High Throughput Screening to Discover Chemical Probes and Pharmacological Agents for Modulating Parkin Activity

 描述(由申请人提供)：许多神经退行性疾病已知与不适当的蛋白质聚集有关。在帕金森氏病(PD)中，运动神经元蛋白聚集体的形成是该病的一个特征。遗传和环境因素都与帕金森病风险增加有关。家族性帕金森病的易感基因有α-突触核蛋白、Parkin、PINK1、LRRK2、DJ等。Parkin基因突变导致早发性常染色体隐性遗传型帕金森氏病(常染色体隐性遗传性青少年帕金森综合征；AR-JP)，占所有隐性传播的早发性帕金森病病例的50%。Parkin蛋白是一种泛素E3连接酶，已被证明可催化多种底物的多泛素化。Parkin似乎是针对错误折叠的蛋白质和受损的线粒体的细胞防御系统的组成部分。环境因素或基因突变对帕金森氏症患者帕金森氏活性的干扰是帕金森氏症的重要机制。我们推测，系统地识别调节控制Parkin线粒体定位的分子通路的小分子探针将有助于揭示可能与帕金森病的发展相关的环境因素类型。此外，这些特定工具化合物的可获得性有助于阐明Parkin线粒体招募和有丝分裂在模型系统中PD的发生和发展中的作用。我们进一步假设，识别可以防止突变的Parkin聚集的小分子抑制剂可能会为调节Parkin聚集的途径产生新的化学探针，并可能为帕金森病提供新的治疗策略。目前还没有特定的药物可以阻止野生型Parkin在去极化时重新募集到线粒体，也没有特定的抑制剂可以阻止Parkin突变体在细胞中的聚集。这项应用的总体目标是开发一套化学探针，以允许开发化学遗传学方法来剖析PINK1和Parkin在帕金森病中的功能。将实施特异性和反筛选试验，以消除非特异性化合物。将开发去卷积分析，以确定在相关的高温超导电池分析中发现的化学探针的分子靶标。

 DESCRIPTION (provided by applicant): Many neurodegenerative diseases are known to be associated with inappropriate protein aggregation. In Parkinson's disease (PD), formation of protein aggregates in motor neurons is a hallmark of the disease. Both genetic and environmental factors have been associated with an increased risk of PD. Several susceptible genes, including alpha-Synuclein, Parkin, PINK1, LRRK2, DJ in familial PD have been identified. Mutations in Parkin are responsible for an early- onset autosomal recessive form (autosomal recessive juvenile parkinsonism; AR-JP) of PD and account for 50% all recessively transmitted early-onset PD cases. Parkin protein is a ubiquitin E3 ligase and has been shown to catalyze polyubiquitination of a variety of substrates. Parkin appears to be an integral component of cellular defense systems against misfolded proteins and damaged mitochondria. Perturbation of the Parkin activity by environmental factors or genetic mutations is the compelling mechanism for a subset of Parkinson's disease. We hypothesized that systematic identification of small molecule probes that modulate the molecular pathways that control Parkin mitochondrial localization will help to unravel the type of environmental factors that may be associated with the development of Parkinson's disease. In addition, availability of such specific tool compounds can help to clarify the roles of Parkin mitochondrial recruitment and mitophagy in the onset and progression of PD in model systems. We further hypothesized that identification of small-molecule inhibitors that can prevent mutant Parkin aggregation may yield nove chemical probes for the pathways that regulates Parkin aggregation and may offer new therapeutic strategies for Parkinson's disease. Currently there are no specific pharmacological agents that can block wild type Parkin recruitment to mitochondria upon depolarization, neither are specific inhibitors that can block aggregation of Parkin mutants in cells. The overall goal of this application is to develop a set of chemical probes to permit development of the chemical genetics approaches to dissect the function of PINK1 and Parkin in Parkinson's disease. Specificity and counter screening assays will be implemented to eliminate non-specific compounds. Deconvolution assays will be developed to allow determination of the molecular targets of the chemical probes discovered in related HTS cell based assays.