Agathisflavone from Schinus terebinthifolia: Structure-Based Analysis of Its Interaction with the Keap1 Kelch Domain

The antioxidant activity of Schinus terebinthifolia fruit extracts was investigated through an integrated in vitro and in silico approach. The extract exhibited high total phenolic (239.03 ± 74.14 mg GAE/100) and flavonoid (661.57 ± 28.09 mg QE/100 g) contents, together with antioxidant activity. Phytochemical characterization identified three major biflavonoids, I6, II8-biapigenin (agathisflavone), I3, II8-binaringenin, and I3′, II8-biapigenin, with agathisflavone identified as the predominant constituent. To investigate the structural basis of the antioxidant activity, the three biflavonoids were subjected to molecular docking against the Kelch domain of the human Keap1 Protein Data Bank (PDB ID: 2FLU) using DOCK6. The receptor was prepared by removing crystallographic water molecules, adding hydrogen atoms, and assigning partial charges, while ligand flexibility was explored using the anchor-and-grow search algorithm. Binding poses were ranked with the Grid Score scoring function, and the docking protocol was validated by receiver operating characteristic (ROC) analysis, yielding an AUC of 0.726, supporting the predictive reliability of the docking workflow. Comparative analysis of the docking poses demonstrated that agathisflavone exhibited the highest interaction complementarity within the Keap1 binding pocket. The best-ranked complex was stabilized by four hydrogen bonds involving Ser39, Gln206, and Ser231, with Ser231 establishing two anchoring interactions, in addition to two T-shaped π–π stacking interactions between the aromatic scaffold of agathisflavone and Tyr201 and Tyr248. This cooperative network of polar and aromatic contacts promotes favorable geometric and electronic complementarity, indicating stable molecular recognition. These findings suggest that agathisflavone may competitively occupy the Nrf2 recognition site on Keap1, providing a structural explanation for the experimentally observed antioxidant activity and identifying this biflavonoid as a promising natural modulator of the Keap1–Nrf2 antioxidant signaling pathway.