Design of Wheat Protein-Derived Keap1-Targeting Antioxidant Peptides: Integrating Intelligent Screening, ProteinMPNN-Guided Sequence Optimization, and TAT-Fused Delivery.

Wheat gliadin-derived peptides show promising antioxidant potential, but their target-guided discovery remains challenging. Here, we established a computation-driven pipeline for Keap1-targeting antioxidant peptides. From 2798 virtual oligopeptides generated using our Peptide_MDI platform, KCENEECTFP and RELVNSTCFA were selected through multitier screening and molecular dynamics simulations. KCENEECTFP exhibited antioxidant activity and modulated the Keap1-Nrf2 pathway in H2O2-stressed IPEC-J2 cells. Structure-guided ProteinMPNN optimization yielded SCDCPSCLYP, which showed enhanced radical scavenging activity, antioxidant enzyme responses, and regulation of Keap1-Nrf2-related genes. SCDCPSCLYP also promoted Nrf2 nuclear translocation and attenuated p65 activation. Surface plasmon resonance confirmed direct Keap1 binding (KD = 7.25 μM), while the cellular thermal shift assay (CETSA) supported Keap1 target engagement in cells. N-terminal TAT fusion further improved cellular uptake and antioxidant efficacy. These findings demonstrate an integrated strategy combining target-guided screening, structure-informed optimization, target validation, and intracellular delivery for the rational development of wheat gliadin-derived antioxidant peptides.