Implementation of a High-Throughput Screen for Identifying Small Molecules to Activate the Keap1-Nrf2-ARE Pathway

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Issue Date
2012-10-08Author
Wu, Kai Connie
McDonald, Peter R.
Liu, Jie Jerry
Chaguturu, Rathnam
Klaassen, Curtis D.
Publisher
Public Library of Science
Type
Article
Article Version
Scholarly/refereed, publisher version
Rights
Copyright: ©Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Show full item recordAbstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01–30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.
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Citation
Wu, K. C., McDonald, P. R., Liu, J. J., Chaguturu, R., & Klaassen, C. D. (2012). Implementation of a High-Throughput Screen for Identifying Small Molecules to Activate the Keap1-Nrf2-ARE Pathway. PLoS ONE, 7(10). http://dx.doi.org/10.1371/journal.pone.0044686
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Except where otherwise noted, this item's license is described as: Copyright: ©Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.