HIV-1 Integrase

HIV-1整合酶

• 批准号: 10486843
• 负责人: stephen h hughes
• 金额: $ 104.89万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10486843
• 关键词: 2019-nCoV; Acquired Immunodeficiency Syndrome; Active Sites; Anti-HIV Agents; Anti-Retroviral Agents; Binding; Binding Sites; Biological Assay; Biomedical Research; Blood; Cells; Chimeric Proteins; Chromatin; Chronic; Clonal Expansion; Collaborations; Complex; Crystallization; Cultured Cells; DNA; Data; Development; Diagnosis; Drug Kinetics; Drug resistance; Generations; Genes; Genome; Goals; HIV; HIV Infections; HIV Integrase; HIV-1; HIV-1 integrase; Human; Human Resources; Individual; Integrase; Integrase Inhibitors; Integration Host Factors; Legal patent; Life Cycle Stages; Ligand Binding Domain; Link; Macaca; Macaca mulatta; Maintenance; Maps; Modeling; Nucleotides; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Play; Process; Proteins; Proviruses; RNA; Reaction; Research; Resistance; Resources; Retroviridae; Role; SIV; Sampling; Spumavirus; Structure; Sulfonamides; Technology; Testing; Time; Tissues; Toxic effect; United States National Institutes of Health; Virion; Virus; Virus Replication; Weight; Work; antiretroviral therapy; gene therapy; improved; integration site; interest; mutant; nanomolar; pandemic disease; prototype; resistant strain; side effect; treatment strategy; viral DNA

The development of new, broadly effective anti-HIV drugs that have little or no toxicity is a high-priority NIH goal for AIDS research. The HIV-1 integration reaction proceeds in two steps, both of which are carried out by integrase (IN). In the first step (3' processing, or 3'P), IN removes a small number of nucleotides (usually 2) from the 3' ends of the linear viral DNA. In the second step, IN inserts the newly trimmed 3' ends of the viral DNA into host DNA; this reaction is called strand transfer, or ST. IN has only one active site that carries out both the 3'P and ST reactions. There are crystal structures of prototype foamy virus IN that show similarities and differences in the way that IN interacts with the viral DNA substrate in the 3'P and ST reactions. Because integration is an essential step in the virus life cycle, IN is an important target for antiretroviral drugs. The four approved anti-IN drugs (raltegravir, elvitegratir, dolutegravir, and bictegravir) all target the ST reaction, and are, for this reason, sometimes called INSTIs. Despite their relatively recent development, INSTIs are potent drugs with few side effects and are becoming increasingly important in antiretroviral therapy (ART). Cabotegravir has been formulated with rilpivirine and this combination has shown promise in long-term treatment strategies. However, as is the case with all anti-HIV drugs, INSTIs select for resistant strains of HIV. We are focusing on developing new anti-IN compounds that show little or no toxicity, and are broadly effective against the known drug-resistant mutants. We have made excellent progress in developing IN inhibitors that have low nanomolar potency in a one-round assay, retain potency against a broad panel of resistant mutants, and show little or no toxicity in cultured cells. Although we continue to make and test IN inhibitors, we have made sufficient progress that we have, with help from the NCI, begun to do pharmacokinetic testing on the best of our compounds. ____There are millions of retroviral integration sites in the host cell genome, but for many retroviruses, integration is far from random; we have been studying how HIV selects its integration sites. In the last few years, we have become increasingly interested in understanding what governs the distribution of HIV integration sites, both in cultured cells