In vitro activity: ELN484228, a phenyl-sulfonamide compound, is a cell-permeable blocker of α-synuclein which is a key protein in Parkinson’s disease. It was identified by a combination of computational and experimental techniques. ELN484228 has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules such as ELN484228 represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.

Kinase Assay: Aggregation of αSyn was assayed in triplicates at 37°C under shaking (300 rpm) in solutions containing 50 μM protein in the absence and presence of tenfold higher concentration of compound ELN484228 in 25 mM Tris buffer pH 7.4, 100 mM NaCl with the addition of 0.01% NaN3. Aliquots were withdrawn on a daily basis and the thioflavin T (ThT) fluorescence signal was recorded after addition of 20 μM of ThT to each aliquot. Fluorescence emission spectra from 460 to 600 nm were then recorded at an excitation wavelength of 446 nm employing a Cary-Eclipse spectrofluorimeter (Varian, Palo Alto CA). Quenching of the ThT fluorescence by the addition of ELN484228 was assayed by incubating pre-formed fibrils with the compound and by comparison of the ThT fluorescence signal before and after the addition of ELN484228, but no significant change in signal was found. The aggregation of αSyn in the presence of ELN484228 was also characterized in the presence of low concentrations of SDS (200 μM) under the same experimental conditions as described above. The time-dependences of the ThT fluorescence signal were fitted to a nucleation-elongation model as previously described. TEM images were obtained using a Philips CEM100 transmission electron microscope. The samples were applied on Formvar-carbon coated nickel grids and stained with 2% (w/v) uranyl acetate.

Cell Assay: Microglia were obtained from cerebral cortices of 1–3 day old neonate mice. A full description of microglia culture methods is provided in the Supporting Information text. Hippocampal neurons were isolated from embryonic day 18 prenatal rat hippocampi and cultured in antibiotic- and serum-free NbActiv4 medium (both from BrainBits, Springfield IL) at 37°C in an atmosphere of 5% CO2, 9% O2 and on glass coverslips coated with poly-lysine. Half of the medium was replaced every 3 to 4 days. Cells were used for the experiments after 21–28 days in vitro.