Exploring insulin aggregation at neutral pH and its inhibition <i>via</i> caffeic acid: a biophysical and bioinformatics analysis

This study aimed to induce fibril formation in human insulin under physiological conditions and to investigate the inhibitory potential of caffeic acid (CA) on these fibrils <i>in-vitro</i>. Various techniques including circular dichroism (CD) spectroscopy, Thioflavin T, ANS fluorescence, Rayleigh light scattering (RLS), and turbidity analysis were conducted to elucidate fibril formation and CA inhibition potential. Fibril formation in insulin was induced by heating (37 °C) and agitation (600 rpm) significantly increased (27.01-fold) the ThT binding after 120 h of incubation. Aggregation also increased turbidity and enhanced RLS fluorescence at 350 nm. Secondary structure analysis revealed at loss of α-helical content and a concomitant increase in β-sheet content in human insulin following aggregation. The presence of varying concentrations of CA resulted in fewer perturbations in the secondary structure of insulin compared with the aggregated insulin sample. Fibril formation was also reduced (80%) in the presence of CA (500 µM). To gain insight, the biophysical interactions between CA and insulin were studied. CA showed moderate affinity (5.54 × 10³ M^-1) towards insulin in static quenching mode. The positive Δ<i>H</i> and Δ<i>S</i> values obtained indicate that the reaction was driven by hydrophobic interactions and the negative value of Δ<i>G</i> indicates a spontaneous reaction between the complexes. Docking analysis showed the interaction of CA with various amino acids of insulin <i>via</i> H-bonds, van der Waals forces, and hydrophobic interactions. Molecular simulations of RMSD, RMSF, and R_g showed that CA formed a stable complex with insulin.