NNRTI induced conformational changes in HIV-1 RT

描述（由申请人提供）：我们研究的主要目标有两个。首先，我们努力定义非核苷类逆转录酶（RT）抑制剂（NNRTI）抑制HIV-1逆转录的多种机制。这些研究具有根本性的重要性，因为它们将有助于发现和/或开发新的NNRTI；它们将有助于确定nnrti与其他类RT抑制剂之间的相互作用；它们将有助于我们了解NNRTI耐药性和超易感性。其次，我们努力识别和表征新的RT抑制剂，如果开发出来，将补充和多样化现有的抗逆转录病毒疗法，并帮助解决对多药耐药HIV-1的抗病毒药物的需求。因此，在本申请中，我们提出了3个目标来解决这些研究目标。我们实验室最近的数据表明，NNRTI调节rt的DNA聚合酶和核糖核酸酶H （RNase H）活性。尽管NNRTI抑制DNA合成的机制已被详细研究，但NNRTI影响酶的RNase H活性的机制尚不清楚。在目标1中，我们建议通过使用最先进的生物物理技术，包括单对荧光共振能量转移和瞬态动力学分析，来阐明这种远距离变构效应的机制。由于RNase H活性被视为药物发现的潜在靶标，我们还将研究nnrti与原型RNase H抑制剂之间的相互作用。由于DNA聚合酶和RNase H的调节可能协同增加药物效力，我们还假设NNRTIs可能优先靶向逆转录过程中对这两种活性都有绝对要求的步骤。因此，在Aim 2中，我们将使用定量PCR来确定HIV-1逆转录过程中对NNRTIs抑制最敏感的步骤。最后，我们的团队最近开发了一种高通量筛选试验，以鉴定抑制HIV-1 RT从DNA引物3'端切除链终止核苷类似物的能力的药效团。从总共筛选的7,265个化合物中，我们确定了3,39-[（3-羧基-4-氧-2,5-环己二烯-1-酰基）亚甲基]双[6-羟基苯甲酸]（APEX-57219）是一个有前途的“先导”化合物。初步的机制分析表明，APEX-57219与模板/引物（T/P）底物竞争以结合HIV-1 RT。在Aim 3中，我们建议进行深入分析以确定APEX-57219的作用机制，并确定其在HIV-1 RT中的结合位点。这些研究将详细了解这种新化合物的生化和病毒学特性，有助于发现和/或开发更有效的T/P竞争RT抑制剂。公共卫生相关性：我们正在进行的研究的主要目标是确定NNRTI抑制HIV-1逆转录的分子机制。此外，我们建议表征一类新的RT抑制剂，称为模板/引物竞争RT抑制剂（TPcRTI）。由于tpcrti表现出一种新的作用机制，预计一旦开发出来，它们将补充和多样化现有的HIV-1治疗策略，更重要的是，为治疗多重耐药HIV-1提供新的途径。

DESCRIPTION (provided by applicant): The primary goals of our research are two-fold. In the first, we endeavor to define the multiple mechanisms by which nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) inhibit HIV-1 reverse transcription. These studies are of fundamental importance because they will assist in the discovery and/or development of new NNRTI; they will help define the interactions between NNRTIs and other classes of RT inhibitors; and they will contribute to our understanding of NNRTI resistance and hyper-susceptibility. In the second, we strive to identify and characterize novel RT inhibitors that, if developed, will complement and diversify existing antiretroviral therapies and help address the need for antiviral agents that are active against multi-drug resistant HIV-1. Accordingly, in this application we propose 3 Aims that address each of these research goals. Recent data from our laboratory demonstrate that NNRTIs modulate both the DNA polymerase and ribonuclease H (RNase H) activities of RT. Whereas the mechanisms of NNRTI inhibition of DNA synthesis have been investigated in detail, the mechanisms by which NNRTIs influence the enzyme's RNase H activity are unknown. In Aim 1 we propose to elucidate the mechanisms for this long-range allosteric effect by using state-of-the-art biophysical techniques that include single-pair fluorescence resonance energy transfer and transient kinetic analyses. Since RNase H activity is viewed as potential target for drug discovery, we will also investigate interactions between NNRTIs and a prototype RNase H inhibitor. Because modulation of both DNA polymerase and RNase H may lead to synergistically increased drug potency, we also hypothesize that NNRTIs may preferentially target steps during reverse transcription that have an absolute requirement for both activities. Therefore, in Aim 2 we will use quantitative PCR to identify the steps during HIV-1 reverse transcription that are most sensitive to inhibition by NNRTIs. Finally, our group recently developed a high throughput screening assay to identify pharmacophores that inhibit the ability of HIV-1 RT to excise chain-terminating nucleoside analogs from the 3'-end of the DNA primer. From a total of 7,265 compounds screened, we identified 3,39-[(3-carboxy-4-oxo-2,5-cyclohexadien-1- ylidene)methylene]bis[6-hydroxy-benzoic acid] (APEX-57219) as a promising "lead" compound. Preliminary mechanistic analyses demonstrate that APEX-57219 competes with the template/primer (T/P) substrate for binding to HIV-1 RT. In Aim 3, we propose in depth analyses to determine the mechanism of action of APEX- 57219, and to identify its binding site in HIV-1 RT. These studies will provide detailed insight into the biochemical and virological properties of this novel compound which could aid in the discovery and/or development of more potent T/P competing RT inhibitors. PUBLIC HEALTH RELEVANCE: The primary goal of our ongoing research is to define the molecular mechanism(s) by which NNRTI inhibit HIV-1 reverse transcription. In addition, we propose to characterize a new class of RT inhibitor termed the template/primer competing RT inhibitor (TPcRTI). Because the TPcRTIs exhibit a novel mechanism of action, it is anticipated that, if developed, they will both complement and diversify existing HIV-1 therapeutic strategies, and more importantly, provide a new avenue for the treatment of multi-drug resistant HIV-1.