Development of a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 protein-protein interaction
Related Experiment Video
Updated: May 6, 2026
Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors
Published on: June 27, 2025
Marjolein Schaap1, Rowena Hancock, Andrew Wilderspin
1University College London, UCL School of Pharmacy, London, United Kingdom.
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.
Insights
Researchers developed a high-throughput assay to find inhibitors of the Keap1-Nrf2 protein interaction. This interaction is key for upregulating antioxidant genes, potentially preventing cancer and degenerative diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Chemoprevention strategies aim to upregulate antioxidant and detoxification genes.
- Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor crucial for this upregulation.
- Inhibition of the Nrf2-Kelch-like ECH associated protein 1 (Keap1) interaction is a target for Nrf2 activation.
Purpose of the Study:
- To develop a high-throughput assay for identifying inhibitors of the Keap1-Nrf2 protein-protein interaction (PPI).
- To provide a screening tool for small molecules that disrupt the Keap1-Nrf2 PPI.
Main Methods:
- Development of a fluorescence (or Förster) resonance energy transfer (FRET) assay.
- Utilizing Yellow fluorescent protein (YFP)-conjugated Keap1 Kelch binding domain and Cyan fluorescent protein (CFP)-conjugated Nrf2-derived peptide.
- Validation of the assay using unlabeled Nrf2-derived peptides.
Main Results:
- A FRET-based assay was successfully developed to monitor the Keap1-Nrf2 PPI.
- The assay demonstrated competition with unlabeled Nrf2 peptides, confirming its validity.
- The assay shows potential as a screening tool for small molecule inhibitors.
Conclusions:
- The developed FRET assay is a viable method for screening inhibitors of the Keap1-Nrf2 PPI.
- This assay can aid in the discovery of novel chemopreventive agents targeting Nrf2 activation.
- Further development could facilitate the identification of small molecules for cancer and degenerative disease prevention.
