Towards HIV eradication using a novel Tat inhibitor

 描述（由申请人提供）：抗逆转录病毒疗法（ART）可有效阻断病毒复制，但无法根除病毒。在接受抑制性治疗的患者中，一小部分潜伏感染的静息记忆性CD 4 +T细胞仍然存在。最受关注的HIV根除策略被称为“休克和杀灭”，该策略试图通过在ART存在下使用抗潜伏剂重新激活病毒来清除病毒库。预期重新激活将通过致细胞病变效应和/或细胞溶解性T淋巴细胞来促进受感染细胞的消除（1）。这种方法具有挑战性，迄今为止尚未成功。现在，我们需要新的治疗药物，针对病毒生命周期的不同阶段，并限制潜伏的艾滋病毒疾病。 病毒蛋白达特结合HIV mRNA并促进病毒转录。阻断达特活性的化合物一直备受追捧;然而，还没有一种进入临床。我们报道了皮质抑素，类固醇样生物碱分离自海绵corticium simplex，代表一类新的抗达特药物候选人。二脱氢皮质抑素A（dCA）有效并选择性地抑制Tat活性，无细胞相关毒性（2）。dCA与达特的RNA结合结构域特异性结合，以低至0.7 pM的EC 50降低感染的培养细胞和原代细胞中的HIV-1 RNA产生。用dCA处理HIV-1感染的细胞可使病毒基因表达进入体外持续深潜伏期的诱导状态，对常用激活剂组（细胞因子HDAC抑制剂、PKC激活剂）的病毒再激活无效。停止dCA治疗不会导致病毒反弹，表明HIV启动子的染色质环境受到表观遗传学抑制。重要的是，dCA废除抗原性病毒再活化从潜伏感染的CD 4 +T原代细胞从患者接受抑制性ART。我们提出了一种替代方法的“休克和杀死”的战略。具体而言，使用Tat抑制剂靶向HIV-1转录，而不是激活内源性潜伏库，我们建议将ART期间发生的残留转录驱动为长期潜伏或深度潜伏。Tat抑制剂与ART组合可以通过阻断正在进行的病毒复制、再活化和补充潜伏病毒库来减小潜伏病毒库的大小。因此， 受感染个体中的记忆T细胞将被稳定，并且长寿命的受感染记忆T细胞的死亡将导致该池随时间的持续衰减，可能在期待已久的灭菌治疗中达到高潮。 在此，我们建议：1）充分表征在dCA存在下在HIV启动子处设置的分子表观遗传事件以及这些修饰如何影响HIV转录活性; 2）建立稳健的潜伏期的原代细胞模型，其将允许确认dCA介导的表观遗传修饰;以及3）理解病毒对dCA的抗性的分子机制。

 DESCRIPTION (provided by applicant): Antiretroviral therapy (ART) effectively blocks viral replication, although it fails to eradicate the virus. A small population of latently infected restng memory CD4+T cells still persists in patients on suppressive therapy. The most explored strategy for HIV eradication is dubbed "shock and kill", which attempts to purge the viral reservoir by reactivating the virus using anti-latency agents in the presence of ART. The expectation is that reactivation will prompt elimination of infected cells by cytopathic effects and/or by cytolytic T-lymphocyte lysis (1). This approach is challenging and has been unsuccessful thus far. Right now, we need new therapeutic agents that target different stages of the virus life cycle and limit latent HIV disease. The viral protein Tat binds HIV mRNA and promotes viral transcription. Compounds that block Tat activity have been highly sought after; however, none is yet in the clinic. We reported that Cortistatins, steroid-like alkaloids isolated from the marine sponge corticium simplex, represent a novel class of anti- Tat drug candidates. Didehydro-Cortistatin A (dCA) potently and selectively inhibits Tat-activity with no cellular associated toxicity (2). dCA binds specifically to the RNA-binding domain of Tat reducing HIV-1 RNA production in infected cultured and primary cells at an EC50 as low as 0.7 pM. Treatment of HIV-1 infected cells with dCA drives viral gene expression into an induced state of persistent deep latency in vitro, refractory to viral reactivation by the usual panel of activators (cytokines HDAC inhibitors, PKC activators). Discontinuation of dCA treatment does not result in viral rebound, suggesting the chromatin environment of the HIV promoter is epigenetically repressed. Importantly, dCA abrogates antigenic virus reactivation from latently infected CD4+T primary cells explanted from patients receiving suppressive ART. We propose an alternative approach to the "shock and kill" strategy. Specifically, using a Tat-inhibitor to target HIV-1 transcription rather than activating the endogenous latent reservoir, we propose to drive the residual transcription that occurs during ART into long-term latency or deep latency. A Tat-inhibitor combined with ART could reduce the size of the latent reservoir pool by blocking ongoing viral replication, reactivation and replenishment of the latent viral reservoir. Thus, the latent pool of cells in an infected individual would be stabilized, and death of the long-lived infected memory T-cells would result in a continuous decay of this pool over time, possibly culminating in the long-awaited sterilizing cure. Here we propose to: 1) fully characterize the molecular epigenetic events set in place at the HIV promoter in the presence of dCA and how these modifications impact HIV transcriptional activity; 2) establish a robust primary cellular model of latency that wll allow for confirmation of dCA mediated epigenetic modifications and 3) understand the molecular mechanism of viral resistance to dCA.