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，我们试图确定非核苷类逆转录酶(RT)抑制剂(NNRTI)抑制HIV-1逆转录的多种机制。这些研究具有重要意义，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与其他类别的RT抑制剂之间的相互作用；它们将有助于我们了解NNRTI的耐药性和超敏性。在第二项研究中，我们致力于识别和表征新型逆转录酶抑制剂，如果开发出来，将补充和多样化现有的抗逆转录病毒疗法，并有助于解决对有效对抗多重耐药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，分别针对这些研究目标。我们实验室的最新数据表明，NNRTI同时调节RT的DNA聚合酶和核糖核酸酶H(RNase H)的活性。虽然NNRTI抑制DNA合成的机制已经被详细研究，但NNRTI影响酶的RNase H活性的机制尚不清楚。在目标1中，我们建议使用最先进的生物物理技术来阐明这种长程变构效应的机制，这些技术包括单对荧光、共振、能量转移和瞬时动力学分析。由于RNase H活性被视为潜在的药物发现目标，我们还将研究NNRTI与一个原型RNase H抑制剂之间的相互作用。由于DNA聚合酶和RNase H的调节可能导致药物效力的协同增加，我们还假设NNRTI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，在目标2中，我们将使用定量PCR来确定HIV-1逆转录过程中对NNRTI抑制最敏感的步骤。最后，我们小组最近开发了一种高通量筛选试验，以确定抑制HIV-1RT从DNA引物3‘端切除链终止核苷类似物的能力的药效团。从筛选出的7,265个化合物中，我们确定了3，39-[(3-羧基-4-氧代-2，5-环己二烯-1-亚甲基)亚甲基]双[6-羟基-苯甲酸](APEX-57219)是一种很有前途的“先导”化合物。初步的机制分析表明，apex-57219与模板/引物(T/P)底物竞争结合HIV-1RT。在目标3中，我们建议进行深入的分析，以确定apex-57219的作用机制，并确定其在HIV-1RT中的结合部位。这些研究将提供对这种新化合物的生化和病毒学性质的详细见解，这可能有助于发现和/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反转录的分子机制(S)。此外，我们还提出了一类新的逆转录酶抑制物，称为模板/引物竞争逆转录酶抑制物(TPcRTI)。由于TPcRTIs展示了一种新的作用机制，预计如果得到开发，它们将补充和多样化现有的艾滋病毒-1治疗战略，更重要的是，为治疗具有多药耐药性的艾滋病毒-1提供一种新的途径。

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.