Fig. 5.

Oleocanthal inhibits polymerization of PHF6 peptide via formation of a Schiff base with the lysine side chain. (a) Negative staining EM of paired helical fibrils formed by PHF6 peptide, similar to K18 and T40. 200 μM PHF6 peptide was incubated in 20 mM MOPS buffer, pH 7.2, 150 mM NaCl and 6.25% DMSO at 37 °C for 24 hrs and then used for negative staining. (b) ThT fluorescence assay of the polymerization of PHF6 peptide in the presence of oleocanthal. Polymerization of 2 μM PHF6 peptide in 20 mM MOPS buffer, pH 7.2 with 0-25 μM of oleocanthal was initiated by addition of equal volume of 300 mM NaCl and 20 mM ThT in 20 mM MOPS buffer, pH 7.2. The assembly was conducted in 96 well plates with 100 μL in each well. (c) Chemical reaction of oleocanthal with N-Bz-Lys-OMe. (d) LC-MS analysis of reaction between oleocanthal and N-Bz-Lys-OMe in methanol before reduction with sodium borohydride. (e) LC-MS analysis of reaction between oleocanthal and N-Bz-Lys-OMe in methanol after reduction with sodium borohydride. (f) Chemical reaction of oleocanthal with PHF6 peptide. (g) LC-MS analysis of reaction between oleocanthal and PHF6 peptide in methanol before reduction with sodium borohydride. (h) LC-MS analysis of reaction between oleocanthal and PHF6 peptide in methanol after reduction with sodium borohydride.