Identifying novel therapeutics for early onset dystonia

早发性扭转肌张力障碍（DYT1）是一种主要的遗传性疾病，引起严重的运动障碍，至少部分原因是基底节区多巴胺能神经传递异常，无神经变性。大多数病例是由编码torsinA基因的特定突变引起的，torsinA是一种位于内质网（ER）和核膜中的AAA+蛋白。最近的研究支持torsinA作为内质网伴侣蛋白的作用，通过分泌途径参与蛋白质的加工，突变的torsinA降低了培养细胞的分泌能力。我们的研究目标是鉴定可能减弱突变体torsinA表达的功能影响的化合物，从而作为DYT1肌张力障碍的潜在治疗策略。为此，我们开发了一种新的筛选方法来识别直接结合野生型torsinA和/或肌张力障碍相关突变形式的小分子。利用这种方法，我们最近确定了一组候选分子，其中一个分子似乎可以显著地使从DYT1患者获得的培养成纤维细胞中报告蛋白——高斯荧光素酶（Gluc）的分泌正常化。拟议的研究旨在进一步表征肌张力障碍的新候选药物，分为三个阶段。在第一阶段，我们将使用相同的筛选方法来调查由来自不同类别的20,000个额外化合物组成的微阵列，以确定结合野生型和/或突变型torsinA的新分子。筛查阳性患者将测试其改善DYT1患者和对照组原代成纤维细胞中葡萄糖报告蛋白分泌的能力。在第二阶段，我们将通过合成新的结构类似物来确定我们的候选药物的结构-活性关系（SAR），这些类似物将在葡萄糖分泌实验中进行测试。在第三阶段，我们将检查哪些蛋白质，除了torsinA，是我们的顶级化合物的细胞靶标。我们的顶级分子的生物素化类似物将被合成，并用于下拉试验，从培养细胞中沉淀目标蛋白质，然后用质谱法进行蛋白质鉴定。通过筛选管道筛选化合物，我们希望建立一套具有良好特征的候选药物，这些候选药物可能适合未来在DYT1动物模型中进行评估。

Early onset torsion dystonia (DYT1) is a dominantly inherited disorder causing severe motor impairment thought to be due, at least in part, to abnormal dopaminergic neurotransmission in the basal ganglia without neurodegeneration. Most cases are caused by a specific mutation in the gene encoding torsinA, an AAA+ protein residing in the endoplasmic reticulum (ER) and nuclear envelope. Recent studies support a role for torsinA as an ER chaperone protein involved in processing proteins through the secretory pathway with mutant torsinA decreasing secretory capacity in cultured cells. The goal of our research is to identify compounds which may attenuate the functional effects of mutant torsinA expression, thereby serving as potential therapeutic strategies for DYT1 dystonia. Towards this end, we have developed a novel screening method to identify small molecules which bind directly to wild-type torsinA and/or the dystonia- related mutant form. Using this approach, we recently identified a set of candidate molecules, one of which appears to significantly normalize secretion of a reporter protein, Gaussia luciferase (Gluc), from cultured fibroblasts obtained from DYT1 patients. The proposed studies are designed to further characterize new drug candidates for dystonia in three stages. In stage one, we will use the same screening method to survey microarrays consisting of 20,000 additional compounds from diverse classes to identify new molecules which bind wild-type and/or mutant torsinA. Screening positives will then be tested for their ability to improve secretion of the Gluc reporter in primary fibroblasts from DYT1 patients and control subjects. In stage two, we will define structure-activity relationships (SAR) for our top candidates by synthesizing new structural analogs which will be tested in the Gluc secretion assay. In stage three, we will examine which proteins, in addition to torsinA, are cellular targets of our top compounds. Biotinylated analogs of our top molecules will be synthesized and used in pull-down assays to precipitate target proteins from cultured cells, followed by mass spectrometry for protein identification. By funneling compounds through the proposed screening pipeline, we hope to establish a set of well characterized drug candidates which may be suitable for future evaluation in DYT1 animal models.