式。在我们实验室最近的工作中，我们证明 AZT 是离体心脏、肝脏和脑线粒体以及离体灌注心脏中胸苷磷酸化的有效抑制剂。我们最近证明 AZT 显着降低灌注心脏中的 TTP 池。根据这项工作，我们提出了另一种假设，其中前药 AZT 抑制胸苷磷酸化，减少 TTP 池，导致脱氧核苷酸池失衡，从而抑制线粒体 DNA 复制并导致线粒体 DNA 耗尽。我们建议继续在几种 AZT 毒性模型中检验这一假设。首先，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。当这些模型产生 AZT 毒性时，我们将测量胸苷和 AZT 磷酸化的速率，确定 AZT 对胸苷磷酸化的影响，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，并测量选定脱氧核苷酸挽救和从头酶的 mRNA 水平。由于 AZT 是胸苷激酶 2 的竞争性抑制剂，因此我们还将测试胸苷和尿苷对逆转 AZT 毒性以及逆转脱氧核苷酸库变化的影响。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，我们将在细胞培养模型中测量与上述相同的参数，包括补充尿苷/胸苷对 AZT 毒性的影响。我们还建议测试以下假设：其他胸苷类似物（例如 d4T）的毒性与类似物三磷酸与天然存在的 dNTP 的比率有关，特别是当细胞分化并进入有丝分裂后时。核苷类似物是HAART的重要组成部分。 这笔赠款的目的是更好地了解世界范围内用于治疗艾滋病的核苷类似物药物的毒性作用。这些信息对于制定治疗这些毒性的策略以及合理设计毒副作用较小的新药非常重要。

项目成果

Metabolism of deoxypyrimidines and deoxypyrimidine antiviral analogs in isolated brain mitochondria.

• DOI: 10.1111/j.1471-4159.2012.07765.x
• 发表时间: 2012-07
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: McCann KA;Williams DW;McKee EE
• 通讯作者: McKee EE

Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity.

• DOI: 10.1385/ct:4:2:155
• 发表时间: 2004-01-01
• 期刊: Cardiovascular toxicology
• 影响因子: 3.2
• 作者: McKee, Edward E;Bentley, Alice T;Susan-Resiga, Delia
• 通讯作者: Susan-Resiga, Delia