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

项目总结/摘要 该项目将针对相关的RNA编辑多蛋白复合物（编辑体） 锥虫病原体（布氏锥虫（Trypanosoma brucei），T.和利什曼原虫属（Leishmania spp.）），它们是导致 非洲昏睡病、恰加斯病和利什曼病的病原体。它将识别抑制剂 这一基本过程，利用FRET为基础的检测灵敏的“混合和测量”的能力， 体外监测T.我们在之前的R21检测开发中开发了布鲁氏菌 格兰特.我们的初步数据表明，编辑体在锥虫中是高度保守的;这些 数据表明，重要的可能性，抑制剂对T。布鲁氏菌编辑体蛋白也将被 有效对抗相关锥虫病原体中的编辑体（T. cruzi和利什曼原虫属（Leishmania spp.）。到 为了解决测定的可行性，我们完成了对具有生物活性的化合物库的中试筛选 （LOPAC 1280），并发现了新的抑制剂，特异性干扰RNA编辑和杀死T。布鲁塞因 体外受这些最近实验的启发，我们建议筛选一个大型化合物库， 与Conrad Prebys Center for Chemical Genomics（Sanford Burnham Prebys）合作 医学发现研究所。在目标1中，将制备编辑体和RNA，同时我们进一步验证， 1536-以及384孔RNA编辑测定，并在HTS活动中实施。目标2将确认RNA 编辑抑制剂，并确定其效力（IC 50），并将评估血流形式的生长抑制 中医布鲁塞。目的3将阐明化学支架的新生构效关系（SAR）， 优化所选择的命中以改善靶特异性和抑制剂的效力。然后，它将调查MOA 和最佳抑制化合物的跨物种效应，然后放大以基准其ADME/T PK属性（& P）。这些抑制性化合物将提供良好表征的化学工具或探针， 剖析编辑体蛋白质的动态组装和可能作为新的优先目标， 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.