Novel mechanism of integrase (IN) resistance to Dolutegravir through epistatic interactions between IN and the nucleocapsid and polypurine tract regions of HIV-1

通过 IN 与 HIV-1 核衣壳和多嘌呤束区域之间的上位相互作用，整合酶 (IN) 对 Dolutegravir 产生耐药性的新机制

• 批准号: 10348121
• 负责人: Atsuko Hachiya
• 金额: $ 50.7万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10348121
• 关键词: Active Sites; Address; Affect; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Catalytic Domain; Clinical; Clinical Trials; Colorado; Complex; Conflict (Psychology); Cryoelectron Microscopy; DNA; Data; Data Set; Dinucleoside Phosphates; Drug Targeting; Drug resistance; Excision; FDA approved; Failure; Generations; Genes; Genetic; Genetic Polymorphism; Goals; HIV; HIV Integrase; HIV therapy; HIV-1; HIV-1 drug resistance; HIV-1 integrase; In Vitro; Individual; Induced Mutation; Infection; Institutes; Integrase; Integrase Inhibitors; Knowledge; Left; Long Terminal Repeats; Modeling; Molecular; Mutation; Nuclear; Nucleocapsid; Patients; Pharmaceutical Preparations; Predisposition; Process; Property; Reaction; Regimen; Reporting; Resistance; Resolution; Ribonuclease H; Serial Passage; Series; Testing; Time; Validation; Virus; Virus Replication; World Health Organization; Zinc Fingers; base; clinical effect; deep sequencing; design; efavirenz; effective therapy; experimental study; improved; inhibitor; innovation; insertion/deletion mutation; low and middle-income countries; mutant; novel; particle; recombinant virus; resistance mechanism; resistance mutation; sound; tool; treatment strategy; viral DNA; viral fitness; viral resistance; virology

PROJECT SUMMARY Integration is essential for HIV-1 replication and is completed by integrase (IN). A class of drugs which inhibit the strand transfer (ST) function of HIV integrase, called IN strand transfer inhibitors (INSTIs), includes approved drugs raltegravir (RAL), elvitegravir (EVG) (1st generation) and dolutegravir (DTG), bictegravir (BIC) (2nd generation). DTG has a higher genetic barrier to resistance than RAL or EVG, and is recommended by the World Health Organization as an alternative to efavirenz in first-line regimens in low- and middle-income countries (LMICs). Selection for DTG resistance is rare, but does exist and is currently not well understood. There is mounting evidence for failure of DTG-based treatment in clinical trials (VIKING-3 study) in the absence of mutations in the targeted IN gene. Our overarching hypothesis is that mutations outside IN can impart drug resistance to IN-targeting drugs through indirect interactions that we call epistatic. The scientific premise for studying these interactions is soundly grounded on two key pieces of evidence: First, in surprising preliminary data from in vitro serial passage experiments in the presence of increasing amounts of DTG, a DTG resistance mutation located outside IN was discovered. Experiments with recombinant viruses validated DTG resistance of this mutation and showed enhanced resistance in the presence of the E157Q IN polymorphism. Moreover, deep sequencing analyses showed that compared to infection by wild-type, mutant–containing viruses resulted in more insertions, deletions, and non-canonical long terminal repeat (LTR) ends in 2-LTR circles and integrated viral DNA. Second, a recent independent study based on similar serial passage experiments, identified changes at the general G-tract region of the 3’-polypurine tract (3’-PPT) in a DTG-resistant virus (Malet et al., 2017). Subsequently, different 3’-PPT changes were reported in a patient that failed DTG therapy. However, the mechanism of DTG resistance through mutations at the 3’-PPT remains unclear due to conflicting hypotheses and lack of experimental validation. Our hypothesis is that mutations outside IN can affect DTG resistance by altering the LTR ends at the INSTI binding site. This hypothesis will be tested by a team of experts that includes PIs Sarafianos (biochemical, virological drug resistance mechanisms), PI Lyumkis (single particle cryo-EM on intasome/drug complexes) and PI Hachiya (virology, drug resistance) with the support by HIV IN experts Hughes (NCI) and Kvaratskhelia (U Colorado), using virological, biochemical, and structural tools to address the aims to investigate the virological, biological, and structural mechanisms of DTG resistance through epistatic interactions. These innovative studies will help elucidate the molecular mechanisms of INSTI resistance through epistatic interactions via mutations that are outside the IN gene and affect the INSTI-binding site. They are significant and will help explain clinical failures to DTG-based regimens in the absence of mutations in IN.

项目摘要 整合对于HIV-1复制是必不可少的，并由整合酶（IN）完成。一类药物，抑制 HIV整合酶的链转移（ST）功能，称为IN链转移抑制剂（INSTI），包括已批准的 药物雷特格韦（RAL）、埃替格韦（EVG）（第一代）和度鲁特韦（DTG）、比替格韦（BIC）（第二代 代）。DTG比RAL或EVG具有更高的耐药遗传屏障，被世界推荐使用 卫生组织在低收入和中等收入国家一线治疗中作为依法韦仑的替代品 （中低收入国家）。DTG抗性的选择是罕见的，但确实存在，目前还没有很好的理解。有 越来越多的证据表明，在缺乏以下因素的情况下，临床试验（维京海盗-3研究）中基于DTG的治疗失败 目标IN基因中的突变。我们的首要假设是，IN以外的突变可以赋予药物 通过我们称之为上位性的间接相互作用对IN靶向药物产生耐药性。科学前提是 研究这些相互作用基于两个关键证据：第一，令人惊讶的初步证据。 来自在增加量的DTG存在下的体外连续传代实验的数据，DTG抗性 发现了位于IN之外的突变。重组病毒的实验验证了 E157 Q IN多态性的存在下，该突变显示出增强的抗性。而且，深 测序分析表明，与野生型感染相比，含突变体的病毒导致 更多的插入、缺失和非典型的长末端重复序列（LTR）以2-LTR环结束，并整合在一起。 病毒DNA其次，最近一项基于类似连续传代实验的独立研究， 在DTG-抗性病毒中3 ′-聚嘌呤段（3 ′-PPT）的一般G-段区域（Malet等，2017年）。 随后，在DTG治疗失败的患者中报告了不同的3 '-PPT变化。但 由于相互矛盾的假设，通过3 '-PPT突变产生DTG抗性的机制仍不清楚 缺乏实验验证。我们的假设是，IN以外的突变可以通过以下方式影响DTG耐药性： 改变LTR末端的FIGI结合位点。这一假设将由一个专家小组进行测试， PI Sarafianos（生物化学、病毒学耐药机制）、PI Lyumkis（单颗粒冷冻EM， Intasome/药物复合物）和PI Hachiya（病毒学，耐药性），由HIV IN专家Hughes (NCI)和Kvaratskhelia（U科罗拉多），使用病毒学，生物化学和结构工具，以解决的目标， 通过上位性研究DTG耐药的病毒学、生物学和结构机制 交互.这些创新性的研究将有助于阐明耐药的分子机制， 通过IN基因外的突变并影响INSTI结合位点的上位相互作用。他们是 显著，并将有助于解释临床失败的DTG为基础的方案在缺乏突变的IN。