Modulation of lysozyme amyloid fibrillation by three structurally similar dihydroxy-coumarins: A comparative analysis

Oligomers/amyloid fibrils arising from protein misfolding are hallmarks of amyloid diseases, therefore inhibiting protein aggregation/amyloid formation is vital for treatment of such diseases. Dihydroxy-coumarins exhibit diverse bioactivities, but the impact of subtle structural variations, particularly the positional arrangement of hydroxyl groups, on their anti-amyloidogenic properties has not been systematically explored. This study compared the ability of three structurally similar dihydroxy-coumarins, daphnetin (7,8-dihydroxy-coumarin, Dap), esculetin (6,7-dihydroxy-coumarin, Esc) and 5,7-dihydroxy-coumarin (Dih), to modulate the aggregation of hen egg white lysozyme (HEWL). Using a combination of biophysical, computational and cellular methods, it was found that all three compounds reduced the final yield of amyloid fibrils and promoted the disassembly of mature fibril in a dose-dependent manner, with Dap exhibiting the most pronounced effect, comparable to epigallocatechin gallate (EGCG). Notably, the relative inhibitory potency followed the order Dap > Esc > Dih, correlating with the specific position of the ortho-dihydroxyl groups on the coumarin scaffold. Spectroscopic and molecular docking analyses suggested that these compounds bound preferentially to aggregation-prone regions of HEWL via hydrogen bonding and hydrophobic interactions, with the C7-C8 dihydroxyl arrangement in Dap facilitating stronger binding affinity and more effective fibril suppression. These findings established a clear structure-activity relationship among dihydroxy-coumarins and identified Dap as a promising candidate for further development as a therapeutic amyloid inhibitor.