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

描述（由申请人提供）：遗传细胞模型将增强神经保护策略的开发，以防止帕金森病中的神经元细胞死亡。用小分子筛选这种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常染色体显性神经变性疾病的建模。我们已经开发了一种突变亨廷顿蛋白在PC 12细胞中表达的诱导型细胞模型。我们已经表明，该模型可以用于中等通量筛选通过测试1040 FDA批准的化合物的NINDS化合物集合。这个画面的命中率大约是0.4 - 0.5%。我们现在建议开发一个类似的遗传细胞模型帕金森氏病，使用突变体α-突触核蛋白的诱导表达。我们有初步的数据表明，过度表达E46 K突变体α-突触核蛋白本身会导致分化的PC 12细胞中的神经元细胞死亡。我们将测试目前的候选神经保护剂，并进行筛选的NINDS化合物收集，以证明中等通量筛选的实用性。在具体目标1中，我们将表征几种诱导表达E46 K α-突触核蛋白的PC 12细胞系，以获得具有低背景表达、高诱导表达和一致毒性的细胞系。我们将优化条件，并获得可靠的检测筛选。在具体目标2中，我们将测试目前被认为是PD神经保护治疗候选药物的化合物，如辅酶Q10，半胱天冬酶抑制剂z-VAD等，以确定它们是否在我们的模型中具有神经保护活性。在特定目标3中，我们将对FDA批准的1040种NINDS化合物和相关化合物进行中等通量筛选，以抑制毒性。将确认所有阳性，并建立剂量-反应曲线以确定近似抑制效力。经过验证的阳性结果可以在未来的PD动物模型中进行测试。帕金森病细胞模型及治疗药物的筛选 项目叙述：帕金森病（PD）是第二常见的神经退行性疾病。目前没有治愈或有效的神经保护治疗。我们的长期目标是在PD模型中开发有效的神经保护剂，以便为PD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PD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.