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

描述(由申请人提供)：通过高效的抗逆转录病毒疗法抗击艾滋病毒感染的巨大成功使艾滋病成为一种可管理的临床实体。然而，核苷逆转录酶抑制剂(NRTI)作为治疗中不可缺少的成分，已显示出严重的不良反应，主要表现为线粒体毒性。这些毒性与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Pol 3的结构研究来阐明NRTIs线粒体毒性的结构基础。具体地说，我们将确定Pol 3在复制过程中的晶体结构，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的线粒体毒性提供分子基础。我们最近获得了含有催化亚基和辅助亚基的人POL3全酶晶体，其衍射率可达3.1E。此外，我们还得到了Pol-3与引物-模板DNA双链的络合物以及Pol-3-DNA与扎西他滨的络合物。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捉到的POL3的原子分辨率快照。比较抑制物与人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。 公共卫生相关性：通过高效的抗逆转录病毒疗法与艾滋病毒感染作斗争取得了巨大成功，使艾滋病成为一个可管理的临床实体。然而，核苷逆转录酶抑制剂(NRTI)作为治疗中不可缺少的成分，已显示出严重的不良反应，主要表现为线粒体毒性。随着艾滋病的持续流行和患者生存时间的显著延长，长期使用NRTI引起的副作用变得越来越常见。研究表明，NRTIs对抑制复制和修复线粒体DNA的人类线粒体DNA聚合酶有不良反应。活跃的DNA复制对细胞器的完整性至关重要。干扰人类POL3活性会导致突变和DNA耗尽，导致肌病、心肌病、乳糖酸中毒和脑病。我们建议对人类线粒体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复制的了解，而且将对设计高效、低毒的抗HIV试剂具有非常重要的价值。

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.