Understanding potent and novel small molecules that target HIV assembly

了解针对 HIV 组装的有效且新颖的小分子

• 批准号: 10077434
• 负责人: JAISRI R LINGAPPA
• 金额: $ 30.89万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10077434
• 关键词: Affinity Chromatography; Anti-Retroviral Agents; Antibodies; Antiviral Agents; Binding; Biochemical; Biological Assay; Biotin; Capsid; Cell Line; Chemicals; Chemistry; Complex; Data; Development; Drug Screening; Drug Targeting; Drug resistance; Enzymes; Event; Funding; Future; Goals; HIV; HIV Infections; HIV-1; HIV-1 drug resistance; HIV-2; Health; Human; Image; Imaging Techniques; In Situ; Infection; Lead; Life Cycle Stages; Ligation; Mass Spectrum Analysis; Modification; Morbidity - disease rate; Multi-Drug Resistance; Mutation; Parents; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Prevalence; Prevention; Production; Proteins; Resistance; Retroviridae; Risk; SIV; Structure-Activity Relationship; T-Lymphocyte; Techniques; Testing; Treatment Failure; Variant; Viral; Virus; analog; antiretroviral therapy; chemical group; crosslink; drug development; drug resistant virus; experimental study; follow-up; gag Gene Products; imaging study; in situ imaging; inhibitor/antagonist; mortality; nanomolar; new therapeutic target; novel; novel therapeutics; pre-exposure prophylaxis; prevent; reconstitution; small molecule; small molecule inhibitor; success; tool; viral resistance; virology

SUMMARY: Antiretroviral drugs are critical for the survival of the ~38 million people who are currently living with HIV-1 infection. These drugs have also been used as pre-exposure prophylaxis to prevent HIV-1 infection; in addition, they are a mainstay of possible HIV-1 cure strategies. However, development of drug resistance threatens to undermine these successes that are critical to the health of many millions of people worldwide. For these reasons, discovery of new antiretroviral targets and compounds that inhibit these targets is critical. Since key HIV-1 enzymes are already targeted by drugs in current use, targets of the future will need to come from poorly understood aspects of the viral life cycle, such as intracellular late events in the viral life cycle, which are critical for virus production. Two decades of studying these events led to identification of a host- catalyzed pathway of putative capsid assembly intermediates that contain the HIV-1 Gag protein and host enzymes. A drug screen that reconstituted this assembly pathway led to discovery of PAV117, an antiretroviral small molecule, and subsequently a more potent analog, PAV206. Preliminary data in the current proposal demonstrate that PAV206 blocks virus production at nanomolar concentrations in T cell lines and HIV-1 infected PBMCs, making it the first potent and selective inhibitor of intracellular late events. Imaging experiments demonstrate that PAV206 colocalizes with the viral protein Gag and also with a host enzyme present in HIV-1 capsid assembly intermediates suggesting that this small molecule targets one or more components of HIV-1 capsid assembly intermediates. This proposal seeks to 1) determine which retroviruses are inhibited by PAV206, 2) identify mutations in HIV-1 that confer PAV206 resistance, and 3) use imaging experiments to determine if PAV206 localizes to a distinct subcellular complex, and 4) use biochemical approaches to probe for PAV206 binding partners. The subcellular localization and binding partner studies will take advantage of PAV206 analogs that are available to us, including one that maintains antiviral activity but contains a biotin tag for antibody recognition and a chemical group for photo-crosslinking. We provide extensive data demonstrating how this analog can be used to great advantage for in situ imaging using the proximity ligation assay (PLA). We also propose to extend these imaging studies using a cutting-edge variation of this technique termed multiplex PLA. Finally, we describe a second analog of PAV206 that allows other tags to be added through click chemistry, thereby expanding options for affinity purification of target proteins and mass spectrometry analysis. With this impressive array of tools, our likelihood of successfully identifying the PAV206 target is high. In conclusion, by advancing our understanding of a novel small molecule inhibitor of HIV-1 late events, studies proposed here will provide an exciting tool for studying HIV-1 assembly and a springboard for generating compounds that could be advanced in the future.

概括： 抗逆转录病毒药物对于目前约 3800 万 HIV-1 感染者的生存至关重要 感染。这些药物还被用作暴露前预防，以预防 HIV-1 感染；在 此外，它们是可能的 HIV-1 治愈策略的支柱。然而，耐药性的发展 这些成功对于全世界数百万人的健康至关重要。 由于这些原因，发现新的抗逆转录病毒靶点和抑制这些靶点的化合物至关重要。 由于关键的 HIV-1 酶已经成为当前使用的药物的目标，因此未来的目标将需要出现 来自对病毒生命周期知之甚少的方面，例如病毒生命周期中的细胞内晚期事件， 这对于病毒的产生至关重要。对这些事件进行了二十年的研究，最终确定了一个宿主—— 含有 HIV-1 Gag 蛋白和宿主的假定衣壳组装中间体的催化途径 酶。重建这条组装途径的药物筛选导致了 PAV117 的发现，这是一种抗逆转录病毒药物 小分子，随后是更有效的类似物 PAV206。当前提案中的初步数据 证明 PAV206 在 T 细胞系和 HIV-1 中以纳摩尔浓度阻断病毒产生 感染 PBMC，使其成为第一个有效的、选择性的细胞内晚期事件抑制剂。影像学 实验表明 PAV206 与病毒蛋白 Gag 以及宿主酶共定位 存在于 HIV-1 衣壳组装中间体中，表明该小分子靶向一个或多个 HIV-1衣壳组装中间体的成分。该提案旨在 1) 确定哪些逆转录病毒 被 PAV206 抑制，2) 识别 HIV-1 中赋予 PAV206 耐药性的突变，以及 3) 使用成像 确定 PAV206 是否定位于不同的亚细胞复合体的实验，以及 4) 使用生化 探测 PAV206 结合伴侣的方法。亚细胞定位和结合伙伴研究将 利用我们现有的 PAV206 类似物，包括一种保持抗病毒活性但 包含用于抗体识别的生物素标签和用于光交联的化学基团。我们提供 大量数据证明了如何使用该模拟来进行原位成像 邻近连接分析（PLA）。我们还建议使用尖端技术扩展这些成像研究 该技术的变体称为多重 PLA。最后，我们描述了 PAV206 的第二个类似物，它允许 通过点击化学添加其他标签，从而扩展目标亲和纯化的选项 蛋白质和质谱分析。有了这一系列令人印象深刻的工具，我们成功的可能性 识别 PAV206 目标较高。总之，通过增进我们对新型小分子的理解 HIV-1 晚期事件抑制剂，此处提出的研究将为研究 HIV-1 组装提供令人兴奋的工具 以及生成未来可能先进的化合物的跳板。