NNRTI induced conformational changes in HIV-1 RT - Equipment Supplement

描述：非核苷类逆转录酶（RT）抑制剂（NNRTIs）是一类重要的治疗药物，广泛用于抗逆转录病毒治疗策略，以治疗和预防HIV-1感染。它们结合在HIV-1 RT中的疏水口袋上，称为nnrti结合口袋（BP），该口袋位于距离酶的聚合酶活性位点约10°的地方。我们目前对nnrti如何抑制HIV-1逆转录，以及NNRTI-BP突变如何赋予抑制剂耐药性的理解，主要是从HIV-1 RT与nnrti复合物的晶体结构中推断出来的。关于NNRTIs如何影响催化相关的rt -模板/引物（T/P）二元和RT-T/P- dntp三元配合物，我们只有有限的知识。重要的是，单分子F共振能量转移研究表明，nnrti与RT的结合会影响RT在T/P底物上的结合取向和滑动动力学。晶体学不能提供洞察生物分子之间的动态相互作用。事实上，nnrti结合的RT-T/P二元配合物只有两种晶体结构可用（而三元配合物则没有），其中一种结构中RT与T/P底物交联。因此，关于nnrti与野生型和突变型RT的结合如何影响，存在一个关键的知识缺口：(i)酶与其底物之间的动态分子间相互作用；（ii） RT-T/P和RT-T/P- dNTP复合物的分子内蛋白质构象变化。本应用程序的主要目标是利用最先进的单分子和整体生物物理方法（在我们的实验室开发）来解决这些知识空白，这些方法可以定量评估HIV-1 RT与其底物之间的动态分子间相互作用以及RT的分子内构象变化。我们预计从这些研究中获得的数据将为nnrti的作用模式和机制提供前所未有的机制见解与NNRTI抗性相关总的来说，这些研究可能会对未来的药物发现工作产生重大影响。

DESCRIPTION: Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are an important therapeutic class of drugs that are widely used in antiretroviral therapy strategies to treat and prevent HIV-1 infection. They bind to a hydrophobic pocket in HIV-1 RT, termed the NNRTI-binding pocket (BP), which is located ~ 10 � away from the polymerase active site of the enzyme. Our current understanding of how NNRTIs inhibit HIV-1 reverse transcription, and how mutations in the NNRTI-BP confer inhibitor resistance, has been largely inferred from crystal structures of HIV-1 RT in complex with NNRTIs. We have only limited knowledge in regard to how NNRTIs affect the catalytically relevant RT-template/primer (T/P) binary and RT-T/P-dNTP ternary complexes. Importantly, single-molecule F�ster resonance energy transfer studies revealed that NNRTI-binding to RT can impact the binding orientation and sliding dynamics of RT on the T/P substrate. Crystallography cannot provide insight into the dynamic interactions between biomolecules. Indeed, there are only 2 crystal structures available of an NNRTI-bound RT-T/P binary complex (and none for the ternary complex) - and in one of these structures RT is cross-linked to the T/P substrate. As such, there is a critical knowledge gap in regard to how NNRTI-binding to wild-type and mutant RT impacts: (i) the dynamic inter-molecular interactions between the enzyme and its substrates; and (ii) the intra-molecular protein conformational changes in RT-T/P and RT-T/P- dNTP complexes. The primary goal of this application is to address these knowledge gaps using state-of-the- art single-molecule and ensemble biophysical approaches (developed in our laboratories) that can quantitatively assess the dynamic inter-molecular interactions between HIV-1 RT and its substrates and the intra-molecular conformational changes in RT. We anticipate that the data derived from these studies will provide unprecedented mechanistic insight into the mode of action of NNRTIs and the mechanisms associated with NNRTI resistance. Collectively these studies may significantly impact future drug discovery efforts.