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.

我们利用酵母开发了一种针对弗里德赖希共济失调（FRDA）的初级、高通量药物筛选试验