Validation and characterization of Tat inhibitors identified through HTS

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 治疗的目的是使病毒储存库的转录沉默，从而使被抑制的HIV启动子极度 很难从延迟中重新激活。使用达特抑制剂双脱氢皮质抑素A（dCA）来证明这一点 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学上受到抑制。dCA定义了一类新的药物，可以沉默， 维持转录失活的HIV启动子，提供了治疗HIV的新方法。 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。达特也因其角色而闻名 神经毒性、神经递质调节、氧化应激、细胞凋亡、血脑屏障破坏， 神经炎症因此，开发达特抑制剂以补充ART引起了极大的兴趣。 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。在结构上不同于dCA的其他临床候选药物， 在临床前管道中需要体现等效的生物活性。 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 控制我们结合适当的反屏幕和丰富的技术，以快速'淘汰'小 非达特特异性的分子。210，240种化合物的HTS由Southern Research完成 (SR)，得到选择性指数>10的两种化合物SRI-43627和SRI-43050，其进一步被纯化。 研究了这一初步的成功促使筛选了另外369，203种化合物， 计数器屏幕29命中以被进一步评估。在本申请中，我们建议执行命中验证， 这些化合物的表征以及作为药物开发的一部分， 复合进展途径。我们提出以下目标： 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。 在这项研究结束时，我们希望（a）已经确定了小分子，将特异性抑制 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， 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, 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.