Nucleotide selectivity and drug resistance by HIV reverse transcriptase

HIV逆转录酶的核苷酸选择性和耐药性

• 批准号: 7930581
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 31.25万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7930581
• 关键词: Active Sites; Attenuated; Binding; Biological Assay; Chemicals; Chemistry; DNA; DNA-Directed DNA Polymerase; Data; Diphosphates; Discrimination; Effectiveness; Enzymes; Equilibrium; Evaluation; Evolution; Fingers; Fluorescence; HIV; HIV Infections; HIV drug resistance; Heart; Kinetics; Label; Measurement; Measures; Methods; Modeling; Molecular Conformation; Monitor; Nucleotides; Pathway interactions; Pharmaceutical Preparations; Play; Positioning Attribute; RNA-Directed DNA Polymerase; Reaction; Resistance; Resistance development; Role; Signal Transduction; Specificity; Structure; Thermodynamics; Work; analog; base; design; enzyme structure; fluorophore; inhibitor/antagonist; insight; mutant; non-nucleoside reverse transcriptase inhibitors; nucleoside analog; nucleoside inhibitor; polymerization; public health relevance; reaction rate (chemical); resistance mechanism; single molecule; stopped-flow fluorescence

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.

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以及耐药性进化的问题的核心。为了继续开发新药并最有效地使用现有药物，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该酶在进化过程中如何变化以提高对核苷的选择性至关重要类似物，同时保留正常核苷酸的足够有效的掺入。关于构象变化在促进 DNA 聚合酶以及其他酶的选择性方面的作用一直存在相当多的争论。新数据表明，构象转换决定 dNTP 是否会被掺入或拒绝。在本提案中，我们将研究 HIV RT 是否遵循这种新的 DNA 聚合酶选择性范例。在初步数据中，我们表明我们可以在手指结构域上用荧光团标记 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构变化率在合并之前。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区分核苷类似物中所起的作用。此外，我们将使用该信号来检查核苷酸结合和掺入动力学的变化，这是核苷类似物抗性的基础。我们还将探索非核苷抑制剂改变核苷酸结合动力学并减弱活性位点化学作用的机制。这些研究将通过结合动力学和结构方法来实现，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观察到的酶结构变化，这些变化被认为是对核苷类似物耐药的原因，并为治疗所需的新药物的设计和评估提供见解。艾滋病毒感染。公共卫生相关性：核苷酸选择性是了解用于治疗 HIV 感染的核苷类似物的有效性以及耐药性演变的核心问题。这项工作将定义通过 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负责核苷类似物耐药性的酶结构变化，并为管理 HIV 感染所需的新药物的设计和评估提供见解。