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

利用针对 RNA 编辑的新型检测方法进行大规模筛选，以鉴定抗锥虫药物

基本信息

批准号：
10092092
负责人：
REZA Salavati SALAVATI
金额：
$ 36.25万
依托单位：
MCGILL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-06 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10092092
关键词：
Acute Address Affect African Trypanosomiasis Benchmarking Binding Biological Assay Blood Circulation Catalogs Catalytic RNA Cells Chagas Disease Chemicals Chronic Collaborations Communicable Diseases Complex Critical Pathways DNA Ligases Data Databases Development Disease Exonuclease Fluorescence Fluorescence Resonance Energy Transfer Future Gene Expression Genomics Grant Growth Growth Inhibitors Human In Vitro Infection Insecta Institutes Leishmania Leishmania major Leishmaniasis Libraries Ligase Mammalian Cell Measures Medical Mind Monitor Monoclonal Antibodies Morphologic artifacts Multiprotein Complexes Parasites Pharmaceutical Preparations Pharmacology Population Powder dose form Process Property Proteins PubChem RNA RNA Binding RNA Editing RNA Processing Reagent Specificity Structure-Activity Relationship Therapeutic Toxic effect Trypanosoma brucei brucei Trypanosoma cruzi Validation analog assay development base chemical genetics counterscreen endonuclease experimental study genetic approach hammerhead ribozyme high throughput screening human DNA improved in vivo inhibitor/antagonist miniaturize mitochondrial genome mouse model novel novel therapeutics pathogen scaffold scale up small molecule libraries tool

项目摘要

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.

项目概要/摘要该项目将针对相关领域中必需的RNA编辑多蛋白复合物（编辑体）锥虫病原体（布氏锥虫、克氏锥虫和利什曼原虫）分别是非洲昏睡病、恰加斯病和利什曼病的病原体。它将识别抑制剂的这一基本过程，利用基于 FRET 的检测能力进行敏感的“混合和测量” 体外监测 T. brucei 的 RNA 编辑活性，这是我们在之前的 R21 测定开发中开发的授予。我们的初步数据表明，锥虫中的编辑体高度保守。这些数据表明，针对布氏锥虫编辑体蛋白发现的抑制剂也将被有效对抗相关锥虫病原体（克氏锥虫和利什曼原虫属）的编辑体。到为了解决测定的可行性，我们完成了药理活性化合物库的初步筛选 (LOPAC1280) 并发现了特异性干扰 RNA 编辑并杀死 T. brucei 的新抑制剂体外。受这些最近实验的启发，我们建议筛选一个大型化合物库编辑体与桑福德伯纳姆普雷比斯康拉德普雷比斯化学基因组学中心合作医学发现研究所。在目标 1 中，将制备编辑体和 RNA，同时我们进一步小型化 1536 孔 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（IC50），并将评估血流形式的生长抑制布氏锥虫。目标 3 将阐明化学支架的新生构效关系 (SAR) 优化选定的命中，以提高抑制剂的靶标特异性和效力。然后将调查 MOA 和最佳抑制化合物的跨物种效应，然后扩大规模以对其 ADME/T 进行基准测试 & PK属性。这些抑制化合物将提供充分表征的化学工具或探针剖析编辑体蛋白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。