Novel mechanisms of HIV resistance to RTIs

HIV对RTIs耐药的新机制

基本信息

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
项目类别：
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

项目摘要

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目标。迄今为止，已批准了12个RT抑制剂（RTI）用于治疗HIV-1感染。其中包括通过充当DNA合成的链末端来阻断HIV-1复制的核苷/潮汐RT抑制剂，而非核苷RT RT抑制剂（NNRTI）则是HIV-1 RT DNA聚合反应的变构抑制剂。尽管包含2种或更多RTI的组合疗法降低了HIV-1感染的发病率和死亡率，但它们的长期疗效受HIV-1的抗药性变体的选择受到限制。需要更好地了解所涉及的机制，以预防和管理耐药性。 HIV-1 RT是由66KDA亚基（P66）和P66衍生的51KDA亚基（p51）组成的异二聚体。 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RNase H结构域（残基428-560）组成。大多数RTI电阻突变鉴定为日期映射到RT的聚合酶结构域。这主要是因为尚未在临床样本中常规分析连接和RNase H结构域。实际上，没有用于患者管理序列RT整个编码区域的基因分型测定。然而，越来越多的证据出现了，这意味着RT抗性中RT聚合酶结构域之外的突变。例如，我们是一项多学科研究的一部分，该研究确定了RT连接域中的N348i突变，该突变赋予了对NRTI和NNRTI的抗性。 N348i在经验丰富的患者中高度普遍，在治疗早期发生（通常是在公认的聚合酶结构域突变之前），并且与赋予AZT耐药性的任何公认的胸苷类似物突变相比，病毒血症的增加相比。在此应用中，我们提出了深入的病毒学，生化和基因型研究，以确定N348I和其他候选突变在HIV-1 RT的C末端突变在RTI抗性中的作用。这将通过3个特定目标来实现。在AIM 1中，我们将通过研究RTI治疗患者的血浆样本中的RTS来研究突变在连接和RNase H结构域中的临床相关性。在AIM 2中，我们将阐明N348i和其他与HIV-1 RT CONTER NRTI和/或NNRTI抗性的C-末端域中N348I和其他与临床相关的突变的分子机制。这些研究将提供有关整个RT分子（不仅是聚合酶结构域）如何赋予耐药性的新见解。在AIM 3中，我们将结合结构 - 活性关系研究与分子建模相结合，以获得结构性的洞察力，以了解突变的结构 - 可能是该酶的活性位点，核酸结合道或NNRTI-结合口袋的远端 - 会产生RTI抗性。除了提供对RTI抗性机制的新见解外，所提出的研究还可能对RTI抗性的基因型和表型测试的未来设计具有重要意义，并确定更有效的RT抑制剂和抑制剂组合。公共卫生相关性：该项目的目的是确定HIV-1逆转录酶（RT）在RT抑制剂抗性中的连接和核糖核酸酶H结构域中的作用。这项研究的结果将及时提供有关HIV-1耐药性研究的快速新兴领域的信息，这些信息可能会提供新的机械见解并影响临床实践中使用的药物耐药性测定的设计和解释。