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

探索 PAF1C 小分子抑制剂作为新型 HIV 潜伏期逆转剂

• 批准号: 10762258
• 负责人: Judd F Hultquist
• 金额: $ 23.3万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10762258
• 关键词: Agonist; Benchmarking; Binding; Biological Assay; Biological Models; Bromodomains and extra-terminal domain inhibitor; C-terminal; Cell Line; Cell model; Cells; Chromatin; Chromatin Remodeling Factor; Collaborations; Complex; Data; Development; Evaluation; Genes; Genetic Transcription; HIV; HIV Infections; HIV tat Protein; HIV-1; HIV/AIDS; Histone Deacetylase Inhibitor; Immune Evasion; In Vitro; Individual; Lead; Licensing; Maintenance; Modeling; Molecular; Molecular Probes; Patients; Penetrance; Peripheral Blood Mononuclear Cell; Persons; Phosphorylation; Phosphotransferases; Positive Transcriptional Elongation Factor B; Protein Kinase C; Provirus Integration; Proviruses; RNA Polymerase II; Regimen; Reporting; Repression; Research Priority; Role; Series; Shock; Site; Specificity; T-Lymphocyte; Tail; Testing; Therapeutic; Transactivation; Transcription Elongation; Transcriptional Regulation; United States National Institutes of Health; Viral; Virus; Withholding Treatment; Work; analog; antiretroviral therapy; design; efficacy testing; experimental study; immune clearance; improved; in vivo; inhibitor; integration site; knock-down; lead candidate; next generation; novel; novel therapeutics; pancreatic differentiation 2 protein; prevent; promoter; reactivation from latency; recruit; research and development; screening; small molecule; small molecule inhibitor; synergism; tat Protein; therapeutic development; viral rebound

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.

项目概要 人类免疫缺陷病毒 (HIV) 在长寿命、潜伏病毒库中的持续存在仍然是导致人类免疫缺陷病毒 (HIV) 死亡的原因之一。 功能性治愈的最大障碍是由具有复制能力的细胞组成。 但转录抑制的原病毒可以逃避免疫清除，甚至在几十年后仍然存在于患者体内 旨在耗尽潜伏病毒库的最早的抗逆转录病毒治疗策略之一被称为“ “休克和杀死”，即通过潜伏期治疗诱导潜伏感染细胞表达病毒 逆转剂 (LRA) 并随后从体内清除，而许多 LRA 已被清除。 据描述，这些药物已被证明在治疗上是站不住脚的，至少部分是由于它们的不完整 外显率和显着的随机性的新 LRA 的开发，以更好地了解 HIV 潜伏期。 和转录调控，以及用于下一代治疗策略，是 NIH HIV/AIDS 的一项研究 高优先级研究主题（NOT-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延伸的活性来抑制转录延伸 因子 b (P-TEFb) 在活跃的 HIV 感染过程中，病毒 Tat 蛋白劫持 P-TEFb 并将其招募到 HIV 感染部位。 新生病毒转录后，P-TEFb 磷酸化 RNA 聚合酶 II (Pol) 的 C 末端尾部。 II），许可延伸最近，我们描述了转录延伸中的一个新参与者，PAF1复合物或 PAF1C。PAF1C 在转录暂停位点结合 RNA Pol II，阻止 P-TEFb 募集和 这实际上是应用转录机制的“停车制动器”，这与最近的发现是一致的。 在我们的初步研究中，PAF1C 充当 HIV 转录的负调节因子和 HIV 潜伏期的正调节因子。 数据，我们报告了一流的 PAF1C 小分子抑制剂的开发和初步表征 我们发现 iPAF1C 显着增强了几种物质的再激活潜力。 细胞系潜伏期模型中存在不同的 LRA，导致整合原病毒处 RNA Pol II 的释放增强 在这个提议中，我们试图检验小分子的假设。 PAF1C 抑制剂通过破坏 PAF1C 成核和释放近端暂停来充当有效的 LRA 首先，我们将测试 iPAF1C 在 HIV 启动子处破坏 PAF1C 的功效和特异性。 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）。 然后，类似物将单独进行测试，并与一系列具有代表性的 LRA 组合进行测试 随后将分析细胞系和原代细胞模型的潜伏期协同组合。 HIV 感染者外周血单核细胞的再激活潜力（目标 2）。 实验将检查潜伏原病毒重新激活的新轴，为潜在原病毒的重新激活提供新的分子探针 了解艾滋病毒潜伏期并制定下一代治疗策略。