and in samples from patients. In the work that was done in cultured cells, we were part of a collaboration that investigated the ability of the host protein HRP2 to replace LEDGF in directing HIV integration to the bodies of highly expressed genes, and we showed that the host factor CPSF6 plays a key role in guiding the preintegration complex to regions of the genome that are gene rich and contain numerous highly expressed genes. LEDGF is a bipartite protein, in which the C-terminus binds IN and the N-terminus binds chromatin. We and others have used integration site analysis to show that there is extensive clonal expansion of HIV-infected cells in patients on ART. We showed in HIV-infected individuals on ART, that more than 40% of the infected cells are in clones and that, in some cases, the integration sites can contribute to this expansion. We also showed that highly expanded clones can carry infectious proviruses, and release virions into the blood. Thus, clonal expansion of infected cells can contribute to the reservoir that has made it impossible to cure HIV infections with the currently available drugs. Although it has been proposed that viral replication plays an essential role in persistence and in the maintenance of the reservoir, even in fully compliant patients on successful ART, we think that the weight of the evidence supports the idea that there are patients in which ART completely blocks viral replication. Thus, it is important to understand the processes that allow infectious proviruses to persist even if viral replication is completely blocked. ____Given the importance of clonal expansion in HIV persistence, we determined how soon after HIV acquisition infected clones can grow large enough to be detected (clones larger than ca.105 cells). We studied 12 individuals sampled in early HIV infection (Fiebig stage III-V/VI) and 5 who were chronically infected. The recently infected individuals were started on ART at or near the time of diagnosis. We isolated more than 6500 independent integration sites from peripheral blood mononuclear cells (PBMC) before ART was initiated and after 0.5-18 years of suppressive ART. Some clones of infected cells could be detected approximately 4 weeks after HIV infection and some of these clones persisted for years. The results help to explain how the reservoir is established early and persists for years. ____Our data provide a better understanding of the generation, maintenance, and persistence of the reservoir that has made it impossible to cure patients with the available anti-HIV drugs. However, there are limitations to the samples that can be obtained from HIV-infected patients. For that reason, we developed a simian immunodeficiency virus (SIV)/rhesus macaque model in collaboration with Dr. Jeffrey Lifson (Leidos Biomedical Research, Inc.) and showed that SIV-infected cells can clonally expand in infected macaques. The technology that we use to isolate large numbers of HIV integration sites was developed as part of the project in which we use redirected HIV integration to map the binding sites of host proteins, both WT and mutant, on chromatin. Although this HIT-Seq technology works well and has allowed us to answer some key questions about where and how important host factors bind to chromatin, we have redirected the personnel and resources that were dedicated to the HIT-Seq project to projects in which we are mapping HIV integration sites in patients and SIV integration sites in macaques. ____ In a collaborative project, we showed that reverse-transcribed SARS-CoV-2 RNA can be integrated into the genomes of cultured human cells. We also presented indirect evidence which suggests that DNA copies of SARS-CoV2 DNA can be