Towards HIV eradication using a novel Tat inhibitor

 描述(申请人提供)：抗逆转录病毒疗法(ART)有效地阻止病毒复制，尽管它无法根除病毒。在接受抑制治疗的患者中，一小部分潜伏感染的静息记忆CD4+T细胞仍然存在。被探索最多的根除艾滋病毒的策略被称为“休克并杀死”，它试图通过在抗逆转录病毒药物存在的情况下使用抗潜伏剂重新激活病毒来清除病毒库。预计重新激活将通过细胞病变效应和/或细胞溶解T淋巴细胞溶解来迅速消除受感染的细胞(1)。这种方法具有挑战性，到目前为止还不成功。目前，我们需要针对病毒生命周期不同阶段的新的治疗剂，并限制潜在的艾滋病毒疾病。病毒蛋白TAT结合HIV mRNA并促进病毒转录。阻断TAT活性的化合物一直备受追捧；然而，目前还没有一种化合物进入临床。我们报道了皮质他汀类类固醇生物碱，它是从海绵Corticum Singex中分离出来的，代表了一类新的抗TAT药物候选药物。二氢皮质抑素A(DCA)有效和选择性地抑制TAT活性，没有细胞相关毒性(2)。DCA特异性结合TAT的RNA结合域，以低至0.7 pM的EC50减少受感染的培养细胞和原代细胞中HIV-1RNA的产生用DCA处理HIV-1感染细胞后，病毒基因的表达在体外进入持续的深潜伏期，不能被通常的激活剂(细胞因子、HDAC抑制剂、PKC激活剂)重新激活。停止DCA治疗并不会导致病毒反弹，这表明HIV启动子的染色质环境受到表观遗传抑制。重要的是，DCA消除了从接受抑制性ART治疗的患者移植的潜伏感染的CD4+T初级细胞中的抗原病毒重新激活。我们提出了一种替代“震慑和杀戮”战略的方法。具体地说，使用TAT抑制剂来靶向HIV-1转录，而不是激活内源潜伏库，我们建议将ART过程中发生的残余转录驱动到长期潜伏期或深潜伏期。TAT抑制剂与ART结合可以通过阻止正在进行的病毒复制、重新激活和补充潜伏病毒库来缩小潜伏病毒库的大小。因此，潜在的资金池 感染者体内的细胞会稳定下来，而长期存活的受感染记忆T细胞的死亡将导致这个池随着时间的推移而不断衰退，最终可能会达到人们期待已久的绝育治疗。在这里，我们建议：1)充分表征在DCA存在的情况下HIV启动子上的分子表观遗传事件以及这些修饰如何影响HIV的转录活性；2)建立一个可靠的原始细胞潜伏期模型，该模型将允许确认DCA介导的表观遗传修饰；3)了解病毒对DCA产生耐药性的分子机制。

 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.