BACKGROUND AND PURPOSE: The aggregation of alpha-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of alpha-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on alpha-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human alpha-syn and in the brains of two A30P alpha-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH: Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 x 3 mg.kg(-1) a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble alpha-synuclein protein levels were measured by Western blot. alpha-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and alpha-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS: In cell lines, oxidative stress induced a robust aggregation of alpha-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with alpha-synuclein inclusions while abolishing the colocalization of alpha-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated alpha-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of alpha-synuclein immunoreactivity and soluble alpha-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS: The results suggest that the PREP may play a role in brain accumulation and aggregation of alpha-synuclein, while KYP-2047 seems to effectively prevent these processes.
We have investigated the effect of regiospecifically introducing substituents in the P2 part of the typical dipeptide derived basic structure of PREP inhibitors. This hitherto unexplored modification type can be used to improve target affinity, selectivity, and physicochemical parameters in drug discovery programs focusing on PREP inhibitors. Biochemical evaluation of the produced inhibitors identified several substituent types that significantly increase target affinity, thereby reducing the need for an electrophilic "warhead" functionality. Pronounced PREP specificity within the group of Clan SC proteases was generally observed. Omission of the P1 electrophilic function did not affect the overall binding mode of three representative compounds, as studied by X-ray crystallography, while the P2 substituents were demonstrated to be accommodated in a cavity of PREP that, to date, has not been probed by inhibitors. Finally, we report on results of selected inhibitors in a SH-SY5Y cellular model of synucleinopathy and demonstrate a significant antiaggregation effect on alpha-synuclein.
Despite its thorough enzymological and biochemical characterization the exact function of prolyl oligopeptidase (PO, E.C. 3.4.21.26) remains unclear. The positive effect of PO inhibitors on learning and memory in animal models for amnesia, enzyme activity measurements in patient samples and (neuro)peptide degradation studies link the enzyme with neurodegenerative disorders. The brain protein alpha-synuclein currently attracts much attention because of its proposed role in the pathology of Parkinson's disease. A fundamental question concerns how the essentially disordered protein is transformed into the highly organized fibrils that are found in Lewy bodies, the hallmarks of Parkinson's disease. Using gel electrophoresis and MALDI TOF/TOF mass spectrometry we investigated the possibility of alpha-synuclein as a PO substrate. We found that in vitro incubation of the protein with PO did not result in truncation of full-length alpha-synuclein. Surprisingly, however, we found an acceleration of the aggregation process of alpha-synuclein using turbidity measurements that was reversed by specific inhibitors of PO enzymatic activity. If PO displays this activity also in vivo, PO inhibitors might have an effect on neurodegenerative disorders through a decrease in the aggregation of alpha-synuclein.
