Figure 2

Multiple HT-CM-SPR screening hit compounds block αSyn misfolding. (a) The impact of compounds on αSyn fibrillization activity, monitored by thioflavin T fluorescence, is shown. Examples are shown of three independent experiments for compounds tested at 300 μM in the αSyn fibrillization assay along with 0.5% DMSO control. Boxed numbers indicate the ratio of experiments demonstrating significant impact. Statistical analyses, (see Supplementary Information) indicate significant differences (p-value < 0.05) from DMSO for 576755 in 3 out of the 3 shown experiments, and for 582032 in 2 out of 3 experiments. Compound 573434 has no impact on αSyn fibrillization in a biochemical assay. (b) Anti-fibrillization activity of compound 576755 at 300 μM is demonstrated in both the standard (left) and seeded-aggregation (right) assays in which αSyn protofibrils are added at the initiation of the assay. (c) A dose response for compound 576755 in the fibrillization assay shows activity as low as 25 μM. For all fibrillization figures each data point represents the mean ± SEM of quadruplicate wells. (d) Dose response of anti-oligomerization activity of three compounds are shown in a split Gaussia luciferase (GLuc) complementation assay executed as described in Methods. GLuc units are in arbitrary light units. The top panel shows the time course of inhibition by 576755 at multiple doses and the bottom panel shows the dose response and IC50s of all three active compounds at t = 24 hours. (e) The impact on oligomerization for all compounds discussed in this manuscript are shown. GLuc units are normalized to the mean of DMSO control. Means ± SD. are shown. (f) The anti-oligomerization activities of the three compounds identified in panel (d) are validated in a FRET based αSyn oligomerization assay executed as described in Methods. Compound structures are also shown in Supplementary Fig. S4.