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

 描述：艾滋病毒感染细胞潜伏库的存在构成了根除病毒的主要障碍。HIV-1潜伏的宿主规模很小，但寿命极长。潜伏感染与无法检测到的病毒基因表达水平有关，似乎不会导致细胞病变。然而，一旦重新激活，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，将重新激活潜伏病毒的假想药物(“抗潜伏期”药物)与目前的抗逆转录病毒药物相结合，将是根除病毒的有效方法。我们发现，苯并三氮唑衍生物可以在潜伏期的体外原代细胞模型中重新激活潜伏的HIV-1，并通过增强从无氧血症患者分离的细胞中激活潜伏的HIV-1 -c细胞因子信号转导。这种重新激活是以一种不寻常的方式实现的，因为它独立于细胞的激活或增殖。因此，我们建议在潜伏的HIV重新激活的背景下，探索病毒重新激活的新机制，作为新的治疗靶点。在R21阶段，我们将研究苯并三唑衍生物是如何介导潜伏的HIV重新激活的，以及这些化合物对病毒和细胞的全球转录有什么影响。在目标1中，我们计划详细表征苯并三氮唑类化合物的作用机理，并确定这些化合物的靶标。初步数据表明，这些化合物抑制了负反馈环，该负反馈环通常在细胞因子信号传递后将STAT5重置为不活跃的基线状态。在目标2中，我们将确定在原代CD4T细胞中使用RNAseq和ChIPseq对全球转录以及HIV-1转录的影响。我们将在苯并三唑衍生物存在的情况下评估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STAT5的占有率来补充这项研究。这些研究将与通过药物化学优化苯并三唑衍生物相补充。这将与葛兰素史克合作完成。最后，我们计划测试在不同c-细胞因子存在的情况下，苯并三氮唑衍生物在从无氧血症患者分离的细胞中重新激活潜伏的艾滋病毒-1的能力。 这些研究将为在赠款的R33阶段在人源化小鼠身上测试这些化合物奠定基础。只有在达到明确的里程碑时，才会进行R33阶段。目标3的目标是在人源化小鼠中反映“休克并杀死”策略，主要目标如下：(A)建立潜伏的艾滋病毒储存库，然后测量在给予目标1中发现的苯并三唑衍生物后艾滋病毒转录的变化；(B)测量可能因病毒重新激活而导致的储存库大小的变化；以及(C)评估由工程细胞毒性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.