Immunomodulatory activity and molecular mechanisms of action of peptides derived from casein hydrolysate by alcalase and flavourzyme based on virtual screening

This study aimed to screen novel immunomodulatory peptides from casein hydrolysates (CH) using alcalase and flavorzyme by virtual screening, and their molecular mechanism were further studied. Based on the primary structural characteristics of immunomodulatory peptides, along with their hydrophobicity and isoelectric point, 3 novel immunomodulatory peptides (ALPMHIR, AMKPWIQPK, NPWDQVKR) were quickly found using virtual screening. These peptides exhibited strong interactions with TLR2/TLR4 through hydrogen bonding and hydrophobic interactions. Molecular docking verified that the key binding sites, such as Ile733, Ala732, and Phe774 in TLR2/TLR4 contributed to docking. Interestingly, the peptide AMKPWIQPK exhibited the strongest immunomodulatory activity and anti-inflammatory activity as 2-way immunomodulatory peptides. Based on western blot analysis and validation using specific inhibitors against MAPK/NF-κB signaling pathways, the results demonstrated that AMKPWIQPK could recognize the TLR2 and TLR4 receptor of the macrophages to upregulate the phospho-IκBα, phospho-p38, and phospho-p65, and further activated the MAPKs/NF-κB signaling pathways to enhance the immunomodulatory activity. These results confirmed that screening and optimizing immunomodulatory peptides by virtual screening and molecular docking were a novel and rapidly feasible method. The peptide AMKPWIQPK was expected to be used as natural-derived immunomodulatory active ingredients in nutritional health care and functional foods.