Signature-based chemical screening for DYT6 dystonia

描述（由申请人提供）：DYT 6肌张力障碍是一种遗传性运动障碍，治疗选择很少。受影响的个人发展不自主的肌肉收缩，主要是上肢和颅区，由于喉肌张力障碍，经常言语缺陷。致病基因最近被鉴定为THAP 1，其编码DNA结合蛋白THAP 1（含死亡相关蛋白[THAP]结构域的死亡相关蛋白-1）。多个研究小组已经独立地将35个THAP 1突变与世界各地遗传多样性人群中的肌张力障碍联系起来，从而揭示了DYT 6是家族性肌张力障碍的重要原因。大多数突变影响已知对THAP 1的DNA结合活性至关重要的残基，提出了DYT 6发病机制可能涉及由于功能性THAP 1蛋白水平不足而导致的异常转录活性的假设。与该假设一致，我们已经检测到相对于对照细胞，在携带DYT 6突变之一的成淋巴细胞中的转录表型。在这个项目中，我们建议开发和试验一种新的检测方法，用于鉴定治疗DYT 6的新候选药物。该检测使用的技术已在很大程度上应用于与异常转录因子活性类似的癌症药物发现，如急性髓性白血病（AML）和尤文肉瘤。使用DNA微阵列，我们将首先定义区分患者和对照细胞的基因表达特征，使用代表18种DYT 6基因型的细胞系来寻找代表DYT 6疾病状态的共同标志物。然后将该特征转化为基于Luminex FlexMAP系统的低成本测定，该系统提供了用于以常规的基于细胞的测定形式捕获和定量来自细胞的靶转录物的自动化方法。在验证Luminex测定法以足够的再现性和Z'因子重现微阵列特征之后，我们将在15，000个小分子的混合集合中试验筛选，以寻找使DYT 6转录表型标准化的化合物。我们预期该项目的潜在结果是：（1）可用于大规模筛选活动以发现新的DYT 6治疗剂的经验证的测定法;（2）可推广至其他形式的遗传性肌张力障碍的已建立的基于特征的筛选平台;和（3）来自中试筛选的候选物，以在DYT 6模型系统中进一步表征。

DESCRIPTION (provided by applicant): DYT6 dystonia is a hereditary movement disorder for which few treatment options exist. Affected individuals develop involuntary muscle contractions, primarily of upper limbs and cranial region with frequent speech defects due to laryngeal dystonia. The causative gene was recently identified as THAP1 which encodes a DNA binding protein, THAP1 (thanatos-associated protein [THAP] domain-containing apoptosis-associated protein- 1). Multiple groups have now independently linked 35 THAP1 mutations to dystonia in genetically diverse populations throughout the world, thereby revealing DYT6 as a substantial cause of familial dystonia. Most mutations impact residues known to be critical for THAP1's DNA binding activity, raising the hypothesis that DYT6 pathogenesis may involve aberrant transcriptional activity due to insufficient levels of functional THAP1 protein. Consistent with that hypothesis, we have detected a transcriptional phenotype in lymphoblasts bearing one of the DYT6 mutations, relative to control cells. In this project we propose to develop and pilot a novel assay for identifying new drug candidates for treating DYT6. The assay uses technology that has been largely applied to drug discovery in cancers similarly linked to aberrant transcription factor activity, such as acute myeloid leukemia (AML) and Ewing sarcoma. Using DNA microarrays, we will first define a gene expression signature distinguishing patient from control cells, using lines representing 18 DYT6 genotypes to find common markers representative of the DYT6 disease state. That signature will then be converted to a low cost assay based on the Luminex FlexMAP" system, which provides an automated method for capturing and quantifying target transcripts from cells in a conventional cell-based assay format. After validating that the Luminex assay recapitulates the microarray signature with sufficient reproducibility and Z' factor, we will pilot the screen in a mixed collection of 15,000 small molecules to seek compounds that normalize the DYT6 transcriptional phenotype. We expect potential outcomes of this project to be: (1) a validated assay that could be used for a large scale screening campaign to find novel DYT6 therapeutics (2) an established signature- based screening platform that could be generalized to other forms of hereditary dystonia; and (3) candidates from the pilot screen to be further characterized in DYT6 model systems.