Screen For Inhibitors of Parkin E3 Autoubiquition

描述（由申请人提供）：神经退行性疾病是维持不同人群健康和生活质量的主要挑战。在这类的各种疾病中，帕金森病（PD）是发病率和预期寿命缩短的主要原因，并且目前正在努力发现新的治疗方法。治疗干预的一种有前途的方法是设计用于增加帕金蛋白的细胞水平的治疗，帕金蛋白是一种已被发现在模型系统中拮抗神经变性的蛋白质，并且与某些形式的PD在遗传上相关。选择和验证的目标，可以操纵，以实现这一效果取决于越来越多的信息有关的parkin调节。药物发现的一个新领域-通过泛素途径酶的蛋白质稳态调节-最近已被证明与寻找抗神经退行性疾病药物有关。事实上，帕金已被确定为环指E3泛素连接酶，其催化泛素化，随后诱导与神经变性相关的各种蛋白质的蛋白酶体降解。此外，它还催化自身的泛素化。因此，帕金自泛素化的选择性抑制剂是 假设它有神经保护作用。在第一阶段，建议建立基于酵母的帕金自泛素化抑制剂筛选试验和帕金底物α-突触核蛋白遍在化的选择性反筛选。E3系统的基本组分（parkin）将在S.酿酒酵母，沿着与p53连接的人帕金蛋白或α-突触核蛋白，以及监测p53活性（β-半乳糖苷酶活化）的报道构建体。然后将重构的E3连接酶功能和报告系统配置并验证为帕金自泛素化抑制剂的高通量筛选。从NCI和学术合作者收集的植物和海洋生物提取物和小分子将被筛选出这种活性的有效抑制剂。在第二阶段，活性提取物的分馏将由测定指导，以鉴定活性成分。由此产生的新型纯化合物将被视为PD治疗的候选药物。模块化测定构建格式将允许评价与各种疾病相关的其他E3。

DESCRIPTION (provided by applicant): Neurodegenerative disease represents a major challenge to the maintenance of health and quality of life in diverse segments of the population. Among the various diseases of this class, Parkinson's disease (PD) is a major cause of morbidity and diminished life expectancy, and there is today an intense effort to discover novel treatments. A promising approach for therapeutic intervention is treatment designed to increase the cellular level of parkin, a protein which has been found to antagonize neurodegeneration in model systems, and which is linked genetically with some forms of PD. The selection and validation of targets that can be manipulated to achieve this effect depends on an increasing amount of information relating to parkin regulation. A novel area for drug discovery -- protein homeostatic regulation via ubiquitin pathway enzymes -- has recently been demonstrated to have relevance to the search for anti-neurodegenerative drugs. Parkin has, in fact, been determined to be a RING-finger E3 ubiquitin ligase that catalyses ubiquitination and, subsequently, induces proteasomal degradation of various proteins associated with neurodegeneration. In addition, it catalyses its own ubiquitination. Thus, selective inhibitors of parkin autoubiquitination are hypothesized to have a neuroprotective effect. In Phase I, it is proposed to establish a yeast-based screening assay for inhibitors of parkin autoubiquitination and a selectivity counter screen for ubiquitination of alpha-synuclein, a parkin substrate. Essential components of the E3 system (parkin) will be cloned and expressed in S. cerevisiae, along with human parkin or alpha-synuclein linked to p53, and a reporter construct that monitors p53 activity (beta-galactosidase activation). The reconstructed E3 ligase function and reporter system will then be configured and validated as a high throughput screen for inhibitors of parkin autoubiquitination. Collections of plant and marine organism extracts and small molecules from the NCI and academic collaborators, will be screened for potent inhibitors of this activity. In Phase II, fractionation of active extracts will be guided by the assay to identify active principles. Novel pure compounds arising from this effort will be considered as development candidates for PD therapy. The modular assay construction format will permit evaluation of other E3s that are associated with a variety of diseases.