Spectroscopic and computational insights into binding of daphnetin with pepsin: Inhibition of pepsin fibrillation and identification of amyloidogenic core region.

Multi-spectroscopic and computational techniques were utilized to investigate the interaction between daphnetin and pepsin, the principal gastric protease, along with daphnetin's inhibitory effect on pepsin aggregation. Fluorescence and UV-vis absorption spectral analyses indicated that daphnetin binds to pepsin via static quenching. Thermodynamic analyses revealed positive ΔH° [+(9.314 ± 1.018) kJ mol] and ΔS° [+(115.487 ± 3.154) J mol] values, indicating the presence of hydrophobic forces, while the negative ΔG° suggests the spontaneous formation of the complex. CD and FT-IR analyses demonstrated a substantial alteration in the secondary structure of pepsin upon daphnetin binding. Daphnetin can inhibit pepsin aggregation as confirmed by thioflavin T (ThT) and Congo Red (CR) binding assays, complemented by circular dichroism (CD) and fluorescence microscopic results. Computational tools (PASTA, WALTZ-DB 2.0, AggreProt 1.0, and TANGO) identified five aggregation-prone regions as key targets. The binding affinity of daphnetin was pH-dependent, highest at pH 4.2, and decreasing at pH 2.0, 7.4, and 9.0. Metal ions (Cu and Zn) were found to increase daphnetin's binding affinity towards pepsin. The enzyme kinetic analysis revealed that daphnetin inhibited pepsin activity through a competitive inhibition mode. These experimental results were further supported by computational analysis, elucidating the dynamics of daphnetin within pepsin network.