Epigenetic HIV Silencing in Macrophages

项目摘要/摘要-表观遗传HIV在巨噬细胞中沉默 抗逆转录病毒疗法(ART)对艾滋病毒感染非常有效，但有很大的局限性， 在没有ART的情况下达到乏力状态(“功能性治疗”)是人们非常感兴趣的一个焦点。一种潜在的方法来 一旦停止抗逆转录病毒疗法，防止持久的病毒库产生复制病毒的关键是让艾滋病毒沉默 这些蓄水池使感染无法“重新点燃”。感染的巨噬细胞系细胞是主要的长期 据信，这是一个病毒可以持续存在的“避难所”，即使病毒在 CNS储集层已被清洗。此外，长期感染的巨噬细胞可能会继续产生低水平的病毒。 病毒，导致艾滋病毒相关的神经认知障碍(手)。因此，让艾滋病毒在体内沉默 在ART抑制的背景下，巨噬细胞可能既有助于“功能治愈”，又能改善手。 艾滋病毒整合到宿主细胞基因组中，并受到积极和消极的表观遗传调控。大有可为 关于T细胞中HIV的表观遗传控制方面的工作已经完成，但对巨噬细胞中的HIV知之甚少。染色质 组织和转录调控是高度细胞和上下文特定的。我们的科学前提是 中枢神经系统中的巨噬细胞不能从表观遗传上抑制艾滋病毒，而是一个长期的储存库。我们 假设巨噬细胞特异性增强子-启动子相互作用调节HIV持续转录 活性，小分子可以重新编程HIV感染的巨噬细胞的表观遗传调节 建立整合的艾滋病毒基因组的长期沉默。为此，我们建立了一个初级 用于高通量筛选表观遗传酶小分子调节子的巨噬细胞模型，以及 为双加氧酶参与组蛋白去甲基化提供初步证据。 在第一阶段，我们将优化初级和次级化验，以进行高通量筛查活动，以确定 HIV-1在原代人巨噬细胞中表达的小分子表观遗传调节；测试 候选表观遗传调节剂，特别是可能防止艾滋病毒沉默的组蛋白和DNA去甲基酶；以及 进一步研究巨噬细胞中调节HIV持续转录的分子机制。同相 2，我们将进行高通量筛选，以开发小分子来沉默巨噬细胞中的HIV-1 行业标准里程碑驱动的药物发现流水线，用于识别生物活性化学类型；验证铅 具有主要HIV分离株和髓系细胞类型的化合物；候选HITS的高级类药物特性 通过药物化学；确定分子靶点并进行作用机理研究。 在第一阶段结束时，我们将拥有一个强大的、高吞吐量的基于初级单元的筛查、正交验证 检测以确认靶上命中，并进一步深入了解调节HIV的巨噬细胞特异性表观遗传因子。 在第二阶段结束时，我们将完成几个结构不同的小分子的高通量筛选 库，识别和验证命中结果，以及选择和提升主要候选人。我们预计这项工作将导致 确定新的小分子作为艾滋病毒沉默方法的基础，重点放在中枢神经系统储存库。

Project Summary/Abstract - Epigenetic HIV Silencing in Macrophages Antiretroviral therapy (ART) is highly effective in HIV infection but has substantial limitations, and strategies to achieve an aviremic state without ART (“functional cure”) are a focus of great interest. A potential approach to prevent persistent viral reservoirs from giving rise to replicating virus once ART is stopped is to silence HIV in these reservoirs so infection cannot “re-ignite”. Infected macrophage lineage cells serve as the main long-term reservoir for HIV in the CNS, which is believed to be a “sanctuary site” where virus can persist even if extra- CNS reservoirs are purged. Furthermore, long-lived infected macrophages may continue to produce low levels of virus, which contributes to HIV-associated neurocognitive disorders (HAND). Thus, silencing HIV in macrophages may both contribute to “functional cure”, and ameliorate HAND in the setting of ART suppression. HIV integrates into the host cell genome and is subject to positive and negative epigenetic regulation. Much work has been done on epigenetic control of HIV in T cells, but less is known in macrophages. Chromatin organization and transcriptional regulation is highly cell and context-specific. Our scientific premise is that macrophages in the CNS fail to epigenetically suppress HIV and serve as a long-term reservoir. We hypothesize that macrophage-specific enhancer-promoter interactions regulate persistent HIV transcription activity, and that small molecules can reprogram epigenetic regulation of HIV infected macrophages to establish long-term silencing of the integrated HIV genome. To this end, we have established a primary macrophage model for high-throughput screening of small molecule modulators of epigenetic enzymes, and provide preliminary evidence for involvement of dioxygenase enzymes involved in histone demethylation. In Phase 1, we will optimize primary & secondary assays for a high-throughput screening campaign to identify small molecule epigenetic regulators of HIV-1 expression in primary human macrophages; test the role of candidate epigenetic modulators, especially histone & DNA demethylases that may prevent HIV silencing; and further investigate molecular mechanisms that regulate persistent HIV transcription in macrophages. In Phase 2, we will perform a high-throughput screen to develop small molecules to silence HIV-1 in macrophages using industry-standard milestone-driven drug discovery pipeline to identify bioactive chemotypes; validate lead compounds with primary HIV isolates & myeloid cell types; advance drug-like properties of candidate hits through medicinal chemistry; and define molecular targets and conduct mechanism of action studies. At the end of Phase I we will have a robust high-throughput primary cell-based screen, orthogonal validation assays to confirm on-target hits, and further insight into macrophage-specific epigenetic factors regulating HIV. At the end of Phase 2, we will complete a high-throughput screen of several structurally diverse small molecule libraries, identify & validate hits, and select and advance lead candidates. We anticipate this work will lead to identification of novel small molecules as a basis for HIV silencing approaches focused on the CNS reservoir.