Protocol for a Steady-State FRET Assay in Cancer Chemoprevention

  • Marjolein C. A. Schaap
  • Andreia M. R. Guimarães
  • Andrew F. Wilderspin
  • Geoffrey WellsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1379)


Cancer chemoprevention is an important strategy to prevent, reverse, or suppress the development of cancer. One of the target pathways that has emerged in recent years is the Keap1-Nrf2-ARE system that regulates the protection of cells against various carcinogens and their metabolites. Increased concentrations of the redox transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces the activation of antioxidant and phase 2 detoxifying genes. Nrf2 is regulated by substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1) that can target Nrf2 for ubiquitination and degradation by the proteasome. The interaction between Nrf2 and Keap1 can be disrupted at the protein–protein interface in order to increase Nrf2 activity for potential therapeutic purposes. This chapter describes a protocol for a steady-state fluorescence or Förster resonance energy transfer (FRET) assay to examine the Keap1–Nrf2 protein–protein interaction (PPI), to investigate the effects of Nrf2 mutations on Keap1 binding and finally to identify potential inhibitors of this PPI. In the assay system Keap1 is conjugated to an YFP protein at the N-terminus whereas an Nrf2-derived 16-mer peptide containing a high-affinity “ETGE” motif is conjugated to a CFP protein at the N-terminus.

Key words

FRET Keap1 Nrf2 Protein–protein interactions Peptide inhibitors Small-molecule inhibitors 



We thank Dr. Edwin Nkansah for kindly providing plasmids and Hei Leung for her contribution to the recombinant protein production. This work was supported by Cancer Research UK (C9344/A10268) and UCL School of Pharmacy.


  1. 1.
    Hayes JD, McMahon M, Chowdhry S et al (2010) Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway. Antioxid Redox Signal 13(11):1713–1748CrossRefPubMedGoogle Scholar
  2. 2.
    Zhang DD, Hannink M (2003) Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress. Mol Cell Biol 23(22):8137–8151PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Dinkova-Kostova AT, Holtzclaw WD, Cole RN et al (2002) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci U S A 99(18):11908–11913PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Kobayashi M, Li L, Iwamoto N et al (2009) The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds. Mol Cell Biol 29(2):493–502PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Tong KI, Katoh Y, Kusunoki H et al (2006) Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model. Mol Cell Biol 26(8):2887–2900PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Itoh K, Wakabayashi N, Katoh Y et al (1999) Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 13(1):76–86PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Ahn, Y.H., Hwang Y, Liu H et al., Electrophilic tuning of the chemoprotective natural product sulforaphane. Proc Natl Acad Sci U S A. 107(21): p. 9590-5.Google Scholar
  8. 8.
    Lo SC, Li X, Henzl MT et al (2006) Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling. EMBO J 25(15):3605–3617PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Hancock R, Bertrand HC, Tsujita T. et al. Peptide inhibitors of the Keap1-Nrf2 protein–protein interaction. Free Radic Biol Med. 52(2): 44-51.Google Scholar
  10. 10.
    Magesh S, Chen Y, Hu L (2012) Small molecule modulators of Keap1-Nrf2-ARE pathway as potential preventive and therapeutic agents. Med Res Rev 32(4):687–726PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Clegg RM (2009) Förster resonance energy transfer—FRET what is it, why do it, and how it’s done. In: Gadella TWJ (ed) Laboratory techniques in biochemistry and molecular biology. Elsevier BV, Urbana, p 38Google Scholar
  12. 12.
    Guimarães AMR. Screening molecular interactions for drug discovery, PhD thesis. 2013, UCL, School of Pharmacy: London.Google Scholar
  13. 13.
    Schaap M, Hancock R, Wilderspin A et al (2013) Development of a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 protein–protein interaction. Protein Sci 22(12):1812–1819PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Shibata T, Ohta T, Tong KI et al (2008) Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy. Proc Natl Acad Sci U S A 105(36):13568–13573PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Qin QP, Peltola O, Pettersson K (2003) Time-resolved fluorescence resonance energy transfer assay for point-of-care testing of urinary albumin. Clin Chem 49(07):1105–1113CrossRefPubMedGoogle Scholar
  16. 16.
    Nkansah E, Shah R, Collie GW et al (2013) Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography. FEBS Lett 587(7):833–839CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Marjolein C. A. Schaap
    • 1
  • Andreia M. R. Guimarães
    • 1
  • Andrew F. Wilderspin
    • 1
  • Geoffrey Wells
    • 1
    Email author
  1. 1.UCL School of PharmacyUniversity College LondonLondonUK

Personalised recommendations