Investigation of virtual screening, inhibition mechanisms of endogenous serine protease inhibitors, and its preservation effects on tilapia

As a vital endogenous enzyme, serine protease (SP) plays a substantive role in the decomposition of fish collagen and the softening of muscle texture. Thus, seeking SP inhibitors could be a promising strategy to retard the quality deterioration of fish meat during preservation. In this work, the influence of SP on the quality of tilapia during cold storage was appraised through correlation analysis. Negative correlations were discerned between SP activity and collagen content as well as texture, and the correlation coefficient with hardness was 0.92, which provided initial evidence that SP accelerated the deterioration of tilapia quality during cold storage. The SP inhibitor was effectively identified via virtual screening technique, and the top 5% of scores were selected for experimental validation to obtain ampelopsin (AMP), which had a good inhibitory effect with IC50 = 3.019 mu M. The mechanism of inhibition was analyzed using the kinetic mechanism and fluorescence spectroscopy. It was discovered that the inhibition of SP by AMP was competitive with K-i = 13.982 mu mol/L and the process was spontaneous. AMP interacts with SP by dynamic quenching, influencing the microenvironment of tryptophan and tyrosine residues in SP. Delta G < 0 proves that the binding process is spontaneous. Molecular docking further suggested that they were mainly combined by hydrogen bonds and hydrophobic interactions. Furthermore, through the preservation verification of AMP on tilapia fillets, it was verified that AMP could inhibit SP activity during cold storage and delay the degradation of fish collagen and texture deterioration.