Binding thermodynamics and kinetics guided optimization of potent Keap1-Nrf2 peptide inhibitors

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 the rational activity improvement of peptide Keap1-Nrf2 inhibitors and to explore the 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 an intracyclic conformation showed a K-d value of 2.8 nM when binding to Keap1 and an 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-Nrf2 PPI inhibitor design. It also provided a useful probe to investigate the Keap1-Nrf2 PPI system.