Signature-based chemical screening for DYT6 dystonia

描述（由申请人提供）：DYT6肌张力障碍是一种遗传性运动障碍，目前很少有治疗选择。受影响的个体出现不随意肌肉收缩，主要是上肢和颅骨区域，由于喉张力障碍而经常出现语言缺陷。最近发现的致病基因是THAP1，它编码一种DNA结合蛋白THAP1 （thanatos-associated protein [THAP] domain containing apoptosis-associated protein- 1）。多个研究小组现在已经独立地将35个THAP1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DYT6是家族性肌张力障碍的一个重要原因。大多数突变影响已知对THAP1 DNA结合活性至关重要的残基，这提出了DYT6发病机制可能涉及由于THAP1功能性蛋白水平不足而导致的异常转录活性的假设。与这一假设相一致，我们在淋巴母细胞中检测到与对照细胞相比，携带DYT6突变之一的转录表型。在这个项目中，我们建议开发和试点一种新的检测方法，以确定治疗DYT6的新候选药物。该检测使用的技术已广泛应用于与异常转录因子活性类似的癌症的药物发现，如急性髓性白血病（AML）和尤文氏肉瘤。使用DNA微阵列，我们将首先定义一个区分患者与对照细胞的基因表达特征，使用代表18个DYT6基因型的细胞系来寻找代表DYT6疾病状态的共同标记。然后，该标记将转换为基于Luminex FlexMAP系统的低成本分析，该系统提供了一种自动方法，以传统的基于细胞的分析格式捕获和定量细胞中的目标转录物。在验证Luminex检测重现了具有足够可重复性和Z因子的微阵列特征后，我们将在15000个小分子的混合集合中进行筛选，以寻找使DYT6转录表型正常化的化合物。我们期望该项目的潜在结果是：(1)可用于大规模筛查活动的验证分析，以发现新的DYT6治疗方法；(2)可推广到其他形式的遗传性肌张力障碍的已建立的基于特征的筛查平台；(3)从试点筛选的候选者在DYT6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.