title: Development of a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 protein-protein interaction.
abstract: One of the strategies proposed for the chemoprevention of degenerative diseases and cancer involves upregulation of antioxidant and free radical detoxification gene products by increasing the intracellular concentration of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This can be achieved by disrupting the interaction between Nrf2 and Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Here, we describe the development of a high-throughput fluorescence (or Förster) resonance energy transfer assay for the identification of inhibitors of the Keap1-Nrf2 protein-protein interaction (PPI). The basis of this assay is the binding of a YFP-conjugated Keap1 Kelch binding domain to a CFP-conjugated Nrf2-derived 16-mer peptide containing a highly conserved "ETGE" motif. The competition aspect of the assay was validated using unlabeled Nrf2-derived 7-mer and 16-mer peptides and has potential as a screening tool for small molecule inhibitors of the PPI. We discuss the development of this assay in the context of other methods used to evaluate this PPI.
authors.affiliation: University College London, UCL School of Pharmacy, London, United Kingdom.
authors.forename: Marjolein
authors.initials: M
authors.lastname: Schaap
authors.forename: Rowena
authors.initials: R
authors.lastname: Hancock
authors.forename: Andrew
authors.initials: A
authors.lastname: Wilderspin
authors.forename: Geoff
authors.initials: G
authors.lastname: Wells
mesh_terms: D020816:Amino Acid Motifs; D001667:Binding, Competitive; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2 / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D013194:Staining and Labeling
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D010455:Peptides
keywords: FRET; Keap1; Nrf2; high-throughput screening; protein-protein interactions