Identification of a novel and high-affinity cyclic peptide targeting Keap1 for inflammation treatment by a combined virtual screening strategy

Inhibition of the Keap1-Nrf2 protein-protein interaction with cyclic peptides represents an attractive strategy to treat inflammation. However, the cyclic peptides for this inhibition are constrained by their low affinity. In this study, the peptides 1-6 were computationally screened using Keap1-pharmacophore modelling, toxicity screening, molecular docking, and interaction analysis. Subsequently, affinity experiments showed that peptide-4 exhibited potent binding affinity for Keap1 (<i>K</i>_d = 7.1 ± 0.2 nM). MD simulations further demonstrated that peptide-4 stably bound to Keap1. Additionally, MTT assays confirmed that peptides 1-6 induced negligible cytotoxicity in RAW264.7 macrophages. <i>In vitro</i> anti-inflammatory experiments indicated that peptide-4 significantly inhibited the mRNA and protein expression of IL-6 and TNFα in LPS-induced RAW264.7 cells. More importantly, experiments in Keap1-knockout RAW264.7 macrophages confirmed that the anti-inflammatory activity of peptide-4 was highly Keap1-dependent. In conclusion, the data demonstrated that the peptide-4 may be a promising candidate cyclic peptide to treat inflammatory disease.