Large-scale screen with a novel assay against RNA editing to identify anti-trypanosomal agents

项目摘要/摘要 该项目将针对相关的必要的RNA编辑多蛋白复合体(编辑体) 锥虫病原体(布氏锥虫、克氏锥虫和利什曼原虫) 非洲昏睡病、恰加斯病和利什曼病的病原体。它将识别出抑制剂 在这一基本过程中，利用基于FRET的检测能力进行敏感的“混合和测量” 布鲁氏毛滴虫RNA编辑活性的体外监测，这是我们在之前的R21试验中开发的 格兰特。我们的初步数据显示，编辑小体在锥虫中高度保守；这些 数据表明，针对布氏锥虫编辑体蛋白的抑制剂的重要可能性也将是 对相关锥虫病原体(克氏锥虫和利什曼原虫)的编辑体有效。至 为了解决化验的可行性，我们完成了药理活性化合物文库的中试筛选 (LOPAC1280)，并发现了新的特异性干扰RNA编辑并杀死布氏毛滴虫的抑制剂 体外培养。在最近这些实验的推动下，我们建议筛选一个大型化合物库来对抗 编辑体与桑福德·伯纳姆·普雷比化学基因组学中心合作 医学探索研究所。在目标1中，将制备编辑小体和RNA，同时我们将进一步微型化以 1536-很好的384井RNA编辑试验，并在HTS活动中实施。AIM 2将确认RNA 编辑抑制剂并确定其效力(IC50)，并将评估对血流形式的生长抑制 布氏毛滴虫属的一种。目标3将阐明化学支架的新生结构-活性关系(SAR)和 优化选定的HITS，以提高靶向特异性和抑制剂的效力。然后，它将调查MOA 和跨物种效应最好的抑制化合物(S)，然后放大他们的ADME/T 主键属性(&P)。这些抑制化合物(S)将提供表征良好的化学工具(S)或探针(S)来 剖析编辑体蛋白的动态组装和可能作为新的优先靶点 针对锥虫病原体的治疗支架。

PROJECT SUMMARY / ABSTRACT This project will target the essential RNA editing multi-protein complex (the editosome) in related trypanosomatid pathogens (Trypanosoma brucei, T. cruzi, and Leishmania spp.), which are the causative agents of African sleeping sickness, Chagas' disease, and leishmaniasis, respectively. It will identify inhibitors of this essential process, utilizing the capacity of a FRET-based assay for a sensitive “mix and measure” in vitro monitoring of RNA editing activity in T. brucei, which we developed in a previous R21 assay development grant. Our preliminary data show that the editosomes are highly conserved among trypanosomatids; these data suggest the important possibility that inhibitors found against T. brucei editosome proteins will also be effective against the editosome in related trypanosomatid pathogens (T. cruzi and Leishmania spp.). To address the assay feasibility, we completed a pilot screen of the library of pharmacologically active compounds (LOPAC1280) and discovered new inhibitors that specifically interfere with RNA editing and kill T. brucei in vitro. Motivated by these recent experiments, we propose to screen a large library of compounds against the editosome in collaboration with Conrad Prebys Center for Chemical Genomics at Sanford Burnham Prebys Medical Discovery Institute. In Aim 1, editosomes and RNAs will be prepared, while we further miniaturize to 1536-well the 384-well RNA editing assay, and implement it in an HTS campaign. Aim 2 will confirm RNA editing inhibitors and determine their potency (IC50), and will assess growth inhibition of the bloodstream forms of T. brucei. Aim 3 will elucidate the nascent structure-activity relationships (SAR) of chemical scaffolds and optimize the selected hits to improve target specificity and potency of inhibitors. It will then investigate the MOA and cross-species effect of the best inhibitory compound(s), followed by scale-up to benchmark their ADME/T & PK properties. These inhibitory compound(s) will provide well-characterized chemical tool(s) or probe(s) to dissect the dynamic assembly of the editosome proteins and preferential targets that may serve as new therapeutic scaffolds against trypanosomatid pathogens.