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.
|Arnold, Jamie J; Bernal, Alejandro; Uche, Uzo et al. (2006) Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity. Anal Biochem 350:214-21|