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

描述（由申请人提供）：高效抗逆转录病毒治疗在对抗HIV感染方面取得了巨大成功，使艾滋病成为一种可管理的临床实体。然而，治疗中不可缺少的核苷类逆转录酶抑制剂（NRTIs）已显示出严重的不良反应，主要表现为线粒体毒性。毒性与NRTIs对人线粒体DNA聚合酶γ (DNA Pol 3)的抑制作用有关。我们建议通过对DNA复制不同阶段捕获的人类Pol 3进行结构研究来阐明NRTIs线粒体毒性的结构基础。具体来说，我们将通过NRTIs zalcitabine和AZT来确定Pol 3在复制过程中的晶体结构和停止复制的Pol 3的结构。这些结构的比较将为NRTIs线粒体毒性研究提供分子基础。我们最近获得了含有催化亚基和辅助亚基的人Pol 3全酶晶体，其衍射分辨率为3.1 E。此外，我们还获得了Pol - 3与引物模板DNA复合体的晶体，以及Pol - 3-DNA与zalcitabine的复合体。在完成拟议的研究后，我们将提供一系列在DNA复制或抑制作用中捕获的Pol 3的原子分辨率快照。比较抑制剂与人类Pol 3的相互作用与HIV逆转录酶的相互作用将为设计高效低毒的抗病毒药物提供宝贵的指导。

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.