Biochemical Mechanisms of Drug Resistance in HIV RT

HIV RT耐药的生化机制

• 批准号: 7760783
• 负责人: WALTER A SCOTT
• 金额: $ 38.25万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760783
• 关键词: 3' -azido-3' -deoxythymidine 5' phosphate; Active Sites; Address; Affect; Binding; Biochemical; Cells; DNA; DNA biosynthesis; Development; Dissociation; Drug resistance; Effectiveness; Enzymes; Excision; HIV; HIV-1; In Vitro; Laboratories; Length; Lentivirus Vector; Life; Measures; Metabolic; Methods; Mutation; N-terminal; Nucleocapsid Proteins; Nucleosides; Nucleotides; Patients; Pharmaceutical Preparations; Positioning Attribute; Process; RNA; RNA-Directed DNA Polymerase; Reaction; Recombinant Proteins; Research; Resistance; Reverse Transcriptase Inhibitors; Ribonuclease H; Role; Site; Therapeutic; Thymidine; Time; Tissues; Viral; Virus; Work; Zidovudine; analog; drug discovery; drug efficacy; enzyme substrate complex; improved; in vivo; insight; mutant; nucleotide analog; public health relevance; reaction rate; research study; therapeutic target; viral DNA

DESCRIPTION (provided by applicant): Nucleoside reverse transcriptase (RT) inhibitors continue to be an important component of therapy against human immunodeficiency virus type 1 (HIV-1). These compounds are phosphorylated by cellular enzymes and are incorporated into DNA by HIV-1 RT leading to chain termination during viral DNA synthesis. The ability of mutants of HIV-1 RT to remove chain-terminating nucleotides from nascent DNA chains (excision activity) is an important mechanism of drug resistance, and mutants with elevated excision activity are often selected during therapy. We propose to develop an analytical approach to determine what influences excision activity in infected cells that will help us understand what drives mutant selection in vivo. Aim 1 is to use purified wild type and mutant HIV-1 recombinant proteins to evaluate the roles of RNA cleavage by the ribonuclease H activity of RT and template fragment dissociation in regulating excision rescue of blocked DNA chains. Aim 2 is to determine the effect of HIV nucleocapsid proteins on the release of secondary ribonuclease H cleavage fragments and excision of chain terminators. Aim 3 is to develop methods to measure intracellular excision activity and to use lentivirus vectors packaged with wild type or mutant RTs to evaluate factors that regulate excision in infected cells. The use of the excision reaction of HIV RT as a therapeutic target is limited by our lack of understanding of the intracellular reaction, including factors that influence timing and rate of the reaction, stability of the enzyme-substrate complexes that carry out secondary ribonuclease H cleavage and chain terminator excision, contribution of viral and cellular factors to these reactions, and accessibility of the viral replication machinery to acceptor substrates and inhibitory molecules. These questions will be addressed by the proposed studies. PUBLIC HEALTH RELEVANCE: Developoment of new therapies against HIV will continue to be necessary because of the rapid mutation rate of this virus that facilitates selection of drug resistance and because of the need to continue therapy in each patient over many years. Research is proposed to characterize intracellular processes that influence the ability of HIV to escape from a class of drugs that blocks viral DNA synthesis, which will provide insight into the potency of these drugs in different tissues and metabolic conditions, the tissue sites where selection of resistance mutants takes place, and how therapeutic strategies can be changed to optimize drug efficacy and avoid selection of resistant mutants.

描述（由申请人提供）：核苷逆转录酶（RT）抑制剂仍然是治疗人类免疫缺陷病毒1型（HIV-1）的重要组成部分。这些化合物被细胞酶磷酸化，并被HIV-1 RT整合到DNA中，导致病毒DNA合成过程中的链终止。HIV-1 RT突变体从新生DNA链中去除链终止核苷酸的能力（切除活性）是耐药性的重要机制，在治疗过程中经常选择具有高切除活性的突变体。我们建议开发一种分析方法来确定是什么影响了感染细胞的切除活性，这将有助于我们了解是什么驱动了体内的突变选择。目的1：利用纯化的野生型和突变型HIV-1重组蛋白，评估RT的核糖核酸酶H活性和模板片段解离对RNA的切割在调节阻断DNA链切除挽救中的作用。目的2是确定HIV核衣壳蛋白对次级核糖核酸酶H裂解片段的释放和链终止子的切除的影响。目的3是开发测量细胞内切除活性的方法，并使用野生型或突变型RTs包装的慢病毒载体来评估感染细胞中调节切除的因素。由于我们缺乏对细胞内反应的了解，包括影响反应时间和速度的因素，进行二次核糖核酸酶H切割和链终止器切除的酶-底物复合物的稳定性，病毒和细胞因子对这些反应的贡献，以及病毒复制机制对受体底物和抑制分子的可及性，将HIV RT切除反应作为治疗靶点的使用受到限制。这些问题将在拟议的研究报告中加以讨论。公共卫生相关性：由于这种病毒的快速突变率促进了耐药性的选择，并且由于需要在每个病人身上持续治疗多年，因此开发针对艾滋病毒的新疗法将继续是必要的。研究人员建议描述影响HIV逃离一类阻断病毒DNA合成的药物的能力的细胞内过程，这将有助于深入了解这些药物在不同组织和代谢条件下的效力，发生耐药性突变体选择的组织部位，以及如何改变治疗策略以优化药物功效并避免耐药性突变体的选择。