Ulcerative colitis (UC) is a chronic relapsing-remitting form of inflammatory bowel disease (IBD) that increases the risk of colorectal cancer, the third most common malignancy in humans. Oxidative stress is a risk factor for the development of UC. The Keap1-Nrf2-ARE pathway is one of the most important defensive mechanisms against oxidative and/or electrophilic stresses. In this study, we identified CPUY192018 as a potent small-molecule inhibitor of the Keap1-Nrf2 PPI, investigated the cyto-protective effects of CPUY192018 on the NCM460 colonic cells and evaluated whether treatment with the inhibitor of the Keap1-Nrf2 PPI exerts protection on an established experimental model of UC induced by dextran sodium sulfate (DSS). Our study clearly demonstrated that CPUY192018 had a cytoprotective effect against DSS in both NCM460 cells and mouse colon via the activation of Nrf2 signaling. These results suggested that activation of Nrf2 by directly inhibiting the Keap1-Nrf2 PPI may be beneficial as a treatment for UC.

Activation of Nrf2 by directly inhibiting the Keap1-Nrf2 Protein-Protein Interaction (PPI) has gained research interest with regard to developing novel agents for treating inflammatory related diseases. In this study, computational methods were used to guide rational activity improvement of peptide Keap1-Nrf2 inhibitors and to explore Keap1 binding cavity. Terminal hydrophobic residues and Tyr residue replacements were introduced into the novel peptides. The experimental values of these peptides further confirmed the potential binding sites explored by the MD simulation. Finally, the most promising peptide 5 with the intracyclic conformation showed a Kd value of 2.8 nM binding to Keap1 and IC50 value of 9.4 nM in the Keap1-Nrf2 PPI fluorescence polarization assay. It proved that the active conformation locking strategy is quite useful in Keap1-Nf2 PPI inhibitor design. It also provided a useful probe to investigate the Keap1-Nrf2 PPI system.

Protein-protein interactions (PPIs) as drug targets have been gaining growing interest, though developing drug-like small molecule PPI inhibitors remains challenging. Peptide PPI inhibitors, which can provide informative data on the PPI interface, are good starting points to develop small molecule modulators. Computational methods combining molecular dynamics simulations and binding energy calculations could give both the structural and the energetic perspective of peptide PPI inhibitors. Herein, we set up a computational workflow to investigate Keap1-Nrf2 peptide PPI inhibitors and predict the activity of novel sequences. Furthermore, we applied this method to investigate p62 peptides as PPI inhibitors of Keap1-Nrf2 and explored the activity change induced by the phosphorylation of serine. Our results showed that because of the unfavorable solvation effects, the binding affinity of the phosphorylated p62 peptide is lower than the Nrf2 ETGE peptide. Our research results not only provide a useful method to investigate the Keap1-Nrf2 peptide inhibitors, but also give a good example to show how to incorporate computational methods into the study of peptide PPI inhibitors. Besides, applying this method to p62 peptides provides a detailed explanation for the expression of cytoprotective Nrf2 targets induced by p62 phosphorylation, which may benefit the further study of the crosstalk between the Keap1-Nrf2 pathway and p62-mediated selective autophagy.