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）：核苷酸的选择性是理解用于治疗HIV感染的核苷类似物的有效性的核心，以及抗药性的演变。为了继续开发新药并最有效地使用可用的药物，了解HIV逆转录酶（RT）在DNA聚合过程中具有核苷酸选择性的机制至关重要。关于构象变化在促进DNA聚合酶以及其他酶的选择性中的作用的争论。新数据表明，构象开关决定了是否将合并或拒绝DNTP。在此提案中，我们将研究HIV RT是否遵循这种新的DNA聚合酶选择性范式。在初步数据中，我们表明，我们可以在手指域上用荧光团标记HIV RT的位置，该位置提供了信号，以监视核苷酸结合的构象变化，并且我们提供数据以定义核苷酸诱导的结构变化的速率，该结构变化的结构变化均已结合。我们将利用这个新信号来建立通过HIV RT来控制选择性的反应途径，并定义酶构象变化对核苷类似物的作用。此外，我们将使用该信号来检查核苷酸结合和掺入核苷类似物的动力学的变化。我们还将探讨非核苷抑制剂改变核苷酸结合和衰减化学在活性位点的机制。这些研究将使用动力学和结构方法的组合，包括停止流量荧光，快速的化学淬灭流和单分子荧光动力学研究。这项工作将通过HIV RT更好地定义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的抗性的酶结构的变化，并为管理HIV感染所需的新药物提供了洞察力。公共卫生相关性：核苷酸的选择性是理解用于治疗HIV感染的核苷类似物的有效性的核心，以及抗药性的演变。这项工作将通过HIV RT定义控制核苷酸选择性的基本步骤，使我们能够更严格地解释导致对核苷类似物的抗性的酶结构的变化，并为管理HIV感染所需的新药物的设计和评估提供见解。