Signature-based chemical screening for DYT6 dystonia

描述(申请人提供)：DYT6肌张力障碍是一种遗传性运动障碍，治疗方案很少。受影响的人会出现不自主的肌肉收缩，主要是上肢和颅骨区域，并因喉部肌张力障碍而经常出现语音缺陷。该致病基因最近被鉴定为THAP1，它编码一种DNA结合蛋白--THAP1(Thanatos-Associated Protein[THAP]Domaining Anotation-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重要原因。大多数突变影响已知的对THAP1DNA结合活性至关重要的残基，这提出了DYT6致病机制可能涉及由于功能THAP1蛋白水平不足而导致转录活性异常的假设。与这一假设一致，我们在携带DYT6突变之一的淋巴母细胞中检测到一种转录表型，相对于对照细胞。在这个项目中，我们建议开发和试验一种新的检测方法来确定治疗DYT6的新药候选。该分析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的药物发现，如急性髓系白血病(AML)和尤文肉瘤。使用DNA微阵列，我们将首先定义一个区分患者和对照细胞的基因表达特征，使用代表18个DYT6基因型的线条来寻找代表DYT6疾病状态的共同标记。然后，该签名将被转换为基于Luminex FlexMAP系统的低成本分析，该系统提供了一种自动化方法，用于以传统的基于细胞的分析格式从细胞中捕获和量化目标转录本。在验证Luminex试验以足够的重复性和Z‘因子重现微阵列签名后，我们将在15,000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。我们预计该项目的潜在成果是：(1)可用于大规模筛查活动以寻找新的DYT6疗法的验证试验；(2)已建立的基于签名的筛查平台，可推广到其他形式的遗传性肌张力障碍；以及(3)来自试点筛查的候选人将在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.