Benzamidine-Mediated Inhibition of Human Lysozyme Aggregation: Differential Ligand Binding in Homologous Proteins

Amyloid fibril formation is a hallmark of several protein misfolding diseases, including systemic hereditary amyloidosis (SHA), in which lysozyme aggregates into plaques, causing inflammation in various tissues. SHA is a rare disease with no current drug treatment options. In our efforts to identify potential therapeutics for SHA, we investigated the inhibitory effects of benzamidine (BEN) on the fibrillation of human lysozyme (HL). Multiple biophysical assays demonstrated BEN’s ability to effectively prevent amyloid formation. Intrinsic fluorescence measurements highlighted BEN’s interaction with HL. We inferred the binding mode of BEN to HL through ITC experiments, molecular docking, and molecular dynamics simulations, confirmed BEN’s binding at the active site, particularly near stretch-2 (residues 52-64), a key region in its anti-amyloidogenic activity. This interaction differed from the previously reported interaction with HEWL. Further, microscopy analyses, including scanning electron microscopy (SEM) and transmission electron microscopy (TEM), further supported these findings by showing reduced fibril formation and alterations in fibril morphology in the presence of BEN. Importantly, BEN exhibited no cytotoxic effects in HEK-293 cells, reinforcing its potential as a therapeutic candidate for amyloidosis. These results provide strong evidence of BEN’s anti-amyloidogenic activity and offer a foundation for future drug development targeting lysozyme amyloidosis.