Friedreich Ataxia High Throughput Drug Screening Assays

描述(由申请人提供)： 弗里德里希共济失调(FRDA)是一种常染色体隐性遗传性神经退行性疾病。FRDA的体征和症状使人联想到线粒体脑肌病，包括进行性四肢共济失调、构音障碍、反射障碍、感觉丧失和肌肉无力。大多数患者存在骨骼畸形和心肌病，30%的患者存在糖耐量受损和糖尿病，偶尔还会出现视力下降和听力损失。症状通常出现在青春期左右，大多数患者在快30岁时只能坐在轮椅上。心肌衰竭是导致过早死亡的最常见原因。FRDA是最常见的遗传性共济失调，在欧洲人群中的患病率约为40,000人中就有1人，目前还没有被证明有效的治疗方法。FRDA是由编码蛋白质Frataxin的FRDA基因突变引起的。虽然Frataxin编码在细胞核中，但它被输入到线粒体基质中。对酵母和小鼠Frataxin同系物以及患者材料的研究表明，由氧化损伤和伴随的线粒体铁积累引起的线粒体功能障碍是FRDA的症状和体征的基础。在酵母模型系统和原代FRDA细胞中使用简单的线粒体功能测量的初步研究支持使用基于细胞的方法对FRDA进行高通量药物筛选的可行性。然后，来自这样一个屏幕的热门化合物可以在最近开发的这种疾病的小鼠模型中进行测试。Hit化合物的蛋白质靶标可以在酵母中使用化学遗传方法来识别，从而允许进一步的药物开发。拟议研究的总体目标是确定FRDA的潜在治疗方法。具体目标是：1)建立高通量、基于细胞的FRDA药物筛选方法。由于线粒体功能障碍是FRDA的体征和症状的基础，我们的检测将旨在筛选能够改善线粒体功能的化合物。我们将在酵母模型系统和原代FRDA细胞中使用适合于96孔格式的线粒体功能测量。2)建立一种用于药物靶标鉴定的化学遗传筛选方法。我们将在酵母模型系统中使用菌落颜色筛选技术，从我们的高通量药物筛选试验中鉴定FRDA的HIT化合物的目标蛋白。

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is an autosomal recessive, inherited neurodegenerative disorder. The signs and symptoms of FRDA are reminiscent of the mitochondrial encephalomyopathies and include progressive ataxia of all four limbs, dysarthria, areflexia, sensory loss, and muscle weakness. Skeletal deformities and cardiomyopathy are found in most patients, impaired glucose tolerance and diabetes mellitus are found in -30% of patients, and reduced visual acuity and hearing loss are occasionally seen. Onset of symptoms usually occurs around puberty and most patients are confined to a wheelchair by their late 20s. Myocardial failure is the most common cause of premature death. FRDA is the most common hereditary ataxia, with a prevalence of approximately 1 in 40,000 in European populations, and there is currently no proven effective treatment. FRDA is caused by mutations in the FRDA gene, which encodes the protein frataxin. Although encoded in the nucleus, frataxin is imported into the mitochondrial matrix. Studies of yeast and murine frataxin homologues, and of patient material, indicate that mitochondrial dysfunction, caused by oxidative damage and concomitant mitochondrial iron accumulation, underlies the signs and symptoms of FRDA. Preliminary studies using simple measures of mitochondrial function in the yeast model system, and in primary FRDA cells, support the feasibility of using a cell-based approach to high-throughput drug screening for FRDA. Hit compounds from such a screen could then be tested in the recently developed mouse models of the disease. The protein targets of hit compounds could be identified using a chemical genetic approach in yeast, allowing further drug development. The overall goal of the proposed research is to identify potential treatments for FRDA. The Specific Aims are: 1) To develop high-throughput, cell-based drug screening assays for FRDA. Because mitochondrial dysfunction underlies the signs and symptoms of FRDA, our assays will be designed to screen for compounds that improve mitochondrial function. We will use measures of mitochondrial function suitable for a 96-well format, in the yeast model system, and in primary FRDA cells. 2) To develop a chemical genetic screening assay for the identification of drug targets. We will use a colony-color screening technique in the yeast model system to identify the target proteins of hit compounds from our high-throughput drug screening assays for FRDA.