Sheep Milk Peptides FAWP, GPGGAW, FGER, and YPF Potently Inhibit Xanthine Oxidase, Offering Natural Hyperuricemia Management

Hyperuricemia, characterized by elevated uric acid levels, is a growing global health concern linked to gout, kidney disease, and cardiovascular issues. Current therapeutic strategies often target xanthine oxidase (XOD), a key enzyme in purine metabolism responsible for uric acid production. While drugs like allopurinol effectively inhibit XOD, they can have side effects, prompting a search for safer, natural alternatives. Food-derived peptides offer a promising avenue due to their bioavailability and lower toxicity, addressing the need for novel, multi-target interventions in metabolic disorders.

Researchers employed an integrated approach combining peptidomics, machine learning, molecular dynamics simulations, and experimental validation to identify XOD-inhibitory peptides from sheep milk. A VHSE-CatBoost model was utilized to screen a library of milk peptides, leading to the identification of four promising candidates: FAWP, GPGGAW, FGER, and YPF. These peptides were then synthesized and subjected to in vitro assays to evaluate their dose-dependent XOD inhibitory activity. Further mechanistic insights were gained through kinetic analysis, thermodynamic studies, UV and CD spectroscopy, molecular docking, and molecular dynamics (MD) simulations to elucidate their binding mechanisms and effects on enzyme structure. Finally, network pharmacology was used to predict potential multi-target effects.

All four identified sheep milk-derived peptides—FAWP, GPGGAW, FGER, and YPF—demonstrated dose-dependent inhibition of xanthine oxidase (XOD). FAWP exhibited the strongest inhibitory activity, with an IC₅₀ of 12.99 mM. Kinetic analysis revealed distinct inhibition mechanisms: FAWP and FGER displayed mixed-type inhibition, suggesting binding to both the free enzyme and the enzyme-substrate complex, while GPGGAW and YPF acted as competitive inhibitors, primarily binding to the active site. Thermodynamic studies confirmed spontaneous binding of FAWP to XOD, indicated by a favorable ΔG of -6.0 kcal/mol. Spectroscopic analyses using UV and CD showed that peptide binding perturbed the aromatic microenvironments of XOD and reduced its α-helix and β-sheet content, suggesting conformational changes. MD simulations further elucidated that FAWP, GPGGAW, and FGER stably occupy the XOD active site through crucial hydrogen bonding and hydrophobic interactions, providing a molecular basis for their inhibitory effects. Network pharmacology predicted that these peptides might exert multi-target effects beyond XOD inhibition, potentially influencing inflammation and renal pathways, which are often dysregulated in hyperuricemia.