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.

我们使用酵母开发了用于弗里德里希共济失调（FRDA）的主要高通量药物筛查测定法 模型系统以及使用主要FRDA成纤维细胞的二级筛选测定法。两种测定是 表型，基于线粒体功能障碍在体征和症状中的关键作用 FRDA。在主要测定中，我们使用一个 可诱导/抑制启动子，然后使用简单的 分光光度读数。我们将测定优化为> 8的S/B值，Z'分数> 0.7。我们 成功地在南方研究所实施了我们的测定 102,000个复合的Ninds库。南部研究所也将进行反屏幕（ 识别所有真正的命中化合物的假阳性）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中的次级测定的次级化合物的优先级，2）确定 作用机理，以及3）确定FRDA治疗剂的潜在靶蛋白和途径。我们的 总体目标是确定FRDA治疗的化合物和细胞靶标。我们的具体目的是： 1。在次级测定中从主屏幕上测试命中化合物。我们将确定效果 FRDA酵母模型中ATP总体产生的HIT化合物。 2。在主要FRDA成纤维细胞中的AIM 1测试化合物。我们将确定化合物的影响 在谷胱甘肽消耗的背景下，四唑次染料还原和生存。 3。测试化合物的作用机理。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。进行YFH1缺失细胞的遗传抑制分析。我们将利用完整的 酵母敲除菌株库，使用遗传抑制剂鉴定其他靶蛋白和途径 分析。我们将在主要FRDA成纤维细胞中确认我们的发现。