In Silico Analysis of Novel DPP-IV Inhibitory Peptides Released from Camel Milk Lactoferrin and the Possible Pathways Involved in Diabetes Protection

Objective: To screen novel dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from camel milk lactoferrin (LF) combined with bioinformatics. Network pharmacology was used to explore the potential mechanism of action of screened peptides on diabetes. Methods: Multiple peptides were generated by simulated enzymatic cleavage of lactoferrin using the BIOPEP website. Screening target peptides by combining peptide databases and molecular docking. Four of them were selected for solid-phase synthesis to verify their DPP-IV inhibitory activity. Molecular docking was performed to analyze the interaction between the peptide and DPP-IV molecules, and the Lineweaver-Burk method was used to analyze the inhibition mode of the peptide. Then, GPQY with stronger inhibition was selected for network pharmacological analysis to predict its potential mechanism of action on diabetes. Furthermore, the active targets of GPQY and diabetes were mined from the Swiss Target Prediction and Gene Cards databases, and the String database was used to obtain protein-protein interaction relationships. The PPI networks were built by Cytoscape 3.9.0 software, and the DAVID database was exploited for enrichment analysis of GO and KEGG signaling pathways for key targets. Results: Two DPP-IV inhibitory peptides were obtained with semi-inhibitory concentration (IC50) values of 348.27±16.11 and 1024.89±19.67 μmol/L. Inhibition pattern analysis indicated competitive inhibition of GPQY and mixed-type inhibition of EACAF. Molecular docking results revealed two peptides bound to the active pocket of DPP-IV through hydrogen bonding, hydrophobic interactions and electrostatic interactions. From the PPI network analysis, GPQY had 25 core-acting targets, including STAT3, MMP9, SRC, and MAPK1. The enrichment results were based on KEGG pathways, which showed that GPQY was involved in the IL-17 signaling pathway, tumor necrosis factor (TNF) signaling pathway, renin-angiotensin system, apoptosis, etc. Conclusion: Camel milk lactoferrin is a good source of DPP-IV inhibitor peptide. GPQY could prevent diabetes and its complications through multiple targets and pathways involved in the inflammatory response and cell proliferation and differentiation.