Understanding potent and novel small molecules that target HIV assembly

总结： 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 感染这些药物也被用作预防HIV-1感染的暴露前预防; 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。 由于目前使用的药物已经靶向了HIV-1的关键酶，因此未来的靶向药物将需要出现。 从对病毒生命周期知之甚少的方面，如病毒生命周期中的细胞内晚期事件， 这对病毒的产生至关重要。经过20年的研究，我们确定了一种宿主- 含有HIV-1 Gag蛋白和宿主的推定衣壳组装中间体的催化途径 内切酶一种重建这种组装途径的药物筛选导致了PAV 117的发现，PAV 117是一种抗逆转录病毒药物， 小分子，随后是更有效的类似物PAV 206。本提案中的初步数据 证明PAV 206在T细胞系和HIV-1中以纳摩尔浓度阻断病毒产生 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 实验证明PAV 206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，表明这种小分子靶向一种或多种 HIV-1衣壳组装中间体的组分。该提案旨在1）确定哪些逆转录病毒 被PAV 206抑制，2）鉴定HIV-1中赋予PAV 206抗性的突变，和3）使用成像 实验以确定PAV 206是否定位于不同的亚细胞复合物，以及4）使用生物化学方法 PAV 206结合配偶体的探测方法。亚细胞定位和结合伴侣的研究将 利用我们可获得的PAV 206类似物，包括保持抗病毒活性但 含有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 大量的数据证明了这种类似物如何能够用于使用 邻位连接测定（PLA）。我们还建议使用最先进的成像技术来扩展这些成像研究。 这种技术的变体称为多重PLA。最后，我们描述了PAV 206的第二种类似物， 通过点击化学添加其他标签，从而扩大了用于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，我们成功地 识别PAV 206目标的概率很高。总之，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 也是一个跳板，用于生产未来可以改进的化合物。

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.