Exploring Small Molecule Inhibitors of PAF1C as Novel HIV Latency Reversal Agents

项目总结 人类免疫缺陷病毒(HIV)在长期潜伏的水库中的持久性仍然是 功能性治愈的最大障碍。潜伏库由具有复制能力的细胞组成， 但转录抑制的前病毒逃避免疫清除，即使在几十年后仍在患者中存在 抗逆转录病毒疗法。最早被设计来耗尽潜在储集层的策略之一被称为 “电击并杀死”，通过潜伏治疗诱导潜伏感染的细胞表达病毒。 反转剂(LRA)，随后从体内清除。而自那以后，许多LRA一直在 这些药物已经被证明在治疗上是站不住脚的，至少部分原因是它们的不完全 外显性和显著的随机性。开发新的LRA以更好地了解HIV潜伏期 转录调控以及在下一代治疗策略中的使用是NIH艾滋病毒/艾滋病的一个重要因素 高优先级研究课题(非OD-20-018)。目前描述的几个LRA致力于提高效率 通过直接或间接增加正转录伸长的活性来改变转录伸长 因子b(P-TEFb)。在活动性HIV感染期间，病毒Tat蛋白劫持P-TEFb并将其招募到 新生的病毒转录。P-TEFb随后使RNA聚合酶II(POL)的C-末端尾部磷酸化 Ii)、许可延伸期。最近，我们描述了转录延长的一个新的参与者，PAF1复合体或 PAF1C。PAF1C在转录暂停部位与RNA Pol II结合，阻止P-TEFb募集和 在转录机器上有效地应用了“停车刹车”。这与最近的发现一致，即 PAF1C作为HIV转录的负调节因子和HIV潜伏期的正调节因子发挥作用。在我们的预赛中 数据，我们报告了一种一流的PAF1C小分子抑制剂的开发和初步表征 成核作用，称为iPAF1C。我们发现iPAF1C显著增强了几个 潜伏期的细胞系模型中不同的LRA，导致整合前体处RNA Pol II的增强释放 和增强的转录延长。在这个提议中，我们试图检验小分子 PAF1C抑制剂通过破坏PAF1C成核和释放近端暂停的LRA而发挥有效的LRA作用 HIV启动子上的RNA Pol II。首先，我们将测试iPAF1C在体外干扰PAF1C的有效性和特异性。 J-Lat单元，使用这些结果作为进一步化合物优化的基准(目标1)。IPAF1C及其领先地位 然后，模拟将单独测试，并与一系列具有代表性的LRA小组一起进行测试 潜伏期的细胞系和原代细胞模型。随后将对协同组合进行分析 艾滋病毒携带者外周血单核细胞的再激活潜力(目标2)。最终，这些 实验将检查潜伏前病毒重新激活的新轴，为 了解艾滋病毒潜伏期和开发下一代治疗策略。

PROJECT SUMMARY The persistence of Human Immunodeficiency Virus (HIV) in long-lived, latent reservoirs remains one of the largest barriers to a functional cure. The latent reservoir consists of cells harboring replication-competent, but transcriptionally inhibited proviruses that evade immune clearance and persist in patients even after decades of antiretroviral therapy. One of the earliest strategies designed to deplete the latent reservoir was referred to as “shock and kill,” whereby latently infected cells would be induced to express the virus by treatment with latency reversing agents (LRAs) and subsequently cleared from the body. While a number of LRAs have since been described, these agents have been proven to be therapeutically untenable, at least in part due to their incomplete penetrance and notable stochasticity. The development of new LRAs for better understanding both HIV latency and transcriptional regulation, as well as for use in next-generation therapeutic strategies, is an NIH HIV/AIDS high priority research topic (NOT-OD-20-018). Several currently described LRAs work to enhance the efficiency of transcriptional elongation by directly or indirectly increasing the activity of positive transcription elongation factor b (P-TEFb). During active HIV infection, the viral Tat protein hijacks P-TEFb and recruits it to sites of nascent viral transcription. P-TEFb subsequently phosphorylates the C-terminal tail of RNA polymerase II (Pol II), licensing elongation. Recently, we described a new player in transcriptional elongation, the PAF1 complex or PAF1C. PAF1C binds to RNA Pol II at sites of transcriptional pausing, preventing P-TEFb recruitment and effectively applying a ‘parking brake’ to the transcriptional machinery. This is consistent with recent findings that PAF1C acts as a negative regulator of HIV transcription and a positive regulator of HIV latency. In our preliminary data, we report the development and initial characterization of a first-in-class small molecule inhibitor of PAF1C nucleation, termed iPAF1C. We show that iPAF1C significantly enhances the reactivation potential of several distinct LRAs in a cell line model of latency, resulting in enhanced release of RNA Pol II at integrated proviruses and enhanced transcriptional elongation. In this proposal, we seek to test the hypothesis that small molecule inhibitors of PAF1C act as effective LRAs by disruption of PAF1C nucleation and release of proximally paused RNA Pol II at the HIV promoter. First, we will test the efficacy and specificity of iPAF1C in disrupting PAF1C in J-Lat cells, using these results as benchmarks for further compound optimization (Aim 1). iPAF1C and its lead analogs will then be tested both individually and in combination with a panel of representative LRAs in a series of cell line and primary cell models of latency. Synergistic combinations will subsequently be analyzed for reactivation potential in peripheral blood mononuclear cells from people living with HIV (Aim 2). Ultimately, these experiments will examine a new axis for reactivation of latent proviruses, providing new molecular probes for the understanding HIV latency and for the development of next-generation curative strategies.