Keap1-Nrf2 Protein-Protein Interaction inhibitors(Japan Tobacco)

Direct inhibition of the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) protein-protein interaction (PPI) represents a critical pathway for enhancing the antioxidant response. Therefore, screening for direct Keap1-Nrf2 PPI inhibitors holds significant potential for addressing oxidative stress-related diseases. This study aims to develop an integrated approach to identify direct Keap1-Nrf2 PPI inhibitors from traditional Chinese medicine (TCM) using Achyranthis bidentatae Radix (ABR) as a case study. The approach incorporated ultrahigh-performance liquid chromatography-quadrupole-orbitrap mass spectrometry analysis, data mining, drug-like property evaluation, molecular docking, chemical structure clustering, molecular dynamics (MD) simulations, in vitro experimental validation, and density functional theory (DFT) calculations. A total of 517 compounds were identified in ABR, of which 248 met the drug-likeness criteria. Additionally, seventeen compounds from six structural clusters were identified as having theoretical Keap1-Nrf2 PPI inhibitory activity. Among these compounds, shidasterone, nortrachelogenin, wogonin, and N-trans-feruloylmethoxytyramine were subjected to experimental evaluation for their Keap1-Nrf2 PPI inhibitory and free radical scavenging activities. MD simulations and DFT calculations demonstrated that these compounds directly inhibited Keap1-Nrf2 PPI through hydrophobic interactions, hydrogen bonds, and salt bridges. Moreover, DFT calculations confirmed that these compounds scavenged free radicals via the hydrogen atom transfer mechanism. In conclusion, the strategy presented herein offers a robust framework for screening direct Keap1-Nrf2 PPI inhibitors with structural diversity from ABR and other TCM sources.