Novel mechanisms of HIV resistance to RTIs

HIV对RTIs耐药的新机制

• 批准号: 8079113
• 负责人: NICOLAS PAUL SLUIS-CREMER
• 金额: $ 32.71万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079113
• 关键词: Active Sites; Anti-Retroviral Agents; Area; Binding; Biochemical; Biological Assay; C-terminal; Chromosome Mapping; Clinical; Code; Combined Modality Therapy; DBL Oncoprotein; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; Distal; Drug resistance; Enzymes; Evolution; Failure; Future; Genotype; Goals; HIV; HIV-1; Health; Infection; Length; Link; Maps; Molecular; Molecular Models; Morbidity - disease rate; Mutation; Nucleic Acid Binding; Nucleosides; Patients; Phenotype; Plasma; Polymerase; RNA-Directed DNA Polymerase; Reaction; Research; Resistance; Reverse Transcriptase Inhibitors; Ribonuclease H; Role; Sampling; Site-Directed Mutagenesis; Structure-Activity Relationship; Testing; Therapeutic; Thymidine; Variant; Viremia; Virus; Zidovudine resistance; analog; antiretroviral therapy; clinical practice; clinically relevant; design; drug development; experience; inhibitor/antagonist; insight; molecular modeling; mortality; non-nucleoside reverse transcriptase inhibitors; novel; polymerization; prevent; recombinant virus; resistance mutation; virology

DESCRIPTION (provided by applicant): HIV-1 reverse transcriptase (RT) is a key target for antiretroviral drug development. To date, 12 RT inhibitors (RTIs) have been approved for the treatment of HIV-1 infection. These include the nucleoside/tide RT inhibitors (NRTI) that block HIV-1 replication by acting as chain-terminators of DNA synthesis, and the nonnucleoside RT inhibitors (NNRTI) that are allosteric inhibitors of HIV-1 RT DNA polymerization reactions. Although combination therapies that contain 2 or more RTI have reduced morbidity and mortality from HIV-1 infection, their long-term efficacy is limited by the selection of drug-resistant variants of HIV-1. A better understanding of the mechanisms involved is needed to prevent and manage drug resistance effectively. HIV-1 RT is a heterodimer composed of a 66kDa subunit (p66), and a p66-derived 51kDa subunit (p51). The catalytically active p66 subunit of RT consists of DNA polymerase (residues 1-315), connection (residues 316-427), and RNase H domains (residues 428-560). Most of the RTI resistance mutations identified to date map to the polymerase domain of RT. This is largely because the connection and RNase H domains have not been routinely analyzed in clinical samples. In fact, none of the genotyping assays available for patient management sequence the entire coding region of RT. However, a growing body of evidence has emerged that implicates mutations outside of the polymerase domain of RT in RTI resistance. For example, we were part of a multi-disciplinary study that identified the N348I mutation in the connection domain of RT that confers resistance to both NRTI and NNRTI. N348I is highly prevalent in RTI-experienced patients, occurs early in therapy (oftentimes before recognized polymerase domain mutations), and is associated with a greater increase in viremia than any of the recognized thymidine analog mutations that confer AZT resistance. In this application, we propose in-depth virology, biochemical and genotypic studies to determine the role of N348I and other candidate mutations in the C-terminal domains of HIV-1 RT in RTI resistance. This will be accomplished through 3 Specific Aims. In Aim 1, we will investigate the clinical relevance of mutations in the connection and RNase H domains by studying RTs in plasma samples from patients on RTI therapy. In Aim 2, we will elucidate the molecular mechanism(s) by which N348I and other clinically-relevant mutations in the C- terminal domains of HIV-1 RT confer NRTI and/or NNRTI resistance. These studies will provide novel insights into how the entire RT molecule (and not just the polymerase domain) functions to confer drug resistance. In Aim 3, we will combine structure-activity relationship studies with molecular modeling to gain structural insight into how mutations - that may be distal to the enzyme's active sites, nucleic acid binding tract or NNRTI- binding pocket - confer RTI resistance. In addition to providing new insights into the mechanisms of RTI resistance, the proposed studies could have important implications for the future design of genotype and phenotype tests for RTI resistance, and for identifying more effective RT inhibitors and inhibitor combinations. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine the role on N348I and other mutations in the connection and ribonuclease H domains of HIV-1 reverse transcriptase (RT) in RT inhibitor resistance. The results from this study will provide timely information on a rapidly emerging area of research in HIV-1 drug resistance that is likely to provide new mechanistic insights and to influence the design and interpretation of drug resistance assays used in clinical practice.

描述（由申请人提供）：HIV-1逆转录酶（RT）是抗逆转录病毒药物开发的关键靶点。迄今为止，已有12种RT抑制剂（RTI）被批准用于治疗HIV-1感染。这些包括通过作为DNA合成的链终止剂来阻断HIV-1复制的核苷/核苷酸RT抑制剂（NRTI），以及作为HIV-1 RT DNA聚合反应的变构抑制剂的非核苷RT抑制剂（NNRTI）。尽管含有2种或2种以上RTI的联合治疗可降低HIV-1感染的发病率和死亡率，但其长期疗效受到HIV-1耐药变异体选择的限制。需要更好地了解所涉及的机制，以有效预防和管理耐药性。HIV-1 RT是由66 kDa亚基（p66）和p66衍生的51 kDa亚基（p51）组成的异源二聚体。RT的催化活性p66亚基由DNA聚合酶（残基1-315）、连接（残基316-427）和RNA酶H结构域（残基428-560）组成。大多数的RTI耐药突变确定的日期映射到RT的聚合酶结构域。这在很大程度上是因为连接和RNase H结构域没有在临床样本中进行常规分析。事实上，没有一种基因分型检测可用于患者管理序列的RT的整个编码区。然而，越来越多的证据表明，RTI耐药的RT聚合酶结构域以外的突变。例如，我们参与了一项多学科研究，该研究确定了RT连接结构域中的N348 I突变，该突变赋予了对NRTI和NNRTI的抗性。N348 I在RTI患者中非常普遍，发生在治疗早期（通常在公认的聚合酶结构域突变之前），与任何公认的胸苷类似物突变（赋予AZT耐药性）相比，与病毒血症增加更大相关。在本申请中，我们提出了深入的病毒学，生物化学和基因型研究，以确定N348 I和其他候选突变的C-末端结构域的HIV-1 RT在RTI抗性的作用。这将通过三个具体目标来实现。在目标1中，我们将通过研究RTI治疗患者血浆样本中的RT来研究连接和RNA酶H结构域突变的临床相关性。在目标2中，我们将阐明N348 I和HIV-1 RT的C末端结构域中的其他临床相关突变赋予NRTI和/或NNRTI抗性的分子机制。这些研究将为整个RT分子（而不仅仅是聚合酶结构域）如何赋予耐药性提供新的见解。在目标3中，我们将结合联合收割机的结构-活性关系的研究与分子建模，以获得结构的洞察力，如何突变-可能是远端的酶的活性位点，核酸结合道或NNRTI-结合口袋-赋予RTI的阻力。除了对RTI耐药机制提供新的见解外，拟议的研究可能对RTI耐药基因型和表型测试的未来设计以及鉴定更有效的RT抑制剂和抑制剂组合具有重要意义。公共卫生相关性：本项目的目的是确定HIV-1逆转录酶（RT）连接区和核糖核酸酶H结构域的N348 I和其他突变在RT抑制剂耐药中的作用。这项研究的结果将提供有关HIV-1耐药性研究领域的及时信息，这可能会提供新的机制见解，并影响临床实践中使用的耐药性检测方法的设计和解释。