Studies of toxicities of HIV RT inhibitors to human mitochondrial DNA polymerase

描述（由申请人提供）：通过高效抗逆转录病毒疗法对抗艾滋病毒感染取得了巨大成功，使艾滋病成为一种可控制的临床实体。然而，核苷逆转录酶抑制剂（NRTIs）作为治疗中不可缺少的成分，却表现出严重的不良反应，主要表现为线粒体毒性。这些毒性与 NRTI 对人类线粒体 DNA 聚合酶 γ (DNA Pol 3) 的抑制作用有关。我们建议通过对在 DNA 复制的各个阶段捕获的人类 Pol 3 进行结构研究来阐明 NRTI 线粒体毒性的结构基础。具体来说，我们将确定复制过程中 Pol 3 的晶体结构以及通过 NRTI 扎西他滨和 AZT 停止复制的 Pol 3 的结构。这些结构的比较将为 NRTIs 线粒体毒性提供分子基础。我们最近获得了人类 Pol 3 全酶晶体，其中含有催化和辅助亚基，衍射分辨率为 3.1 E。此外，我们还获得了 Pol 3 与引物模板 DNA 双链体复合物的晶体，以及 Pol 3-DNA 与扎西他滨的复合物。完成拟议的研究后，我们将提供一系列 DNA 复制或抑制作用中 Pol 3 的原子分辨率快照。比较抑制剂与人 Pol 3 和 HIV 逆转录酶的相互作用将为设计高效低毒的抗病毒试剂提供宝贵的指导。 公共健康相关性：通过高效抗逆转录病毒疗法对抗艾滋病毒感染取得了巨大成功，使艾滋病成为一种可控制的临床疾病。然而，核苷逆转录酶抑制剂（NRTIs）作为治疗中不可缺少的成分，却表现出严重的不良反应，主要表现为线粒体毒性。随着艾滋病流行的持续以及患者生存时间的显着延长，长期使用 NRTI 引起的副作用变得越来越普遍。研究表明，NRTI 会因抑制复制和修复线粒体 DNA 的人类线粒体 DNA 聚合酶而产生不良反应。活跃的 DNA 复制对于细胞器的完整性至关重要。干扰人类 Pol 3 活性会导致突变和 DNA 耗竭，从而导致肌病、心肌病、乳糖酸中毒和脑病。我们计划对人线粒体DNA聚合酶的NRTIs抑制机制进行结构研究。这项研究的结果不仅将增加我们对线粒体 DNA 复制的理解，而且对于帮助设计高效、低毒的抗 HIV 试剂也具有不可估量的价值。

DESCRIPTION (provided by applicant): Tremendous success fighting HIV infection with highly active antiretroviral therapy turns AIDS into a manageable clinical entity. However, nucleoside reverse transcriptase inhibitors (NRTIs), the indispensable component of the treatment, have displayed severe adverse reaction mostly manifested as mitochondrial toxicities. The toxicities have been implicated to NRTIs inhibitory effect on human mitochondrial DNA polymerase gamma (DNA Pol 3). We propose to elucidate structural basis of NRTIs mitochondrial toxicity by conducting structural studies of human Pol 3 captured at various stages of DNA replication. Specifically, we will determine crystal structures of Pol 3 during replication and structures of a stalled replicating Pol 3 by NRTIs zalcitabine and AZT. Comparison of these structures will provide molecular basis for NRTIs mitochondrial toxicity. We have recently obtained crystals of human Pol 3 holoenzyme containing the catalytic and accessory subunits that diffracted to 3.1 E resolution. In addition, we have obtained crystals of Pol 3 complexed with a primer-template DNA duplex, as well as a complex of Pol 3-DNA with zalcitabine. Upon completion of the proposed studies, we will provide an array of atomic-resolution snapshots of Pol 3 caught in action of DNA replication or inhibition. Comparison of inhibitor interactions with human Pol 3 with that of HIV reverse transcriptase will provide invaluable guidance in design high potency and low toxic antiviral reagents. PUBLIC HELATH RELEVANCE: Tremendous success fighting HIV infection with highly active antiretroviral therapy turns AIDS into a manageable clinical entity. However, nucleoside reverse transcriptase inhibitors (NRTIs), the indispensable component of the treatment, have displayed severe adverse reaction mostly manifested as mitochondrial toxicities. As AIDS epidemic continues and significantly increased patients survival duration, the side effects due to long-term usage of NRTIs become increasingly more common. Studies have implicated the NRTIs adverse reaction to inhibition of human mitochondrial DNA polymerase that replicates and repairs mitochondrial DNA. Active DNA replication is critical to the integrity of the organelle. Interference with human Pol 3 activity causes mutations and DNA depletion, resulting myopathy, cardiomyopathy, lactose acidosis and encephalopathy. We propose to conduct structural studies of NRTIs inhibitory mechanism of human mitochondrial DNA polymerase. The results of the study will not only increase our understanding of mitochondrial DNA replication, but also will be invaluable to aid design of high potency and low toxic anti- HIV reagents.