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

项目摘要 人类免疫缺陷病毒（HIV）在长期潜伏的宿主中的持续存在仍然是 功能性治疗的最大障碍潜在的储存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，甚至在患者体内持续数十年， 抗逆转录病毒疗法最早的一种旨在耗尽潜在水库的策略被称为 “休克和杀死”，其中潜伏感染的细胞将被诱导表达病毒的治疗与潜伏 逆转剂（LRA），随后从体内清除。虽然一些LRA已经被 尽管已经描述了这些药剂，但是已经证明这些药剂在治疗上是站不住脚的，至少部分是由于它们的不完全的化学性质。 随机性和显著的随机性。开发新的LRA以更好地了解HIV潜伏期和 和转录调控，以及用于下一代治疗策略，是一个NIH艾滋病毒/艾滋病 高优先级研究课题（NOT-OD-20-018）。目前描述的几种LRA工作以提高效率 通过直接或间接地增加正转录延伸的活性， 因子B（P-TEF B）。在活动性HIV感染期间，病毒达特蛋白劫持P-TEFb并将其募集到 新生病毒转录P-TEFb随后磷酸化RNA聚合酶II（Pol 二）、许可延长。最近，我们描述了转录延伸中的一个新参与者，即PAF 1复合物或 PAF1C。PAF 1C在转录暂停位点与RNA Pol II结合，阻止P-TEFb募集， 有效地对转录机器施加“停车制动”。这与最近的研究结果一致， PAF 1C作为HIV转录的负调节因子和HIV潜伏期的正调节因子。在我们的初步调查中 数据，我们报告的发展和初步表征的一流的小分子抑制剂PAF 1C 成核，称为iPAF 1C。我们发现，iPAF 1C显著增强了几个细胞的再活化潜力。 在潜伏期的细胞系模型中存在不同的LRA，导致整合的前病毒中RNA Pol II的释放增强 和增强的转录延伸。在这个提议中，我们试图验证小分子 PAF 1C抑制剂通过破坏PAF 1C成核和释放近端暂停的 RNA Pol II位于HIV启动子处。首先，我们将测试iPAF 1C在破坏PAF 1C中的有效性和特异性。 J-Lat细胞，使用这些结果作为进一步化合物优化的基准（目标1）。iPAF 1C及其电极导线 然后将单独测试类似物，并与一系列代表性LRA组合测试类似物 细胞系和原代细胞模型的潜伏期。随后将分析协同组合， HIV感染者外周血单核细胞的再活化潜力（Aim 2）。最终，这些 实验将检查一个新的轴重新激活潜伏的原病毒，提供新的分子探针， 了解艾滋病毒潜伏期和下一代治疗策略的发展。

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.