High Throughout Assay to Probe UCH-L1 Ligase Inhibitors

描述（由申请人提供）：帕金森病（PD）的特征是存在路易小体（胞质神经元包涵体）和黑质多巴胺能神经元的显著损失，a-突触核蛋白被确定为路易小体的主要纤维成分，因此将该蛋白的积累与PD的发病机制联系起来。不能调节a-突触核蛋白的浓度，例如PD发病机制的功能障碍。不能调节a-突触核蛋白的浓度，例如降解过程的功能障碍，也可能导致蛋白质的积聚和纤维性颤动。一个与帕金森病相关的基因PARK5参与蛋白酶体降解途径，它是一种泛素C端水解酶（UCH-L 1），水解泛素C端酯和酰胺，被认为在多泛素和/或泛素化蛋白水解肽的加工中起关键作用。一种罕见的UCH l1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（s18y）被表明可以降低患帕金森病的风险。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的联系的挑战。UCH-L 1，尤其是那些与PD易感性相关的变异，会导致a-突触核蛋白在培养细胞中积累，这一效应无法用其已知的水解酶活性来解释。UCH-L1表现出第二种二聚化依赖的泛素连接酶活性。与PD风险降低相关的UCH-L1多态性变异（s18y）连接酶活性降低，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，而蛋白酶体蛋白降解是分子（“分子探针”）在细胞培养和PD动物模型中用来干扰UCH-L1连接酶活性的关键过程。这种“化学遗传”策略是对理解蛋白质功能的传统遗传方法（例如，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PD治疗方法的潜在先导化合物。下面的程序将寻找具有以下活性的探针：(1)fuch - l1二聚化抑制剂，(2)UCH-L1连接酶活性抑制剂，(3)UCH-L1表达的抑制因子和激活因子。

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.