RNA as the catalyst for screening drugs against trypanosomatids

描述(申请人提供)：三种相关的锥虫病原体，布氏锥虫、克氏锥虫和利什曼原虫，分别是非洲昏睡病、沙加1病和利什曼病的病原体。这些重要的病原体影响着全球的大量人口，并导致大量死亡。治疗这些疾病的药物是不合适的，因为它们毒性很大，效果不是很好，而且对这些药物已经产生了抗药性。锥虫线粒体基因的表达需要一种特定形式的RNA编辑，这是这些生物特有的，通过这种编辑，前体mRNA序列通常会因尿苷核苷酸的插入和缺失而发生广泛的变化。编辑后的mRNAs被翻译成氧化磷酸化系统的组成部分。我们发现线粒体RNA编辑在布氏锥虫致病阶段是必不可少的，这一发现为抗锥虫药物提供了一个新的有希望的靶点。编辑是由尚未完全定义或表征的多蛋白复合体(编辑小体)催化的。该项目建议产生化合物来表征编辑体蛋白，并确定它们作为药物靶标的潜力，这些药物将有效地对抗三种相关的锥虫病原体。我们计划利用我们在RNA生物化学方面的专业知识，调整我们基于核酶的检测方法，以高通量筛选抗编辑小体的化合物。具体目标1概述了利用荧光共振能量转移(FRET)开发高通量屏幕的计划，以报告编辑小体的基本功能。特定目的2将描述用于验证在第一个目的中观察到的抑制的特异性的二次检测。这些研究将有可能识别可能选择性干扰编辑体组装和/或功能的化合物，并表征RNA编辑的各种相互作用和反应阶段。他们还将为抗锥虫病药物的开发做出贡献。

DESCRIPTION (provided by applicant): The three related trypanosomatid pathogens, Trypanosoma brucei spec., Trypanosoma cruzi, and Leishmania spec., are the causative agents of African sleeping sickness, Chagas1 disease, and Leishmaniasis, respectively. These important pathogens affect large populations globally and cause numerous deaths. The drugs for these diseases are unsuitable since they are very toxic, not very effective, and resistance to these drugs has developed. Mitochondrial gene expression in trypanosomatids requires a particular form of RNA editing, unique to these organisms, by which precursor mRNA sequences are changed, often extensively, by the insertion and the deletion of uridine nucleotides. The edited mRNAs are translated into components of the oxidative phosphorylation system. Our finding that mitochondrial RNA editing is essential in the disease-causing stage of T. brucei has suggested a new promising target for anti- trypanosomatid drugs. Editing is catalyzed by a multiprotein complex (editosome) that has not yet been fully defined or characterized. This project proposes to generate chemical compounds to characterize the editosome proteins and determine their potential as targets for drugs that will be effective against the three related trypanosomatid pathogens. We are planning to use our expertise in the RNA biochemistry and adapt our ribozyme based assay for high throughput screening of compounds against the editosome. Specific Aim 1 outlines the plan to develop a high throughput screen using fluorescence resonance energy transfer (FRET) to report on the essential functions of the editosome. Specific Aim 2 will describe the secondary assays to validate the specificity of inhibition observed in the first aim. These studies will potentially identify compounds that may selectively perturb editosome assembly and/or function and characterize the various interactions and reaction stages of the RNA editing. They will also contribute to development of drugs against trypanosomatids.