integrated in a small number of cells in some patient-derived tissues._____PATENTS LINKED TO THIS PROJECT: (1) Zhao XZ, Smith S, Metifiot M, Johnson B, Marchand C, Hughes SH, Pommier Y, Burke TR Jr: Compounds for Inhibiting Drug-Resistant Strains of HIV-1 Integrase. U.S. Patent #9,676,771 B2, issued June 13, 2017. (2) Zhao XZ, Smith S, Metifiot M, Johnson B, Marchand C, Hughes SH, Pommier Y, Burke TR Jr: Compounds for Inhibiting Drug-Resistant Strains of HIV-1 Integrase. U.S. Patent #10,208,305, issued February 19, 2019. (3) Burke TR, Hughes SH, Johnson B, Pommier Y, Smith SJ, Vu B, Zhao XZ (submitted in 2011): HIV Integrase Inhibitory Oxoisoindoline Sulfonamides. Patent pending: PCT/US2012/048169 (PC application). (4) Zhao XZ, Burke TR, Hughes SH, Johnson B, Marchand CR, Metifiot MA, Pommier Y, Smith SJ (submitted in 2013): Hydroxylamide-Containing Compounds with Improved Efficacy against Raltegravir-Resistant Strains of HIV-1 Integrase. Tracking number: E-093-2013/0-US-01 (US application).

几乎没有毒性或没有毒性的新的，广泛有效的抗HIV药物的发展是艾滋病研究的高优先级NIH目标。 HIV-1整合反应以两个步骤进行，这两步均由整合酶（IN）进行。在第一步（3'加工或3'p）中，从线性病毒DNA的3'末端去除少量核苷酸（通常为2）。在第二步中，在将病毒DNA的新修剪3'末端插入到宿主DNA中；该反应称为链转移或st。在IN只有一个活跃的位点可以同时执行3'P和ST反应。有原型泡沫病毒的晶体结构在与3'p和ST反应中与病毒DNA底物相互作用的方式上显示出相似性和差异。由于整合是病毒生命周期中必不可少的一步，因此IN是抗逆转录病毒药物的重要靶标。四种批准的抗内毒药（Raltegravir，Elvitegratir，Dolutegravir和Bictegravir）均针对ST反应，因此，有时被称为Instis。尽管它们的发展相对较新，但研究所是有效的药物，副作用很少，并且在抗逆转录病毒疗法（ART）中变得越来越重要。 Cabotegravir已配制了Rilpivirine，这种组合在长期治疗策略中表现出了希望。但是，与所有抗HIV药物一样，研究所选择HIV的抗性菌株。我们专注于开发几乎没有毒性或没有毒性的新型抗内部化合物，并且对已知的抗药性突变体具有广泛有效的作用。我们在单轮测定中具有低纳摩尔效力的抑制剂中发展，对广泛的抗性突变体保持效力，并且在培养细胞中几乎没有毒性。尽管我们继续在抑制剂中进行和测试，但我们取得了足够的进步，在NCI的帮助下，我们开始在我们的最佳化合物上进行药代动力学测试。 ____是宿主细胞基因组中数百万个逆转录病毒整合位点，但对于许多逆转录病毒，整合远非随机。我们一直在研究HIV如何选择其整合位点。在过去的几年中，我们对了解培养细胞和患者样本中的艾滋病毒整合位点分布的原因变得越来越感兴趣。在培养细胞中完成的工作中，我们是一项合作的一部分，该合作研究了宿主蛋白HRP2取代LedGF将HIV整合引导到高度表达基因的身体中的能力，我们表明，宿主因子CPSF6在指导基因组的前基因组中具有许多高度表达基因的基因组的预聚综合体中起着关键作用。 LEDGF是一种两分蛋白，其中C末端结合在其中，N末端结合染色质。我们和其他人使用整合位点分析表明，ART患者的HIV感染细胞有广泛的克隆扩张。我们在HIV感染的ART的个体中表明，超过40％的感染细胞在克隆中，在某些情况下，整合位点可以有助于这种扩张。我们还表明，高度扩展的克隆可以携带感染性病毒，并将病毒体释放到血液中。因此，受感染细胞的克隆扩张可以促进储层，这使得无法用当前可用的药物治愈HIV感染。尽管已经提出，病毒复制在持久性和维持储层中起着至关重要的作用，即使在完全合规的成功艺术患者中，我们认为证据的重量也支持这样一种观念，即有一些患者完全阻止病毒复制。因此，重要的是要了解允许传染病病毒持续存在的过程，即使病毒复制被完全阻止。 ____赋予克隆膨胀在HIV持久性中的重要性，我们确定了艾滋病毒获取后的克隆后多久能够生长足够大以至于被检测到（克隆大于Ca.105细胞）。我们研究了12个人在早期的HIV感染（Fiebig III-V/VI期）和5​​例长期感染的人中采样。最近感染的个体是在诊断时或接近诊断时开始从事艺术的。我们在启动ART之前和0.5 - 18年的抑制性艺术后，我们从外周血单核细胞（PBMC）中分离了6500多个独立的整合位点。艾滋病毒感染后约4周，可以检测到一些感染细胞的克隆，其中一些克隆持续了多年。结果有助于解释如何尽早建立储层并持续多年。 ____库提供了对水库的产生，维护和持久性的更好理解，这使得无法治愈患者使用可用的抗HIV药物。但是，可以从HIV感染的患者那里获得的样本存在局限性。因此，我们与Jeffrey Lifson博士（Leidos Biomedical Research，Inc。）合作开发了猿猴免疫缺陷病毒（SIV）/恒河猴模型，并表明SIV感染的细胞可以在受感染的猕猴中延伸。我们用来隔离大量HIV整合位点的技术是在该项目的一部分中开发的，在该项目中，我们使用重定向的HIV积分在染色质上绘制WT和突变体的宿主蛋白的结合位点。尽管该HIT-SEQ技术效果很好，并使我们能够回答有关宿主因素与染色质有关的重要问题，但我们已经将专门针对HIT-Seq项目的人员和资源重定向到我们正在Macaques中的患者和SIV集成站点中绘制HIV集成站点的项目。 ____在一个协作项目中，我们表明可以将反向转录的SARS-COV-2 RNA整合到培养的人类细胞的基因组中。 We also presented indirect evidence which suggests that DNA copies of SARS-CoV2 DNA can be integrated in a small number of cells in some patient-derived tissues._____PATENTS LINKED TO THIS PROJECT: (1) Zhao XZ, Smith S, Metifiot M, Johnson B, Marchand C, Hughes SH, Pommier Y, Burke TR Jr: Compounds for Inhibiting Drug-Resistant Strains of HIV-1 Integrase.美国专利＃9,676,771 B2，2017年6月13日发行。（2）Zhao XZ，Smith S，Metifiot M，Johnson B，Marchand C，Hughes SH，Hughes SH，Pommier Y，Burke TR JR：用于抑制HIV-1集成酶抑制药物抗药性的化合物。美国专利＃10,208,305，于2019年2月19日颁发。（3）Burke TR，Hughes SH，Johnson B，Pommier Y，Smith SJ，Vu B，Zhao XZ（2011年提交）：HIV集成酶抑制氧化酶氧异氧释糖苷磺氨硫酰胺。专利待处理：PCT/US2012/048169（PC应用程序）。 （4）Zhao XZ，Burke TR，Hughes SH，Johnson B，Marchand CR，Metifiot MA，Pommier Y，Smith SJ（2013年提交）：含羟基酰胺的化合物，具有提高疗效的HIV-1集成酶抗性菌株的功效。跟踪号：E-093-2013/0-US-01（US应用程序）。