Integrated discovery and mechanistic characterization of xanthine oxidase inhibitory peptides from sheep Milk

Hyperuricemia is rising worldwide, with xanthine oxidase (XOD) as a key therapeutic target. This study integrated peptidomics, machine learning, molecular dynamics simulations, and experimental validation to discover XOD-inhibitory peptides from sheep milk. A VHSE-CatBoost model screened milk peptides and identified four candidates: FAWP, GPGGAW, FGER, and YPF. All peptides inhibited XOD dose-dependently, with FAWP showing the strongest activity (IC₅₀ = 12.99 mM). Kinetic analysis showed mixed-type inhibition for FAWP and FGER, and competitive inhibition for GPGGAW and YPF. Thermodynamics demonstrated spontaneous FAWP-XOD binding (ΔG = -6.0 kcal/mol). UV and CD spectra indicated perturbation of aromatic microenvironments and reduced α-helix/β-sheet content in XOD. MD simulations showed that FAWP, GPGGAW, and FGER stably occupy the active site through hydrogen bonding and hydrophobic interactions. Network pharmacology further suggested multi-target effects on inflammation and renal pathways. These sheep milk-derived peptides offer promising natural agents for hyperuricemia management.