Mitochondrial Nucleotide Carriers of NRTI Metabolites

描述(由申请人提供)： 核苷逆转录酶抑制剂(NRTI)是高效抗病毒治疗的关键成分。磷酸化衍生物(NRTI-P)模拟HIV RT的细胞dNTP底物，但产生链终止，从而限制逆转录病毒的扩增。NRTI产生类似于线粒体(Mito)遗传病患者的可逆副作用，包括心肌病、肌病、神经病和乳酸酸中毒，这些副作用与NRTI诱导的各种组织中mito DNA的丢失有关。毒性被归因于抑制DNA聚合酶g，干扰有丝分裂DNA的维持。另一种或附加的有丝分裂损伤机制也可能在起作用。无论机制如何，NRTI的毒性依赖于MITO的输入。我们已经分离出编码MITO代谢物载体家族(OMC)孤儿成员的新的人类基因和cDNA。5个携带核苷酸(NT)携带者特有的特征，包括一个假定的腺嘌呤NT转运体(ANT)，两个假定的CoA转运体，以及一个非特异的核苷酸交换子(OMC27)。在蛋白脂体放射化学通量分析中，我们发现纯化的OMC27及其酵母同源基因能够运输NRTI-P的AZT-TP和-DP、DDL-TP和ddC-TP。阻断同源酵母基因可保护AZT诱导的有丝分裂功能障碍。本项目研究了OMC27在NRTI介导的毒性中的作用，目的是了解NRTI进入基质的机制，并识别具有已知抗病毒活性的核苷类似物，这些类似物有可能通过丝裂原排斥和降低丝裂原毒性来改善治疗指数。我们将检验这些假设，即OMC27(和酵母同源物)是NRTI-P的高亲和力转运体，这是NRTI-P有丝分裂摄取的主要途径；OMC27的表达对于NRTI诱导的有丝分裂失败至关重要；并且从NRTI抗病毒活性与MC介导的摄取活性的比率可能明显看出NRTI的有用治疗指数；使用互补的体外、酵母和哺乳动物细胞以及体内小鼠研究。

DESCRIPTION (provided by applicant): Nucleoside reverse transcriptase inhibitors (NRTI) are critical components of highly active anti-viral therapies. Phosphorylated derivatives (NRTI-P) mimic the cellular dNTP substrates for HIV RT, but produce chain termination, thereby limiting retroviral amplification. NRTIs produce reversible side effects that resemble those of patients with mitochondrial (mito) genetic disorders, including cardiomyopathy, myopathy, neuropathy and lactic acidosis, and these correlate with NRTI-induced loss of mito DNA in various tissues. Toxicity has been attributed to inhibition of DNA polymerase g, interfering with mito DNA maintenance. Alternative or additional mechanisms of mito damage may also be at play. Regardless of mechanism, NRTI toxicity is dependent on import into mito. We have isolated novel human genes and cDNAs that encode orphan members of the mito metabolite carrier family (OMC). Five carry a signature specific to nucleotide (nt) carriers, including a putative adenine nt transporter (ANT), two putative CoA transporters, and a non-specific exchanger of nucleotides (OMC27). We have shown that purified OMC27 and its yeast ortholog transport NRTI-P's AZT-TP and -DP, ddl-TP, and ddC-TP in proteoliposome radiochemical flux assays. Disruption of the orthologous yeast gene protects against AZT-induced mito dysfunction. This project examines the role of OMC27 in NRTI-mediated toxicity, with goals of understanding mechanisms by which NRTIs gain entry to the matrix, and identifying nucleoside analogs with known anti-viral activity that offer potential for improved therapeutic index by virture of mito exclusion and reduced mito toxicity. We will test the hypotheses that OMC27 (and yeast ortholog) are highly affinity transporters of NRTI-P, that this represents the primary pathway for mito uptake of NRTI-P; that OMC27 expression is critical to NRTI-induced mito failure; and that a useful therapeutic index for NRTIs may be evident from the ratio of NRTI anti-viral to MC-mediated uptake activities; using complementary in vitro, yeast and mammalian cell, and in vivo mouse studies.