Discovery of a small-molecule inhibitor and cellular probe of Keap1-Nrf2 protein-protein interaction

Bioorg Med Chem Lett. 2013 May 15;23(10):3039-43. doi: 10.1016/j.bmcl.2013.03.013. Epub 2013 Mar 14.


A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1-Nrf2 protein-protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure-activity relationships support its use as a lead for our ongoing optimization.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Crystallography, X-Ray
  • Dose-Response Relationship, Drug
  • Drug Discovery*
  • Fluorescence Polarization
  • High-Throughput Screening Assays
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Isoquinolines / chemistry
  • Isoquinolines / pharmacology*
  • Kelch-Like ECH-Associated Protein 1
  • Models, Molecular
  • Molecular Imaging*
  • Molecular Probes / chemistry
  • Molecular Probes / pharmacology*
  • Molecular Structure
  • NF-E2-Related Factor 2 / metabolism*
  • Phthalimides / chemistry
  • Phthalimides / pharmacology*
  • Protein Binding / drug effects
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology*
  • Structure-Activity Relationship


  • Intracellular Signaling Peptides and Proteins
  • Isoquinolines
  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • LH601A
  • Molecular Probes
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • Phthalimides
  • Small Molecule Libraries