Carboxymethylated-k-casein: A convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation

Reduced and carboxymethylated-κ-casein (RCM-κ-CN) is a milk-derived amyloidogenic protein that readily undergoes nucleation-dependent aggregation and amyloid fibril formation via a similar pathway to disease-specific amyloidogenic peptides like amyloid beta (Aβ), which is associated with Alzheimer’s disease. In this study, a series of flavonoids, many known to be inhibitors of Aβ fibril formation, were screened for their ability to inhibit RCM-κ-CN fibrilisation, and the results were compared with literature data on Aβ inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-κ-CN fibril formation. IC50 values were between 10- and 200-fold higher with RCM-κ-CN than literature results for Aβ fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-κ-CN assay make it an economic alternative first screen for Aβ inhibitory activity, especially for use with large compound libraries.