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进化的核心问题。为了继续开发新药物并最有效地利用现有药物，了解HIV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在进化过程中如何变化以增加对核苷类似物的选择性，同时保持正常核苷酸的充分有效结合将是至关重要的。构象变化对DNA聚合酶和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，一个构象开关决定了dNTP是被纳入还是被拒绝。在这个提议中，我们将研究HIV RT是否遵循这种DNA聚合酶选择性的新范式。在初步数据中，我们表明我们可以用手指结构域上的荧光团标记HIV RT，该位置提供信号以监测核苷酸结合时的构象变化，并且我们提供数据来定义核苷酸诱导的结合前结构变化的速率。我们将利用这一新信号来建立控制HIV RT选择性的反应途径，并定义酶构象变化在对核苷类似物的歧视中所起的作用。此外，我们将使用该信号来检查核苷酸结合和结合的动力学变化，这些变化是对核苷类似物产生抗性的基础。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反应的机制。这些研究将采用动力学和结构相结合的方法，包括停止流动荧光，快速化学猝灭流动和单分子荧光动力学研究。这项工作将更好地定义控制HIV RT核苷酸选择性的反应，使我们能够更严格地解释被认为对核苷类似物产生抗性的酶结构的观察变化，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艾滋病毒感染的核苷类似物的有效性和耐药性演变的核心问题。这项工作将定义控制HIV RT核苷酸选择性的基本步骤，使我们能够更严格地解释对核苷类似物耐药的酶结构的观察变化，并为管理HIV感染所需的新药的设计和评估提供见解。

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.