Development and characterization of HIV-1 Tat degraders

摘要 尽管有有效的抗逆转录病毒治疗（ART），潜伏的前病毒可以重新启动病毒的生产， 刺激或治疗中断。病毒达特蛋白增强HIV-1转录物的延伸 启动子，控制潜伏期和活性病毒生产之间的转换。闭锁功能性治疗 目的是使病毒库的转录沉默，使抑制HIV启动子变得非常困难。 从潜伏状态中恢复使用达特抑制剂双脱氢皮质抑素A（dCA）来证明这一概念。 将dCA与ART组合抑制转录并阻断治疗中断后的病毒反弹，因为 启动子在表观遗传上受到抑制。dCA定义了一类新的药物，可以沉默和维持 一种无转录活性的HIV启动子，为治疗HIV提供了一种新的方法。 达特是非常有吸引力的治疗干预靶点，因为：1）在病毒复制早期表达; 2)无细胞同源物; 3）达特抑制剂阻断病毒扩增所必需的反馈环; 4）表观遗传 修饰在HIV启动子处积累，使得再活化不太可能。达特也因其在 神经毒性、血脑屏障破坏和神经炎症。因此，对这一问题的巨大兴趣 开发达特抑制剂以补充ART。 推进dCA进入临床试验的主要障碍是生产大量dCA的成本。 分子由于其复杂的结构。其他临床候选药物，在结构上与dCA不同， 在临床前管道中需要等效的生物活性。 我们优化了基于细胞的达特反式激活测定以用于高通量筛选（HTS），其中dCA作为 控制我们结合了适当的反筛和大量的技术来快速丢弃小分子 不是达特特有的369，203种化合物的HTS由Southern Research（SR）完成， 三种化合物，TT-44951、TT-44881和TT-44863，治疗指数（TI）为51.2 - 181.1 和良好的化学性能。在此应用中，我们建议执行命中验证和表征 在化合物进展途径中，由Thimble Therapeutics生成的化学合成类似物 (CPP)，以扩大我们的小组达特抑制剂商业化这类新的化合物。我们建议 以下目标： 具体目标1。基于达特HIV-1 LTR反式激活的破坏来抑制达特抑制剂。 具体目标2。描述选定命中的作用机制。 在这项研究的最后，我们希望：（a）已经确定了小分子，将特异性抑制 在基于细胞的测定中的达特;（B）具有足够的代谢稳定性和PK特性，用于将来的 在动物模型中进行药理学评估，并最终在人体临床试验中进行。

Abstract Despite effective antiretroviral therapy (ART), latent proviruses can reinitiate viral production upon cell stimulation or treatment interruption. The viral Tat protein enhances transcript elongation from the HIV-1 promoter, controlling the switch between latency and active viral production. The block-and-lock functional cure aims at the transcriptional silencing of the viral reservoir, rendering suppressed HIV promoters extremely difficult to reactivate from latency. The Tat inhibitor, didehydro-cortistatin A (dCA) was used to prove this concept. Combining dCA with ART inhibits transcription and blocks viral rebound upon treatment interruption, as the promoter becomes epigenetically repressed. dCA defines a novel class of drugs that can silence and maintain a transcriptionally inactive HIV promoter, offering a novel approach in the treatment of HIV. Tat is very attractive target for therapeutic intervention because: 1) is expressed early during virus replication; 2) no cellular homologs; 3) Tat inhibitors block the feedback loop necessary for viral amplification; 4) epigenetic modifications accumulate at the HIV promoter rendering reactivation less likely. Tat is also known for its role in neurotoxicity, blood-brain barrier disruption, and neuroinflammation. Thus, the immense interest in the development of Tat inhibitors to complement ART. The major hurdle towards advancing dCA into clinical trials is the cost of producing large quantities of this molecule due to its complex structure. Additional clinical candidates, structurally distinct from dCA, that embody equivalent bioactivity are needed in the pre-clinical pipeline. We optimized a cell-based Tat transactivation assay to use in high throughput screening (HTS), with dCA as control. We combined appropriate counter-screens and a wealth of techniques to quickly discard small molecules that are not Tat specific. The HTS of 369,203 compounds was completed by Southern Research (SR), yielding three compounds, TT-44951, TT-44881 and TT-44863, with therapeutic index (TI) varying from 51.2 to 181.1 and good chemical properties. In this application, we propose to perform hit validation and characterization of chemically synthesized analogs generated by Thimble Therapeutics, during the compound progression pathway (CPP), to expand our panel of Tat inhibitors to commercialize this novel class of compounds. We propose the following aims: Specific Aim 1. Validate Tat inhibitors based on disruption of Tat HIV-1 LTR transactivation. Specific Aim 2. Characterize the mechanism of action of selected hits. At the end of this study we expect to: (a) have identified small molecules that will specifically inhibit Tat in cell-based assays; (b) have adequate metabolic stability and PK properties for future pharmacological assessment in animal models and eventually in human clinical trials.