Cell Model of Parkinson's Disease and Screening for Therapeutic Compounds

描述(申请人提供)：神经保护策略的发展，以防止帕金森病的神经细胞死亡将通过遗传细胞模型得到加强。用小分子筛选这样的细胞模型可以确定药物开发的主要候选者。突变蛋白在培养物中的表达可用于建立常染色体显性遗传性神经退行性疾病的模型。我们建立了突变的亨廷顿蛋白在PC12细胞中可诱导表达的细胞模型。我们通过测试FDA批准的1040种化合物的NINDS化合物集合，表明该模型可以用于中等吞吐量的筛选。该屏幕的命中率约为0.4-0.5%。我们现在建议开发一个类似的帕金森氏病遗传细胞模型，使用突变的α-突触核蛋白的诱导表达。我们有初步数据表明，过度表达E46K突变体α-突触核蛋白本身就会导致分化的PC12细胞中神经细胞死亡。我们将测试目前候选的神经保护剂，并进行NINDS化合物收集的筛选，以演示中等吞吐量筛选的实用性。在具体目标1中，我们将对几个诱导表达E46Kα-突触核蛋白的PC12细胞株进行鉴定，以获得低背景表达、高诱导表达和一致毒性的株系。我们将优化条件，获得可靠的筛选方法。在具体目标2中，我们将测试目前被认为是帕金森病神经保护治疗候选化合物，如辅酶Q10、半胱氨酸天冬氨酸氨基转移酶抑制剂z-VAD等，以确定它们在我们的模型中是否具有神经保护活性。在具体目标3中，我们将对FDA批准的1040个NINDS化合物集合和相关化合物进行中等通量筛选，以抑制毒性。所有阳性结果都将得到确认，并将建立剂量-反应曲线，以确定近似的抑制效力。经过验证的阳性HITS将在未来用于PD动物模型的测试。帕金森病的细胞模型和治疗化合物的筛选 项目简介：帕金森病(PD)是第二种最常见的神经退行性疾病。目前还没有治愈或有效的神经保护治疗方法。我们的长期目标是在帕金森病模型中开发有效的神经保护剂，为帕金森病的临床治疗试验做准备。我们提出的研究有可能找到具有神经保护作用的化合物，并进一步用于帕金森病的临床前和临床试验。

DESCRIPTION (provided by applicant): The development of neuroprotective strategies to prevent neuronal cell death in Parkinson's disease would be enhanced by genetic cell models. Screening of such a cell model with small molecules could identify lead candidates for drug development. Expression of mutant proteins in culture can be used to model autosomal dominant neurodegenerative diseases. We have developed an inducible cell model expression of mutant huntingtin in PC12 cells. We have shown that this model can be used for medium throughput screens by testing the NINDS compound collection of 1040 FDA approved compounds. The hit rate in this screen was approximately 0.4 - 0.5%. We now propose to develop a similar genetic cell model for Parkinson's disease, using inducible expression of mutant alpha-synuclein. We have preliminary data indicating that over expression E46K mutant alpha-synuclein itself causes neuronal cell death in differentiated PC12 cells. We will test current candidate neuroprotective agents, and perform a screen of the NINDS compound collection to demonstrate the utility for moderate throughput screens. In Specific Aim 1, we will characterize several PC12 cell lines inducibly expressing E46K alpha-synuclein, in order to obtain lines with low background expression, high inducible expression, and consistent toxicity. We will optimize conditions and get a reliable assay for screening. In Specific Aim 2, we will test compounds currently considered candidates for neuroprotective therapy for PD, such as Co-enzyme Q10, caspase inhibitor z-VAD and others to determine whether they have neuroprotective activity in our model. In Specific Aim 3, we will carry out a medium throughput screen of the 1040 NINDS compound collection of FDA-approved and related compounds, for inhibition of toxicity. All positives will be confirmed, and dose- response curve will be established to determine approximate inhibitory potencies. Validated positive hits could then be taken to testing in PD animal models in future.Cell Model of Parkinson's Disease and Screening for Therapeutic Compounds Project Narrative: Parkinson's disease (PD) is the second most common neurodegenerative disease. There is currently no cure or effective neuroprotective treatment. Our long-term goal is to develop effective neuroprotective agents in PD models in order to prepare for clinical therapeutic trials in PD. Our proposed research has potential to find compounds which are neuroprotective and further be taken to preclinical and clinical trials of PD.