Drug and drug target identification for Friedreich ataxia

我们利用酵母菌建立了一种初步的、高通量的弗里德里希共济失调(FRDA)药物筛选试验 模型系统，以及使用原代FRDA成纤维细胞的二次筛选试验。两种检测方法都是 表型和基于线粒体功能障碍的关键作用的体征和症状 FRDA。在初步实验中，我们关闭了酵母Frataxin同系物Yfhlp的表达。 可诱导/可抑制启动子，然后在96孔板中使用简单的 分光光度读数。我们优化了我们的检测方法，使S/B值为&gt；8，Z‘值为&gt；0.7。我们 在南方研究所成功实施了我们的检测，该研究所正在进行筛选 包含102,000种化合物的NINDS文库。南方研究所还将进行反审查(至 识别假阳性)和所有真正的命中化合物的剂量-反应分析。这份提案描述了 我们的计划是：1)根据酵母和人体细胞中的二次化验确定HIT化合物的优先顺序，2)确定 作用机制，以及3)确定FRDA疗法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.在二次酵母菌试验中测试初筛的Hit化合物。我们将确定其影响 在FRDA的酵母模型中，HIT化合物对总体ATP产量的影响。 2.在原代FRDA成纤维细胞中检测Aim 1化合物。我们将确定化合物的影响 四唑盐染料还原和谷胱甘肽耗竭背景下的生存。 3.测试化合物的作用机制。我们将利用完整的酵母文库 确定目标蛋白和途径的基因敲除菌株。我们将在初级FRDA中确认我们的发现 成纤维细胞。..。 4.对Yfh1缺失细胞进行基因抑制分析。我们将利用完整的 酵母基因敲除菌株文库用于鉴定额外的靶蛋白和利用基因抑制因子的途径 分析。我们将在原代FRDA成纤维细胞中证实我们的发现。

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, Yfhlp, 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 secondaryyeast 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.