Inhibitory effect of silymarin on amyloid formation in ALS-associated hSOD1 P66R mutant

The aberrant aggregation of human superoxide dismutase 1 (hSOD1) into β-sheet-rich amyloid fibrils is a crucial process in the pathogenesis of amyotrophic lateral sclerosis (ALS), enhancing motor neuron degeneration and disease progression. The P66R mutation in SOD1 destabilizes local structure and promotes β-sheet-driven fibrillation, which makes it a suitable model for exploring approaches for reducing pathogenic aggregation. Here, we evaluate silymarin, a polyphenolic compound with known antioxidant and neuroprotective properties, for its potential to inhibit P66R-hSOD1 aggregation. ThT fluorescence and transmission electron microscopy analyses demonstrate a significant decrease in amyloid fibril formation in the presence of silymarin; in addition, FTIR spectroscopy confirms the suppression of β-sheet formation. Fluorescence quenching and ANS binding assays indicate a moderate-affinity binding between silymarin and the mutant protein, along with a reduction in surface hydrophobicity. Hemolysis assays confirm its protective effect against membrane damage induced by aggregates, while molecular docking and dynamic simulations indicate that silymarin stabilizes aggregation-prone areas with hydrogen bonding and hydrophobic interactions, thereby promoting compact conformations and reducing solvent-exposed surfaces. The findings identified silymarin as an effective anti-amyloidogenic agent that reduces β-sheet accumulation and fibril formation while also decreasing cytotoxicity, highlighting its potential as a therapeutic candidate for ALS.