Novel peptides with xanthine oxidase inhibitory activity identified from macadamia nuts: integrated in silico and in vitro analysis

Peptides with xanthine oxidase (XOD)-inhibitory activities are potential dietary interventions for the treatment of hyperuricemia and gout. In this study, Macadamia integrifolia antimicrobial protein 2 (MiAMP2) was chosen for in silico proteolysis with pepsin, trypsin and chymotrypsin through ExPASy PeptideCutter, and the obtained peptides were further screened by water solubility, ADMET prediction and molecular docking. Four novel peptides, namely RPLY, PGPR, HGGR and GPY, were supposed to be non-toxic and have XOD-inhibitory potential. Molecular docking analysis showed that these peptides were able to bind to the active sites (such as Leu873, Phe914 and Thr1010) of XOD through interactions mainly including conventional hydrogen bond, alkyl and pi-interaction. These peptides were further synthesized to verify their in vitro XOD-inhibitory activities, and the IC50 values of PGPR, GPY and HGGR were shown to be 24.84 ± 0.02, 30.44 ± 0.33 and 24.89 ± 0.19 mM, respectively. Kinetic experiments demonstrated that PGPR and HGGR are mixed-type inhibitors, and GPY is a competitive inhibitor toward XOD with the inhibitory constant (Ki) values ranging from 1.09 to 8.98. Our results suggested that MiAMP2 is an important source of bioactive peptides for the management of hyperuricemia.