Novel mechanisms of HIV resistance to RTIs

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（RTIs）被批准用于治疗HIV-1感染。其中包括核苷/潮汐RT抑制剂（NRTI），它通过作为DNA合成的链终止子来阻断HIV-1的复制，以及非核苷RT抑制剂（NNRTI），它是HIV-1 RT DNA聚合反应的变构抑制剂。尽管含有2种或更多RTI的联合治疗降低了HIV-1感染的发病率和死亡率，但其长期疗效受到HIV-1耐药变体选择的限制。为了有效地预防和管理耐药性，需要更好地了解所涉及的机制。HIV-1 RT是由66kDa亚基（p66）和p66衍生的51kDa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RNase H结构域（残基428-560）组成。迄今为止发现的大多数RTI耐药突变都与rt的聚合酶结构域有关。这主要是因为在临床样本中没有常规分析连接和RNase H结构域。事实上，没有一种可用于患者管理的基因分型分析对RT的整个编码区进行测序。然而，越来越多的证据表明，在RTI耐药性中，RT的聚合酶结构域外的突变可能存在。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，该突变赋予对NRTI和NNRTI的抗性。N348I在有rti经历的患者中非常普遍，发生在治疗早期（通常在公认的聚合酶结构域突变之前），并且与任何公认的胸苷类似物突变（赋予AZT耐药性）相比，病毒血症的增加更大。在这项申请中，我们提出深入的病毒学、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端域在RTI抗性中的作用。这将通过三个具体目标来实现。在Aim 1中，我们将通过研究接受RTI治疗的患者血浆样本中的RTs来研究连接和RNase H结构域突变的临床相关性。在Aim 2中，我们将阐明N348I和HIV-1 RT C端域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）如何发挥作用以赋予耐药性提供新的见解。在Aim 3中，我们将结合结构-活性关系研究与分子建模，以获得结构上的洞察如何突变-可能是远端的酶的活性位点，核酸结合束或NNRTI结合口袋-赋予RTI抗性。除了为RTI耐药机制提供新的见解外，所提出的研究可能对未来设计RTI耐药的基因型和表型测试，以及确定更有效的RT抑制剂和抑制剂组合具有重要意义。公共卫生相关性：该项目的目标是确定N348I和其他突变在HIV-1逆转录酶（RT）连接和核糖核酸酶H结构域中的作用。这项研究的结果将为HIV-1耐药性这一迅速出现的研究领域提供及时的信息，这可能提供新的机制见解，并影响临床实践中使用的耐药性分析的设计和解释。

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.