High Throughout Assay to Probe UCH-L1 Ligase Inhibitors

用于探测 UCH-L1 连接酶抑制剂的高通量检测

• 批准号: 6834684
• 负责人: PETER T LANSBURY
• 金额: $ 20万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6834684
• 关键词: Parkinson' s disease; alpha synuclein; chemical genetics; dimer; drug discovery /isolation; enzyme activity; enzyme induction /repression; enzyme inhibitors; enzyme structure; gene expression; genetic polymorphism; genetic susceptibility; high throughput technology; hydrolase; ligase; molecular probes; nanotechnology; small molecule; technology /technique development; ubiquitin

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is characterized by the presence of Lewy bodies (the cytoplasmic neuronal inclusions) and the significant loss of dopaminergic neurons in the substantia nigra, a-synuclein was identified as one major fibril component of the Lewy bodies, thus linked the accumulation of this protein to the pathogenesis of PD. Failure to regulate the concentration of a-synuclein, for example by dysfunction of the pathogenesis of PD. Failure to regulate the concentration of a-synuclein, for example by dysfunction of degradation process, can also contribute to the build-up and consequently fibrillation of the protein. A gene, PARK5, has been linked to PD are involved in proteasomal degradation pathway and it is an ubiquitin C terminal hydrolase (UCH-L 1) that hydrolyzes C-terminal ester and amides of ubiquitin and is believed to play a key role in processing polyubiquitin and/or ubiquity lated proteolytic peptide. A rare mutation (193M) of UCH L 1 that yields a 50% reduction in its hydrolytic activity has been tentatively linked to a rare early onset form of PD, at the same time a polymorphism of the enzyme (S 18Y) was indicated to reduce the risk of PD. The assumption that each enzyme expresses a single enzymatic activity in vivo, however, is challenged by the linkage of UCH-L 1 to PD. UCH-L 1, especially those variants linked to higher susceptibility to PD, causes the accumulation of a-synuclein in cultured cells, an effect that cannot be explained by its recognized hydrolase activity. UCH-L1 exhibits a second, dimerization-dependent, ubiquityl ligase activity. The polymorphic variant of UCH-L1 that is associated with decreased PD risk (S 18Y) has reduced ligase activity, but comparable hydrolase activity as the wild-type enzyme. Thus the ligase activity, as welt as the hydrolase activity of UCH-L1 may play a role in proteasomal protein degradation, a critical process for molecules ("molecular probes") that can be used to perturb UCH-L1 ligase activity in cell culture and animal models of PD. This "chemical genetic" strategy is complementary to traditional genetic approaches (e.g., knockouts and trasngenics) for understanding protein function but has a distinct advantage in that the probes are potential lead compounds for the development of novel PD therapetutics. The program detailed below will seek probes with the following activities: (1) inhibitors ofUCH-L1 dimerization, (2) inhibitors of UCH-L1 ligase activity, and (3) repressors and activators of UCH-L1 expression.

描述（由申请人提供）：帕金森病（PD）的特征是存在路易体（细胞质神经元内含物）和黑质中多巴胺能神经元的显着损失，α-突触核蛋白被确定为路易体的一种主要原纤维成分。路易体，因此将该蛋白的积累与帕金森病的发病机制联系起来。无法调节 a-突触核蛋白的浓度，例如通过 PD 发病机制的功能障碍。未能调节α-突触核蛋白的浓度，例如由于降解过程的功能障碍，也可能导致蛋白质的积聚，从而导致纤维化。与 PD 相关的基因 PARK5 参与蛋白酶体降解途径，它是一种泛素 C 末端水解酶 (UCH-L 1)，可水解泛素 C 末端酯和酰胺，并被认为在加工过程中发挥关键作用多聚泛素和/或泛素化蛋白水解肽。 UCH L 1 的罕见突变 (193M) 导致其水解活性降低 50%，目前已初步将其与一种罕见的早发性帕金森病联系起来，同时该酶的多态性 (S 18Y) 被表明可降低其水解活性。帕金森病的风险。然而，每种酶在体内表达单一酶活性的假设受到 UCH-L 1 与 PD 连接的挑战。 UCH-L 1，尤其是那些与 PD 较高易感性相关的变体，会导致培养细胞中 a-突触核蛋白的积累，这种效应无法用其公认的水解酶活性来解释。 UCH-L1 表现出第二种二聚依赖性泛素连接酶活性。与 PD 风险降低相关的 UCH-L1 多态性变体 (S 18Y) 具有降低的连接酶活性，但水解酶活性与野生型酶相当。因此，连接酶活性以及 UCH-L1 的水解酶活性可能在蛋白酶体蛋白降解中发挥作用，这是分子（“分子探针”）的关键过程，可用于扰乱细胞培养物中的 UCH-L1 连接酶活性，并且PD动物模型。这种“化学遗传”策略是对了解蛋白质功能的传统遗传方法（例如基因敲除和转基因）的补充，但具有明显的优势，因为探针是开发新型帕金森病治疗药物的潜在先导化合物。下面详述的计划将寻找具有以下活性的探针：(1)UCH-L1二聚化的抑制剂，(2)UCH-L1连接酶活性的抑制剂，以及(3)UCH-L1表达的阻遏物和激活物。