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患有HIV-1的3800万人的生存至关重要 感染。这些药物也已被用作预防前预防，以防止HIV-1感染。在 此外，它们是可能的HIV-1治疗策略的支柱。但是，耐药性的发展 威胁要破坏这些成功对全球数百万人的健康至关重要的成功。 由于这些原因，发现抑制这些靶标的新的抗逆转录病毒靶标和化合物至关重要。 由于关键的HIV-1酶已经是当前用途的药物的目标，因此未来的目标将需要到 从病毒生命周期的知识较低的各个方面，例如病毒生命周期中的细胞内晚期事件， 这对于病毒生产至关重要。研究这些事件的二十年导致了宿主的识别 - 推定的衣壳组件中间体的催化途径，其中包含HIV-1 GAG蛋白和宿主 酶。重建该组装途径的药物屏幕导致发现PAV117，抗逆转录病毒 小分子，然后是一个更有效的类似物PAV206。当前建议中的初步数据 证明PAV206在T细胞系和HIV-1中以纳摩尔浓度阻止病毒产生 感染的PBMC，使其成为细胞内晚期事件的第一个有效和选择性抑制剂。成像 实验表明，PAV206与病毒蛋白GAG共定位，并与宿主酶共定位 在HIV-1 CAPSID组件中间存在​​中间体，这表明该小分子靶向一个或多个 HIV-1衣壳组件中间体的组件。该建议寻求1）确定哪些逆转录病毒 被PAV206抑制，2）识别HIV-1中赋予PAV206电阻的突变，3）使用成像 实验确定PAV206是否本地化为不同的亚细胞复合物，4）使用生化 探测PAV206绑定伙伴的方法。亚细胞定位和结合伙伴研究将 利用我们可用的PAV206类似物，包括维持抗病毒活动但 包含用于抗体识别的生物素标签和用于光合链接的化学组。我们提供 广泛的数据，说明如何使用此模拟来使用该模拟来实现原位成像 接近连接测定法（PLA）。我们还建议使用尖端扩展这些成像研究 该技术的变化称为多重PLA。最后，我们描述了PAV206的第二个类似物，该模拟允许 通过点击化学添加的其他标签，从而扩展了目标净化目标的选项 蛋白质和质谱分析。有了这些令人印象深刻的工具，我们成功的可能性 识别PAV206目标很高。总之，通过促进我们对新型小分子的理解 HIV-1后期事件的抑制剂，此处提出的研究将为研究HIV-1组装提供令人兴奋的工具 以及用于生成将来可能采用的化合物的跳板。