High Throughout Assay to Probe UCH-L1 Ligase Inhibitors

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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.