A family of compounds that reactivate latent HIV without T cell activation

 描述：HIV感染细胞的潜在储存库的存在构成了病毒根除的主要障碍。HIV-1潜伏库很小，但寿命极长。潜伏性感染与检测不到的病毒基因表达水平相关，并且似乎是非细胞病变的。然而，在重新激活后，潜伏病毒进入活跃的复制模式，在这种模式下，它们完全有能力传播和诱导疾病。该领域目前的想法是，将能够重新激活潜伏病毒的假想药物（“抗潜伏”药物）与当今的抗逆转录病毒药物相结合，将是根除病毒的有效方法。我们已经发现，苯并三唑衍生物可以在潜伏期的体外原代细胞模型中和从病毒血症患者分离的细胞中通过增强 cytokine signaling.这种再活化是以一种不寻常的方式实现的，因为它不依赖于细胞活化或增殖。因此，我们建议探索这种新的病毒再激活机制，作为一种新的治疗靶点的背景下，潜伏的艾滋病毒的再激活。 在R21阶段，我们将研究苯并三唑衍生物如何介导潜伏的HIV的再活化，以及这些化合物对病毒和细胞的整体转录的影响。在目标1中，我们计划详细描述苯并三唑衍生物的作用机制，并确定这些化合物的靶点。初步数据表明，这些化合物抑制负反馈回路，该负反馈回路通常在细胞因子信号传导后将STAT 5重置为无活性的基线状态。在目标2中，我们将确定在原代CD 4 T细胞中使用RNAseq和ChIPseq对全局转录以及HIV-1转录的影响。我们将评估在苯并三唑衍生物存在下RNApol-II与HIV-1 LTR以及细胞启动子的结合。我们将通过分析宿主和HIV基因组中的STAT 5占有率来补充这项研究。这些研究将补充通过药物化学优化苯并三唑衍生物。这将与GlaxoSmithKline合作完成。最后，我们计划测试苯并三唑衍生物的能力，重新激活潜伏的HIV-1病毒血症患者分离的细胞中存在的不同的抗病毒细胞因子。 这些研究将为在赠款的R33阶段在人源化小鼠中测试这些化合物奠定基础。 R33阶段只有在实现明确的里程碑后才能进行。目标3的目标是在人源化小鼠中反映“休克和杀死”策略，其主要目标如下：（a）建立潜伏的HIV储库，然后测量施用目标1中发现的苯并三唑衍生物后HIV转录的变化;（B）测量可能由病毒再活化引起的储库大小的变化;和（c）评价由工程化细胞毒性T淋巴细胞组成的效应臂。

 DESCRIPTION: The existence of latent reservoirs of HIV-infected cells constitutes the major impediment towards viral eradication. HIV-1 latent reservoirs are small, but extremely long-lived. Latent infection is associated with undetectable levels of viral gene expression and appears to be non-cytopathic. However, upon reactivation, latent viruses enter an active mode of replication in which they are fully competent for spread and induction of disease. The current thinking in the field is that a combination of hypothetical drugs that will reactivate latent viruss (``anti-latency'' drugs), with present-day antiretroviral drugs, will be an effective approach towad viral eradication. We have found that benzotriazole derivatives can reactivate latent HIV-1 in an in vitro primary cell model of latency and in cells isolated from aviremic patients by potentiating c cytokine signaling. This reactivation is achieved in an unusual way in that it is independent on cellular activation or proliferation. We therefore propose to explore this novel mechanism of viral reactivation as a novel therapeutic target in the context of reactivation of latent HIV. In he R21 phase, we will study how benzotriazole derivatives mediate reactivation of latent HIV and what are the effects of these compounds on global transcription, both viral and cellular. In Aim 1, we plan to characterize in detail the mechanism of action of benzotriazole derivatives as well as identify the target for these compounds. Preliminary data suggest that these compounds suppress a negative feed-back loop that normally resets STAT5 to an inactive, baseline state after cytokine signaling. In Aim 2, we will determine the effects on global transcription as well a HIV-1 transcription using both RNAseq and ChIPseq in primary CD4 T cells. We will evaluate RNApol-II binding to the HIV-1 LTR as well as cellular promoters in the presence of benzotriazole derivatives. We will complement this study with an analysis of STAT5 occupancy across host and HIV genomes. These studies will be complemented with the optimization of benzotriazole derivatives via medicinal chemistry. This will be done in collaboration with GlaxoSmithKline. Finally, we plan to test the ability of benzotriazole derivatives to reactivate latent HIV-1 in cells isolated from aviremic patients in the presence of different c-cytokines. These studies will set the stage for testing of these compounds in humanized mice during the R33 phase of the grant. The R33 phase will be undertaken only if well-defined milestones are achieved. The goal of Aim 3 is to mirror a "shock and kill" strategy in humanized mice, with the following primary goals of (a) establishing a latent HIV reservoir and then to measure changes in HIV transcription upon administration of benzotriazole derivatives found in Aim 1; (b) measuring changes in reservoir size that may result from viral reactivation; and (c) evaluating an effector arm consisting of engineered cytotoxic T lymphocytes.