title: Optimization of fluorescently labeled Nrf2 peptide probes and the development of a fluorescence polarization assay for the discovery of inhibitors of Keap1-Nrf2 interaction.
abstract: Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z' factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.
authors.affiliation: Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.
authors.forename: Daigo
authors.initials: D
authors.lastname: Inoyama
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.forename: Xinyi
authors.initials: X
authors.lastname: Huang
authors.forename: Lesa J
authors.initials: LJ
authors.lastname: Beamer
authors.forename: Ah-Ng Tony
authors.initials: AN
authors.lastname: Kong
authors.forename: Longqin
authors.initials: L
authors.lastname: Hu
mesh_terms: D001667:Binding, Competitive; D004353:Drug Evaluation, Preclinical; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; 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*; D011485:Protein Binding / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; D010455:Peptides