<p>Parkinson’s disease (PD) is a detrimental neurodegenerative disorder characterized by the presence of large protein aggregates in the brain called Lewy bodies, which are primarily composed of the protein α-synuclein (αSyn). Due to the dysregulation of αSyn levels in PD, controlling its levels through the manipulation of protein degradation pathways has been suggested as a therapeutic avenue for the treatment of PD and related diseases. Although αSyn is known to be degraded through the autophagy and proteasome pathways, it is one of only a few known substrates of the ubiquitin-independent proteasome pathway, which utilizes the 20S core particle of the proteasome (20S CP) to degrade proteins. We therefore hypothesize that small molecule stimulation of the 20S CP will enhance αSyn degradation and reduce αSyn pathology, providing a therapeutic benefit in PD models.</p><p>We began our studies by developing a fluorescence resonance energy transfer (FRET) reporter assay to monitor 20S CP activity and screen for small molecule stimulators. This assay provides a greater dynamic range to detect 20S CP stimulation compared to the most commonly used assay to monitor proteasome activity. Using the FRET assay, we were able to identify a number of novel 20S CP stimulators that differ in structure as well as potency and degree of stimulation. We next evaluated the ability of four small molecule stimulators to enhance protein degradation by the 20S CP in a biochemical assay using 15 different purified proteins. These 15 proteins include known substrates of the 20S CP and vary in size and degree of disorder. From this assay, we demonstrate that a 20S CP stimulator is likely to enhance the degradation of highly disordered proteins, such as αSyn, but the effect on other protein levels appears to be distinct for each stimulator. Two of our more potent stimulators, AM-404 and miconazole, were used with the proteasome inhibitor bortezomib for subsequent studies in HEK-293T cells in which we corroborated the results of our biochemical assay. While both AM-404 and miconazole were shown to impact highly disordered proteins, there was not much overlap between the proteins shown to be affected by each stimulator. Due to the distinct effect of each stimulator on protein regulation by the 20S CP, this study indicates the potential of tailoring a small molecule 20S CP stimulator to enhance the degradation of particular substrates.</p><p>Since AM-404 and miconazole were shown to impact 20S CP activity in different ways, we next evaluated whether either stimulator would be able to prevent the αSyn-induced inhibition of the 20S CP. High levels of αSyn have been shown to lead to proteasome impairment in biochemical and cell studies. We confirm 20S CP impairment in the presence of micromolar amounts of αSyn, and we demonstrate that miconazole, but not AM-404, is effective at maintaining 20S CP activity in the presence of increasing concentrations of αSyn. We also show that αSyn-overexpressing PC12 cells (PC12 C4 cells) display reduced proteasome activity compared to the parent cell line. Miconazole and AM-404 increased proteasome activity in PC12 C4 cells, which were more sensitive to 20S CP stimulation than non-transfected PC12 cells, but miconazole was shown to be more effective at modulating αSyn phosphorylated at Ser129 in PC12 C4 cells. </p><p>Our results reveal the dynamic nature of the 20S CP and the ways in which its activity can be modulated to affect protein levels. While AM-404 is effective at stimulating the 20S CP to enhance the degradation of some proteins, miconazole was shown to be more efficient at modulating αSyn levels and impacting αSyn pathology, as it relates to 20S CP impairment. While the results described here mark the beginning of an exciting area of study, we do demonstrate the therapeutic potential of 20S CP stimulation to combat PD. </p><div><br></div>