Validation and characterization of Tat inhibitors identified through HTS

通过 HTS 鉴定的 Tat 抑制剂的验证和表征

基本信息

批准号：
10468812
负责人：
Susana T Valente
金额：
$ 23.13万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-12 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10468812
关键词：
Affinity Animal Model Anti-HIV Agents Anti-Retroviral Agents Apical Apoptosis Automobile Driving Biochemical Biological Assay CD4 Positive T Lymphocytes Calorimetry Cell Line Cell model Cells Chemicals Chimeric Proteins Clinical Clinical Trials Collaborations Complement Complex Confocal Microscopy DNA Development Disease remission Electrophoretic Mobility Shift Assay Epigenetic Process Feedback Fluorescence Future Gene Silencing Genetic Transcription Genome Goals HIV HIV-1 Hela Cells Human In Vitro Individual Interruption Lead Letters Luciferases Metabolic Modification Molecular Computations Neurotransmitters Nuclear Oxidative Stress Pathway interactions Peripheral Blood Mononuclear Cell Pharmaceutical Preparations Pharmacology Phase Production Property Proviruses RNA Research Role Structure Structure-Activity Relationship Sum System TNF gene Techniques Testing Tetanus Helper Peptide Therapeutic Intervention Titrations Toxic effect Transactivation Transcript Triage Tryptophan Validation Viral Viral Proteins Viral reservoir Virus Virus Replication analog antiretroviral therapy base blood-brain barrier disruption chemical synthesis chromatin immunoprecipitation clinical candidate cortistatin cost cost effective counterscreen cytotoxicity detection limit drug development follow-up high throughput screening high-throughput drug screening humanized mouse in vitro testing indexing inhibitor interest molecular dynamics mouse model neuroinflammation neurotoxicity novel novel drug class novel strategies particle pre-clinical promoter scaffold small molecule success targeted treatment tat Protein viral rebound viral resistance

项目摘要

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.

抽象的尽管有史刺激或治疗中断。病毒TAT蛋白增强了HIV-1的转录伸长启动子，控制潜伏期和活动病毒产生之间的切换。阻止锁的功能 CURE的目的旨在使病毒疗效的转录沉默使抑制艾滋病毒启动子极度抑制难以从延迟中重新激活。使用TAT抑制剂Didehydro-Cortistatin A（DCA）证明这一点概念。将DCA与ART结合，抑制转录并在治疗中断时阻止病毒反弹，随着启动子的表观反射。 DCA定义了一类新型药物，这些药物可以沉默，并且维持转录的无活性HIV启动子，为艾滋病毒治疗提供了一种新的方法。 TAT是治疗干预的非常有吸引力的靶标，因为：1）在病毒复制期间早期表达； 2）无细胞同源物； 3）TAT抑制剂阻止病毒扩增所需的反馈回路； 4）表观遗传学在HIV启动子渲染的重新激活下积累的修饰可能较小。塔特也以其角色而闻名在神经毒性，神经递质调节，氧化应激，凋亡，血脑屏障破坏和神经炎症。这是对TAT抑制剂的发展对完成艺术的巨大兴趣。将DCA推进临床试验的主要障碍是生产大量的成本分子由于其复杂的结构。其他在结构上与DCA不同的临床候选者，在临床前管道中需要体现同等生物活性。我们优化了基于细胞的TAT反式激活测定法，以在高吞吐量筛选（HTS）中使用DCA作为控制。我们结合了适当的柜台和丰富的技术，以快速“清除”小型不是特异性的分子。南方研究完成了210,240种化合物的HTS （SR），产生两种化合物，SRI-43627和SRI-43050，具有选择性指数> 10 调查。这一最初的成功促使屏幕又获得了369,203种化合物，从而产生计数器屏幕29命中以进一步评估。在此应用程序中，我们建议执行HIT验证和这些化合物的表征以及由SR合成的类似物作为药物开发的一部分复合进程途径。我们提出以下目标：具体目标1。基于TAT HIV-1 LTR反式激活的破坏来验证TAT抑制剂。特定目的2。表征选定命中的作用机理。在这项研究结束时，我们期望（a）确定了小分子，这些分子会特别抑制基于细胞的测定中的TAT。（b）具有足够的代谢稳定性和未来的PK特性动物模型和人类临床试验中的药理评估。