Validation and characterization of Tat inhibitors identified through HTS

摘要 尽管有效的抗逆转录病毒疗法(ART)，潜伏的前病毒可以在细胞上重新启动病毒的产生 刺激或治疗中断。病毒Tat蛋白增强HIV-1转录本的延长 启动子，控制潜伏和活跃的病毒生产之间的切换。块加锁函数 CURE的目标是使病毒储存库的转录沉默，从而极大地抑制艾滋病毒启动子 很难从延迟中重新激活。TAT抑制剂二氢皮质酮A(DCA)被用来证明这一点 概念。将DCA与ART结合，在治疗中断时抑制转录并阻止病毒反弹， 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，它可以沉默和 保持转录上不活跃的HIV启动子，为HIV的治疗提供了一种新的方法。 TAT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)没有细胞同源物；3)TAT抑制剂阻断了病毒扩增所需的反馈回路；4)表观遗传学 修饰累积在HIV启动子上，使得重新激活的可能性降低。TAT还以其作用而闻名 在神经毒性、神经递质调节、氧化应激、细胞凋亡、血脑屏障破坏以及 神经炎。因此，开发补充抗逆转录病毒治疗的TAT抑制剂的巨大兴趣。 推动DCA进入临床试验的主要障碍是大量生产这种药物的成本 分子，由于其复杂的结构。其他临床候选对象，在结构上与DCA不同， 在临床前的管道中需要体现同等的生物活性。 我们优化了一种基于细胞的TAT反式激活试验，用于高通量筛查(HTS)，以DCA为 控制力。我们结合了适当的反筛选技术和丰富的技术，快速地将小的 不是TAT特异性的分子。南方研究公司完成了210,240个化合物的HTS (SR)，得到两个化合物SRI-43627和SRI-43050，其选择性指数&gt；10进一步 调查过了。这一初步的成功促使我们又筛选出了369,203个化合物，产生了 计数器屏幕有29个命中，有待进一步评估。在此应用程序中，我们建议执行命中验证和 作为药物开发的一部分，SR合成的这些化合物和类似物的表征 复合进展途径。我们提出以下目标： 具体目的1.基于对TAT HIV-1 LTR反式激活的干扰来验证TAT抑制剂。 具体目标2.描述选定命中的作用机制。 在这项研究结束时，我们预计(A)已经确定了能够特异性抑制的小分子 TAT在基于细胞的分析中。(B)具有足够的代谢稳定性和未来的PK属性 在动物模型中以及最终在人类临床试验中进行的药理学评估。

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, neurotransmitter modulation, oxidative stress, apoptosis, 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 ‘weed out’ small molecules that are not Tat specific. The HTS of 210,240 compounds was completed by Southern Research (SR), yielding two compounds, SRI-43627 and SRI-43050 with a selectivity index >10 that were further investigated. This initial success prompted the screen of an additional 369,203 compounds, yielding upon counter screen 29 hits to be further evaluated. In this application, we propose to perform hit validation and characterization of these compounds as well as analogs synthesized by SR as part of drug development during the compound progression pathway. 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.