Drug and drug target identification for Friedreich ataxia

Friedreich 共济失调的药物和药物靶点鉴定

• 批准号: 7571979
• 负责人: ROBERT B WILSON
• 金额: $ 7.88万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7571979
• 关键词: Biochemical Genetics; Biological Assay; Cells; Dose; Drug Delivery Systems; Dyes; Fibroblasts; Friedreich Ataxia; Genetic; Glutathione; Goals; Hereditary Spastic Paraparesis; Homologous Gene; Human; Huntington Disease; Knock-out; Knowledge; Libraries; Mitochondria; Parkinson Disease; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Preclinical Drug Evaluation; Process; Production; Proteins; Research Institute; Role; Score; Screening procedure; Signs and Symptoms; Testing; Tetrazolium; Therapeutic; Yeast Model System; Yeasts; base; cellular targeting; compound 30; frataxin; mitochondrial dysfunction; nervous system disorder; promoter; response

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.

我们开发了一个初级的，高通量的药物筛选试验弗里德赖希共济失调（FRDA）使用酵母 模型系统，以及使用原代FRDA成纤维细胞的二次筛选测定。了两种试验 表型和基于线粒体功能障碍的关键作用，在体征和症状， FRDA。在最初的试验中，我们使用一种抑制剂关闭了酵母共济失调蛋白同源物Yfhlp的表达。 诱导型/阻遏型启动子，然后在96孔板中使用简单的 分光光度读数我们将我们的测定优化为S/B值> 8，Z'得分> 0.7。我们 在南方研究所成功地实施了我们的检测，该研究所正在进行筛选 102，000个化合物的NINDS库。南方研究所还将进行反屏幕（以 识别假阳性）和所有真正命中化合物的剂量反应分析。该提案描述了 我们的计划是：1）根据酵母和人类细胞中的二级检测，优先考虑命中化合物，2）确定 作用机制，和3）确定FRDA治疗的潜在靶蛋白和途径。我们 总体目标是鉴定用于治疗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来自初步筛选的命中化合物。我们将确定 在FRDA酵母模型中，命中化合物对整体ATP产生的影响。 2.在原代FRDA成纤维细胞中测试来自Aim 1的化合物。我们将确定化合物的效果 四唑染料还原和谷胱甘肽耗竭情况下的存活率。 3.测试化合物的作用机制。我们将利用完整的酵母库 敲除菌株以确定靶蛋白和途径。我们将在主要FRDA中确认我们的发现 成纤维细胞... 4.对Yfh 1缺失细胞进行遗传抑制分析。我们将充分利用 酵母敲除菌株文库，以使用遗传抑制因子鉴定另外的靶蛋白和途径 分析.我们将在原代FRDA成纤维细胞中证实我们的发现。