Nucleotide selectivity and drug resistance by HIV reverse transcriptase

描述（由申请人提供）：核苷酸选择性是理解用于治疗HIV感染的核苷类似物的有效性和耐药性演变问题的核心。为了继续开发新的药物和最有效地利用现有的药物，这将是至关重要的，以了解艾滋病毒逆转录酶（RT）在DNA聚合过程中实现核苷酸选择性的机制，以及酶如何在进化中变化，以增加对核苷类似物的选择性，同时保持足够有效的正常核苷酸的掺入。关于构象变化对DNA聚合酶以及其他酶的选择性的作用，一直存在相当大的争论。新的数据表明，构象开关决定了dNTP是否会被纳入或拒绝。在这个提议中，我们将调查HIV RT是否遵循DNA聚合酶选择性的新范式。在初步的数据，我们表明，我们可以标记HIV RT与荧光团的手指结构域的位置，提供了一个信号，以监测核苷酸结合后的构象变化，我们目前的数据来定义的核苷酸诱导的结构变化率之前纳入。我们将利用这一新的信号，建立由HIV RT的选择性反应的途径，并确定酶的构象变化对核苷类似物的歧视所发挥的作用。此外，我们将使用这个信号来检查核苷酸结合和掺入的动态变化，这些变化是对核苷类似物耐药的基础。我们还将探讨非核苷抑制剂改变核苷酸结合动力学和减弱活性位点化学的机制。这些研究将使用动力学和结构方法的组合来实现，包括停流荧光、快速化学猝灭流和单分子荧光动力学研究。这项工作将更好地定义HIV RT控制核苷酸选择性的反应，使我们能够更严格地解释被认为是对核苷类似物耐药的酶结构变化，并为设计和评估管理HIV感染所需的新药提供见解。公共卫生相关性：核苷酸选择性是理解用于治疗HIV感染的核苷类似物的有效性以及耐药性演变问题的核心。这项工作将定义由HIV RT控制核苷酸选择性的基本步骤，使我们能够更严格地解释观察到的酶结构变化，负责对核苷类似物的耐药性，并为设计和评估管理HIV感染所需的新药提供见解。

DESCRIPTION (provided by applicant): Nucleotide selectivity is at the heart of the problem of understanding the effectiveness of nucleoside analogs used to treat HIV infections, and the evolution of resistance. In order to continue to develop new drugs and most efficiently use the ones available, it will be critical to understand the mechanisms by which HIV reverse transcriptase (RT) achieves nucleotide selectivity during DNA polymerization and how the enzyme changes in evolving to increase selectivity against nucleoside analogs while retaining sufficiently efficient incorporation of normal nucleotides. There has been considerable debate over the role of conformational changes in contributing to the selectivity of DNA polymerases as well as for other enzymes. New data suggest that a conformational switch dictates whether a dNTP will be incorporated or rejected. In this proposal we will investigate whether HIV RT follows this new paradigm for DNA polymerase selectivity. In preliminary data, we show that we can label HIV RT with a fluorophore on the fingers domain in a position that provides a signal to monitor the conformational changes upon nucleotide binding, and we present data to define the rates of nucleotide-induced changes in structure that precede incorporation. We will exploit this new signal to establish the pathway of reactions governing selectivity by HIV RT and define the role played by enzyme conformational changes in discrimination against nucleoside analogs. In addition, we will use this signal to examine changes in the dynamics of nucleotide binding and incorporation that underlie resistance to nucleoside analogs. We will also explore the mechanism by which nonnucleoside inhibitors alter dynamics of nucleotide binding and attenuate chemistry at the active site. These studies will be achieved using a combination of kinetic and structural methods, including stopped-flow fluorescence, rapid chemical quench-flow and single molecule fluorescence kinetic studies. This work will define better the reactions governing nucleotide selectivity by HIV RT, allow us to more rigorously interpret observed changes in enzyme structure thought to be responsible for resistance to nucleoside analogs, and provide insights to into the design and evaluation of new drugs needed to manage HIV infections. PUBLIC HEALTH RELEVANCE: Nucleotide selectivity is at the heart of the problem of understanding the effectiveness of nucleoside analogs used to treat HIV infections, and the evolution of resistance. This work will define the elementary steps governing nucleotide selectivity by HIV RT, allow us to more rigorously interpret observed changes in enzyme structure responsible for resistance to nucleoside analogs, and provide insights to into the design and evaluation of new drugs needed to manage HIV infections.