Insights into the structural and biophysical mechanisms of benzamidine-driven inhibition of human lysozyme aggregation.

Amyloid fibril formation is a hallmark of several protein misfolding diseases, including systemic hereditary amyloidosis (SHA), in which lysozyme aggregates into plaques, causing inflammation to 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 human lysozyme fibrillation (HLF). Multiple biophysical assays demonstrated BEN's ability to prevent the lysozyme fibrillation. Intrinsic fluorescence measurements highlighted BEN's interaction with human native lysozyme (HNL). We inferred that the binding mode of BEN to HNL through ITC, molecular docking, and molecular dynamics simulations, confirmed BEN's binding at the active site, near stretch-2 (residues 52-64). The circular dichroism (CD) analysis revealed alterations in the secondary structure of HNL and HLF upon BEN's treatment. In HLF, an increase in BEN's concentration resulted in a concentration-dependent reduction in β-sheet content. SEM and TEM analyses revealed reduced fibril formation and significant alterations in fibril morphology in the presence of BEN. Importantly, BEN exhibited no cytotoxic effects in HEK-293 cells. These results provide strong evidence of BEN's anti- amyloidogenic activity and offer a foundation for future drug development targeting lysozyme amyloidosis.