Modulation of BRD4 to epigenetically suppress HIV

摘要 HIV病毒DNA整合到宿主细胞基因组中并建立稳定的潜伏感染， 找到艾滋病治愈方法的主要障碍。HIV前病毒在细胞库中的表达受宿主表观遗传学的调节 和转录机制。确定针对宿主机器的有效方法， 加强艾滋病毒潜伏期是一个重要的目标。BRD4是属于溴结构域（BD）的表观遗传阅读器。 和末端外结构域（ET）蛋白家族（BET）。通过BD，BRD4结合乙酰赖氨酸（KAc）残基 在染色质组蛋白中，作为一个支架平台，通过蛋白质-蛋白质 相互作用（PPI）来调节基因转录，包括HIV。通过泛BET抑制剂调节BET/BRD 4 (JQ1)靶向BD中经典的KAc结合位点的HIV-B11已经显示出激活HIV转录。最近 我的小组和其他人的研究表明，BRD4在功能上是多功能的，它在基因转录上的活性 是由特定的蛋白质组成的使用结构辅助设计，我们的研究（1，2）已经确定 一种前导小分子（ZL 0580）和几种不同于JQ 1但诱导HIV转录的类似物 通过BRD4抑制。我们证明，ZL 0580诱导抑制两种转录活性 和潜伏的HIV在多种细胞模型，包括J-Lat，CD4 T细胞和骨髓细胞/小胶质细胞。对接分析 结合模式和机制研究表明，与JQ1不同，ZL 0580靶向一个独特的新区域， BRD4 BD1结合[非乙酰基赖氨酸（KAc）位点]，并通过抑制 达特反式激活和通过在HIV LTR处诱导抑制性染色质结构。根据这些小说 研究结果，我们的中心假设是，宿主BRD4及其相关的表观遗传机制可以被调节， 来抑制艾滋病病毒，导致艾滋病病毒的潜伏期。除了ZL 0580，我们正在进行的研究还发现了另一种 类似物（YL0255），其在结构上接近ZL 0580并诱导更强的HIV抑制。在本申请中， 我们将以ZL 0580和YL0255作为两种新型分子探针，系统地研究BRD 4的调控机制 HIV表观遗传抑制在目标1中，我们将确定BRD4是否是一种选择性蛋白靶点， ZL0580/YL0255和潜在的结构基础。在目标2中，我们将阐明分子机制， ZL0580/YL0255对BRD4的调节诱导HIV表观遗传抑制。在目标3中，为了证明 在概念上，我们将检查ZL 0580/YL0255对BRD 4的调节是否诱导体内潜伏HIV的抑制。 总的来说，这些研究对于更好地理解HIV表观遗传调控和潜伏期至关重要， 为将来开发诱导HIV抑制和/或沉默的新策略奠定了重要基础。

ABSTRACT Integration of HIV viral DNA into host cell genomes and establishment of stable latent infection have posed a major obstacle for finding an HIV cure. HIV proviral expression in cell reservoirs is regulated by host epigenetic and transcriptional mechanisms. Identification of effective approaches targeting host machineries to disrupt or enforce HIV latency is a significant goal. BRD4 is an epigenetic reader that belongs to the bromodomain (BD) and extra-terminal domain (ET) protein family (BET). Via the BDs, BRD4 binds to acetyl-lysine (KAc) residues in chromatin histones and serves as a scaffolding platform for recruiting partner proteins through protein-protein interactions (PPI) to regulate gene transcription, including HIV. Modulation of BET/BRD4 by a pan-BET inhibitor (JQ1), which targets the classic KAc binding site in BDs, has been shown to activate HIV transcription. Recent studies by my group and others indicate that BRD4 is functionally versatile, and its activity on gene transcription is tailored by specific partner proteins it engages. Using structure-aided design, our studies (1, 2) have identified a lead small molecule (ZL0580) and several analogs that are distinct from JQ1 but induce HIV transcriptional suppression through BRD4. We demonstrate that ZL0580 induces suppression of both transcriptionally active and latent HIV in multiple cell models, including J-Lat, CD4 T cells and myeloid cells/microglia. Docking analyses of binding modes and mechanistic investigations indicate that unlike JQ1, ZL0580 targets a distinct new region of BRD4 BD1 for binding [non-acetyl-lysine (KAc) site] and induces HIV transcriptional suppression by inhibiting Tat transactivation and by inducing a repressive chromatin structure at the HIV LTR. Based on these novel findings, our central hypothesis is that host BRD4 and its associated epigenetic machinery can be modulated to repress HIV, leading to enforced HIV latency. Other than ZL0580, our ongoing research has identified another analog (YL0255) that is structurally close to ZL0580 and induces stronger HIV suppression. In this application, we will use ZL0580 and YL0255 as two novel molecular probes to systemically investigate modulation of BRD4 in HIV epigenetic suppression. In Aim 1, we will determine if BRD4 is a selective protein target for ZL0580/YL0255 and the underlying structural basis. In Aim 2, we will elucidate the molecular mechanisms by which modulation of BRD4 by ZL0580/YL0255 induces HIV epigenetic suppression. In Aim 3, as a proof of concept, we will examine if modulation of BRD4 by ZL0580/YL0255 induces repression of latent HIV in vivo. Collectively, these studies are critical for better understanding HIV epigenetic regulation and latency and will lay critical groundwork for developing novel strategies for inducing HIV suppression and/or silencing in future.