Looking for a generic inhibitor of amyloid-like fibril formation among flavone derivatives

Abstract

A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid.

Keywords: Alpha-synuclein; Amyloid; Amyloid beta; Fibril; Flavone; Inhibitor; Protein aggregation.

Figure 1. The best flavone inhibitors of insulin amyloid-like fibril formation.

Figure 2. Concentration dependence of Abeta (A), and alpha-synuclein (B) aggregation kinetics.

Raw data at different peptide concentrations is represented by scatter plots of different colors; fitting is represented by red curves. Inserts show concentration dependences of t₅₀ values and also serves as color-code legends.

Figure 3. Effect of flavones on the aggregation of alpha-synuclein.

Each flavone derivative is represented by different color. Relative ThT fluorescence intensities are shown in light colors (A) and relative halftimes of aggregation in dark colors (B).

Figure 4. Aggregation of alpha-synuclein in the presence of flavones followed by Congo Red differential spectra.

The ligand/protein ratio was 3:2.

Figure 5. AFM images of alpha synuclein aggregates.

The ligand/protein ratio was 3:2.

Figure 6. Effect of flavones on the aggregation of Abeta.

Each flavone derivative is represented by different color. Relative ThT fluorescence intensities are shown in light colors (A) and relative halftimes of aggregation in dark colors (B).

Figure 7. Aggregation of Abeta in the presence of flavones followed by Congo Red differential spectra.

Abeta concentration was 15 µ M. The ligand/protein ratio was 7:1.

Figure 8. AFM images of Abeta aggregates. Abeta concentration was 15 µM.

The ligand/protein ratio was 7:1.

Figure 9. Aggregation of alpha-synuclein (A–C) and Abeta (D–F) in presence of EGCG.

Illustrated by relative ThT intensity and t₅₀ (A and D), Congo Red differential spectra (B and E) and AFM images of final aggregates (C and F). Abeta concentration used for Congo Red and AFM studies was 15 µM. The EGCG/Abeta ratio was 7:1, and EGCG/alpha-synuclein ratio was 3:2.