Understanding potent and novel small molecules that target HIV assembly

摘要： 抗逆转录病毒药物对目前约3800万艾滋病毒携带者的生存至关重要 感染。这些药物也被用作暴露前预防，以防止艾滋病毒-1感染； 此外，它们是可能的HIV-1治疗策略的中流砥柱。然而，耐药性的发展 有可能破坏这些对全世界数百万人的健康至关重要的成功。 出于这些原因，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靶点，未来的靶点将需要出现 从鲜为人知的病毒生命周期方面，如病毒生命周期中的细胞内晚期事件， 它们对病毒的产生至关重要。对这些事件进行了20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 酵素。重新构建这种组装途径的药物筛选导致了抗逆转录病毒PAV117的发现 小分子，然后是更强大的类似物，PAV206。本提案中的初步数据 证明PAV206以纳摩尔浓度阻止T细胞系和HIV-1病毒的产生 感染PBMC，使其成为第一个有效和选择性的细胞内晚期事件抑制剂。成象 实验证明，PAV206与病毒蛋白Gag和宿主酶共定位 在HIV-1衣壳组装中间体中的存在表明这种小分子针对一个或多个 HIV-1衣壳组装中间体的成分。这项建议试图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，包括一种保持抗病毒活性的类似物，但 包含一个用于抗体识别的生物素标签和一个用于光交联的化学基团。我们提供 大量数据展示了这种模拟如何用于使用现场成像 邻近结扎试验(PLA)。我们还建议使用尖端技术来扩展这些成像研究 这项技术的变体被称为多路聚乳酸。最后，我们描述了PAV206的第二个模拟，它允许 通过点击化学添加的其他标签，从而扩大了靶的亲和纯化的选择 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 确定PAV206目标是很高的。总而言之，通过推进我们对一种新型小分子的理解 HIV-1晚期事件的抑制剂，这里提出的研究将为研究HIV-1组装提供一个令人兴奋的工具 以及一个产生化合物的跳板，这些化合物在未来可能会得到改进。

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.