RNA as the catalyst for screening drugs against trypanosomatids

描述（由申请人提供）：三种相关的锥虫病病原体，布鲁氏锥虫，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。这些重要病原体影响全球大量人口，并造成大量死亡。治疗这些疾病的药物是不合适的，因为它们毒性很大，不是很有效，而且对这些药物的耐药性已经产生。锥虫线粒体基因的表达需要一种特殊形式的RNA编辑，这是这些生物体独有的，通过这种编辑，前体mRNA序列通常通过尿苷核苷酸的插入和删除而发生广泛改变。编辑后的mrna被翻译成氧化磷酸化系统的组成部分。我们发现线粒体RNA编辑在布鲁氏锥虫致病阶段至关重要，这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物（编辑体）催化的。本项目拟生成化合物来表征编辑体蛋白，并确定其作为药物靶点的潜力，这些药物将对三种相关的锥虫病原体有效。我们计划利用我们在RNA生物化学方面的专业知识，并调整我们基于核酶的检测方法，以高通量筛选针对编辑体的化合物。具体目标1概述了利用荧光共振能量转移（FRET）开发高通量筛选的计划，以报告编辑体的基本功能。特异性目标2将描述二级分析，以验证第一个目标中观察到的抑制的特异性。这些研究将潜在地确定可能选择性地干扰编辑体组装和/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。它们还将有助于开发抗锥虫的药物。

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.