Discovery of potent Keap1-Nrf2 protein-protein interaction inhibitor based on molecular binding determinants analysis

J Med Chem. 2014 Mar 27;57(6):2736-45. doi: 10.1021/jm5000529. Epub 2014 Feb 21.


Keap1 is known to mediate the ubiquitination of Nrf2, a master regulator of the antioxidant response. Directly interrupting the Keap1-Nrf2 interaction has been emerged as a promising strategy to develop novel class of antioxidant, antiinflammatory, and anticancer agents. On the basis of the molecular binding determinants analysis of Keap1, we successfully designed and characterized the most potent protein-protein interaction (PPI) inhibitor of Keap1-Nrf2, compound 2, with K(D) value of 3.59 nM binding to Keap1 for the first time to single-digit nanomolar. Compound 2 can effectively disrupt the Nrf2-Keap1 interaction with an EC50 of 28.6 nM in the fluorescence polarization assay. It can also activate the Nrf2 transcription activity in the cell-based ARE-luciferase reporter assay in a dose-dependent manner. The qRT-PCR results of Nrf2 transcription targets gave the consistent results. These results confirm direct and highly efficient interruption of the Keap1-Nrf2 PPI can be fully achieved by small molecules.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Inflammatory Agents, Non-Steroidal / chemical synthesis
  • Anti-Inflammatory Agents, Non-Steroidal / pharmacology
  • Cell Membrane Permeability
  • Computational Biology
  • Computer Simulation
  • Crystallography, X-Ray
  • Dose-Response Relationship, Drug
  • Drug Discovery
  • Electrochemistry
  • Humans
  • Hydrogen Bonding
  • Interferometry
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / drug effects*
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Kelch-Like ECH-Associated Protein 1
  • Luciferases / genetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Conformation
  • NF-E2-Related Factor 2 / chemistry
  • NF-E2-Related Factor 2 / drug effects*
  • NF-E2-Related Factor 2 / metabolism
  • Oxidative Stress / drug effects
  • Protein Binding
  • RNA / biosynthesis
  • RNA / genetics
  • Real-Time Polymerase Chain Reaction
  • Small Molecule Libraries
  • Transcription, Genetic


  • Anti-Inflammatory Agents, Non-Steroidal
  • Intracellular Signaling Peptides and Proteins
  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • Small Molecule Libraries
  • RNA
  • Luciferases