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种逆转录酶抑制剂(RTI)被批准用于治疗HIV-1感染。其中包括核苷/肽RT抑制剂(NRTI)，它通过作为DNA合成链的终止剂来阻止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。尽管包含2种或2种以上RTI的联合疗法降低了HIV-1感染的发病率和死亡率，但其长期疗效受到对HIV-1耐药变种的选择的限制。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-1RT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)和核糖核酸酶H区(残基428-560)组成。到目前为止发现的大多数RTI耐药突变都映射到RT的聚合酶区域。这在很大程度上是因为连接和RNaseH结构域没有在临床样本中进行常规分析。事实上，没有一种可用于患者管理的基因分型方法对RT的整个编码区进行了测序。然而，越来越多的证据表明RT聚合酶区域外的突变与RTI耐药性有关。例如，我们参与了一项多学科研究，该研究确定了RT连接区域中的N348I突变，该突变对NRTI和NNRTI都具有耐药性。N348I在有RTI经验的患者中高度流行，发生在治疗的早期(通常在可识别的聚合酶区域突变之前)，与导致AZT耐药性的任何已知的胸苷类似物突变相比，与病毒血症的增加有关。在这一应用中，我们建议进行深入的病毒学、生化和基因分型研究，以确定N348I和HIV-1RT C-末端结构域的其他候选突变在RTI耐药性中的作用。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RNaseH结构域突变的临床相关性。在目标2中，我们将阐明N348I和其他临床相关的HIV-1RT C端区突变导致NRTI和/或NNRTI耐药的分子机制(S)。这些研究将为整个RT分子(而不仅仅是聚合酶区域)如何产生耐药性提供新的见解。在目标3中，我们将把结构-活性关系研究与分子建模相结合，以获得结构上的洞察--可能位于酶活性位点、核酸结合区或NNRTI结合口袋的突变--如何赋予RTI耐药性。除了对RTI耐药机制提供新的见解外，拟议的研究还可能对未来RTI耐药的基因型和表型测试的设计以及寻找更有效的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)的连接和核糖核酸酶H区域在RT抑制剂耐药中的作用。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.