Drug and drug target identification for Friedreich ataxia

描述（由申请人提供）：我们使用酵母模型系统开发了弗里德里希共济失调（FRDA）的初级高通量药物筛选试验，以及使用初级FRDA成纤维细胞的二级筛选试验。这两种检测都是表型的，并基于线粒体功能障碍在FRDA体征和症状中的关键作用。在初步实验中，我们使用可诱导/可抑制启动子关闭酵母fraataxin同源物Yfh1p的表达，然后在96孔板中使用简单的分光光度法读取线粒体功能。优化后的S/B值为> 8，Z′值为> 0.7。我们在南方研究所成功实施了我们的分析，该研究所正在筛选102,000个化合物的NINDS文库。南方研究所还将对所有真正受到打击的化合物进行反筛选（以识别假阳性）和剂量反应分析。该提案描述了我们的计划：1)根据酵母和人类细胞的二次分析确定击中化合物的优先级，2)确定作用机制，3)确定FRDA治疗的潜在靶蛋白和途径。我们的总体目标是确定治疗FRDA的化合物和细胞靶点。我们的具体目标是：1；在二次酵母试验中测试从初级筛选中获得的命中化合物。我们将在FRDA酵母模型中确定hit化合物对总ATP产生的影响。2. 在原代FRDA成纤维细胞中检测来自Aim 1的化合物。我们将确定在谷胱甘肽耗竭的情况下，化合物对四氮唑染料还原和生存的影响。3. 测试化合物的作用机制。我们将利用酵母敲除菌株的完整文库来确定目标蛋白和途径。我们将在原代FRDA成纤维细胞中证实我们的发现。4. 对Yfh1缺失细胞进行基因抑制分析。我们将利用酵母敲除菌株的完整文库，利用遗传抑制因子分析来确定额外的靶蛋白和途径。我们将在原代FRDA成纤维细胞中证实我们的发现。

DESCRIPTION (provided by applicant): We developed a primary, high-throughput drug-screening assay for Friedreich ataxia (FRDA) using a yeast model system, as well as a secondary screening assay using primary FRDA fibroblasts. Both assays are phenotypic and are based on the critical role of mitochondrial dysfunction in the signs and symptoms of FRDA. In the primary assay we turn off the expression of the yeast frataxin homologue, Yfh1p, using an inducible/repressible promoter, and then follow mitochondrial function in 96-well plates using a simple spectrophotometric readout. We optimized our assay to S/B values of > 8, and Z' scores of > 0.7. We successfully implemented our assay at the Southern Research Institute, which is in the process of screening the 102,000-compound NINDS library. The Southern Research Institute will also perform counter-screens (to identify false-positives) and dose-response analyses of all bona fide hit compounds. This proposal describes our plan to: 1) prioritize hit compounds based on secondary assays in yeast and human cells, 2) determine mechanisms of action, and 3) identify potential target proteins and pathways for FRDA therapeutics. Our overall goal is to identify compounds and cellular targets for the treatment of FRDA. Our Specific Aims are: 1. To test hit compounds from the primary screen in a secondary yeast assay. We will determine the effects of hit compounds on overall ATP production in the yeast model of FRDA. 2. To test compounds from Aim 1 in primary FRDA fibroblasts. We will determine the effects of compounds on tetrazolium dye reduction and on survival in the context of glutathione depletion. 3. To test compounds for mechanisms of action. We will take advantage of the complete library of yeast knockout strains to determine target proteins and pathways. We will confirm our findings in primary FRDA fibroblasts. 4. To perform a genetic suppressor analysis of Yfh1 -depleted cells. We will take advantage of the complete library of yeast knockout strains to identify additional target proteins and pathways using genetic suppressor analysis. We will confirm our findings in primary FRDA fibroblasts.