Schaap, M;
Hancock, R;
Wilderspin, A;
Wells, G;
(2013)
Development of a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 protein-protein interaction.
Protein Science
, 22
(12)
1812 - 1819.
10.1002/pro.2384.
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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.
| Type: | Article |
|---|---|
| Title: | Development of a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 protein-protein interaction |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1002/pro.2384 |
| Publisher version: | http://dx.doi.org/10.1002/pro.2384 |
| Language: | English |
| Additional information: | © 2013 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. |
| Keywords: | FRET, Keap1, Nrf2, high-throughput screening, protein-protein interactions |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy > Pharma and Bio Chemistry |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/1410304 |
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