Figure 1: Tolcapone and tafamidis effects over WT, V122I-TTR and A25T-TTR aggregation and stability.

(a) Chemical structures of the commercially available drugs tolcapone (Tasmar, CAS 134308-13-17) and tafamidis. Anti-amyloidogenic effect of tolcapone and tafamidis on (b) WT-TTR and (c) V122I-TTR and (d) anti-amyloidogenic effect of tolcapone on A25T-TTR. TTR solutions were incubated with several concentrations of test molecules and aggregation was induced by acidification. Turbidity at 340 nm was used to monitor TTR aggregation and fibril formation. At the end of the assays, the percentage of TTR aggregation for each experimental condition was calculated with respect to the TTR turbidity values obtained in the absence of compounds (vehicle only). (e) WT-TTR was incubated in the presence or absence of 10 molar equivalents of tolcapone. Urea was added to promote protein unfolding at the indicated final concentrations. (f) WT-TTR was incubated with several concentrations of tolcapone (from 0.25 to 10 molar equivalents with respect to WT-TTR) and denaturation promoted by addition of urea at a final concentration of 6.5 M. In both assays e and f, after 72 h incubation at RT, Trp fluorescence intensity (355/335 nm) was measured as a sensor of tetramer integrity. TTR stabilization effect induced by tolcapone binding was calculated with respect to the tryptophan fluorescence emission of the protein in the absence of the compound (vehicle only). In all panels, error bars indicate the standard error of the mean (s.e.m.; n=3).