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

• Published in: Bioorganic & medicinal chemistry letters Vol. 23; no. 10; pp. 3039 - 3043
• Main Authors: Hu, Longqin, Magesh, Sadagopan, Chen, Lin, Wang, Lili, Lewis, Timothy A., Chen, Yu, Khodier, Carol, Inoyama, Daigo, Beamer, Lesa J., Emge, Thomas J., Shen, Jian, Kerrigan, John E., Kong, Ah-Ng Tony, Dandapani, Sivaraman, Palmer, Michelle, Schreiber, Stuart L., Munoz, Benito
• Format: Journal Article
• Language: English
• Published: OXFORD Elsevier Ltd 15.05.2013; Elsevier
• Subjects: Chemistry; Chemistry, Medicinal; Chemistry, Organic; chromatography; cis-trans isomers; Crystallography, X-Ray; Dose-Response Relationship, Drug; Drug Discovery; fluorescence; Fluorescence Polarization; High-Throughput Screening Assays; Humans; Inhibitors of protein–protein interaction; Intracellular Signaling Peptides and Proteins - metabolism; Isoquinolines - chemistry; Isoquinolines - pharmacology; Keap1; Kelch-Like ECH-Associated Protein 1; Life Sciences & Biomedicine; Models, Molecular; Molecular Imaging; Molecular Probes - chemistry; Molecular Probes - pharmacology; Molecular Structure; NF-E2-Related Factor 2 - metabolism; Nrf2; Nrf2 activation; Pharmacology & Pharmacy; Phthalimides - chemistry; Phthalimides - pharmacology; Physical Sciences; Protein Binding - drug effects; protein-protein interactions; reporter genes; Science & Technology; Small Molecule Libraries - chemistry; Small Molecule Libraries - pharmacology; stereochemistry; stereoisomers; Structure-Activity Relationship; structure-activity relationships; X-ray diffraction; Keap1; Nrf2 activation; Nrf2; Inhibitors of protein–protein interaction; TARGET; Inhibitors of protein-protein interaction; PATHWAY; BARDOXOLONE METHYL; MODULATORS; KIDNEY-FUNCTION
• ISSN: 0960-894X; 1464-3405
• DOI: 10.1016/j.bmcl.2013.03.013

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