Virtual screening and validation of dipeptidyl peptidase-IV inhibitory peptides from goat milk: Insights from molecular dynamics simulations and in vitro experiment

Goat milk, distinguished by its high β-casein content and superior digestibility compared with bovine milk, represents a promising source of bioactive peptides. Dipeptidyl peptidase-IV (DPP-4) inhibitors are vital for managing type 2 diabetes mellitus by enhancing incretin-mediated glycemic control. Naturally derived DPP-4 inhibitory peptides are of interest as food-compatible candidates for functional ingredient development. This study aimed to identify and characterize DPP-4 inhibitory peptides from goat milk proteins through an integrated computational and experimental approach. Eleven major goat milk proteins were subjected to in silico hydrolysis using alcalase, pepsin, and trypsin, generating 319 peptides (3-15 amino acids). Virtual screening with ToxinPred, PeptideRanker, StackDPPIV, and KarmaDock prioritized nontoxic bioactive candidates, followed by molecular docking and 500-ns Gaussian accelerated molecular dynamics simulations using AMBER 22 to elucidate binding and conformational dynamics with DPP-4 (Protein Data Bank identification code: 4A5S). High-performance liquid chromatography validated the presence of 3 top peptides-MMSF (from α-lactalbumin), MPFPK, and GPFPIL (from β-casein)-in the <3-kDa hydrolysate fraction, with retention times closely matching standards (MPFPK: 8.923 vs. 8.856 min; MMSF: 11.003 vs. 10.912 min; GPFPIL: 12.406 vs. 12.334 min). In vitro assays confirmed competitive inhibition, with half-maximal inhibitory concentration values of 0.69 mM (MMSF), 0.32 mM (MPFPK), and 2.83 mM (GPFPIL), and negligible cytotoxicity (>95% cell viability at 10 mM in Caco-2 cells). Docking revealed interactions with critical DPP-4 residues (H740, Y631, Y547), with MPFPK forming the most extensive hydrogen bond network (5 bonds). Molecular dynamics simulations indicated enhanced DPP-4 stability (reduced root mean square deviation), increased radius of gyration and solvent-accessible surface area, and distinct flap region (H235-E255) perturbations; GPFPIL induced a stable open conformation, MPFPK caused dynamic open-closed transitions, and MMSF disrupted β-sheet stability. Molecular mechanics Poisson-Boltzmann surface area analysis confirmed the superior binding affinity of MPFPK. These findings establish goat milk as a valuable source of DPP-4 inhibitory peptides, with MPFPK demonstrating exceptional potency and stability. This integrated framework provides a scalable approach for discovering food-derived bioactives, advancing the development of natural therapeutics and functional foods for management of type 2 diabetes.