Construing the Interactions of Coumarin Derivatives with the Main Protease of Stomach Pepsin Using Spectroscopic and Computational Analyses: Insights into the Binding Thermodynamics, Antifibrillation Studies, and Enzymatic Assay

Multispectroscopic and computational techniques were employed to study the interaction of umbelliferone (UMB)/4-methyl-umbelliferone (4-MU) with the main protease of the stomach, pepsin. Additionally, the potential inhibitory effects of these coumarin derivatives were investigated on pepsin aggregation. Results from steady-state fluorescence, excited-state fluorescence lifetime, and UV-vis absorption analyses indicated that the binding of UMB and 4-MU with pepsin followed a static quenching mechanism. CD and FT-IR experiments further provide information on the conformational changes in pepsin during ligand binding. The thermodynamic analysis indicated positive Delta H and Delta S, suggesting that hydrophobic forces played a dominant role with a negative Delta G implying the spontaneous complexation. In addition, UMB/4-MU exhibited a promising inhibitory effect toward protein fibrillation, which was verified through Thioflavin T (ThT) and Congo red (CR) binding assays along with imaging studies that include fluorescence microscopy and field emission scanning electron microscopy (FESEM). Kinetic parameters demonstrated that pepsin activity was inhibited by UMB and 4-MU through an uncompetitive mode of inhibition. Molecular docking and molecular dynamics (MD) simulations offered insight into the binding affinity and flexibility of pepsin upon complexation with UMB and 4-MU.