Validation and characterization of Tat inhibitors identified through HTS

摘要 尽管抗逆转录病毒疗法 (ART) 有效，但潜伏的原病毒仍可在细胞上重新启动病毒产生 刺激或治疗中断。病毒 Tat 蛋白增强 HIV-1 转录本延伸 启动子，控制潜伏期和活跃病毒生产之间的转换。块锁功能 治愈的目的是病毒库的转录沉默，从而极大地抑制 HIV 启动子 很难从延迟中重新激活。 Tat 抑制剂二脱氢皮质抑素 A (dCA) 被用来证明这一点 概念。将 dCA 与 ART 相结合，抑制转录并阻止治疗中断时的病毒反弹， 因为启动子受到表观遗传抑制。 dCA 定义了一类新型药物，可以沉默和 维持转录失活的 HIV 启动子，为治疗 HIV 提供了一种新方法。 Tat 是治疗干预非常有吸引力的靶点，因为：1）在病毒复制的早期表达； 2) 没有细胞同源物； 3）Tat抑制剂阻断病毒扩增所需的反馈回路； 4）表观遗传 修饰在 HIV 启动子处累积，导致重新激活的可能性降低。 Tat 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、血脑屏障破坏等 神经炎症。因此，人们对开发 Tat 抑制剂以补充 ART 产生了极大的兴趣。 将 dCA 推进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。其他临床候选药物在结构上与 dCA 不同， 临床前管道中需要体现同等的生物活性。 我们优化了基于细胞的 Tat 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掩护和丰富的技术来快速“淘汰”小 非 Tat 特异性分子。 Southern Research 完成了 210,240 种化合物的 HTS (SR)，产生两种化合物，SRI-43627 和 SRI-43050，其选择性指数 > 10，进一步 调查了。这一初步成功促使我们筛选了另外 369,203 种化合物，结果 计数器屏幕 29 个点击有待进一步评估。在此应用程序中，我们建议执行命中验证并 这些化合物以及 SR 合成的类似物的表征，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. 根据 Tat HIV-1 LTR 反式激活的破坏来验证 Tat 抑制剂。 具体目标 2. 描述所选命中的作用机制。 在本研究结束时，我们期望 (a) 鉴定出能够特异性抑制的小分子 基于细胞的检测中的 Tat。 (b) 具有足够的代谢稳定性和 PK 特性以供未来使用 动物模型和最终人体临床试验中的药理学评估。

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.