Abstract
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.
Highlights
Oxidative stress is the consequence of imbalanced production of reactive oxygen species (ROS) and the ability of cells to detoxify these ROS, which can result in ROS-induced tissue damage
The present study describes the development, implementation, and validation of a high-throughput screen to identify activators of the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2-antioxidant response elements (ARE) pathway
Using a luciferase-based assay driven by AREs in the promoter region of the luciferase gene, the AREc32 cells provides a rapid and convenient quantification of
Summary
Oxidative stress is the consequence of imbalanced production of reactive oxygen species (ROS) and the ability of cells to detoxify these ROS, which can result in ROS-induced tissue damage. The Kelch-like ECH-associated protein 1 (Keap1) - nuclear factor, erythroid derived 2, like 2 (Nrf2) pathway serves as one of the major protective mechanisms in cells in response to oxidative/ electrophilic stress. Nrf heterodimerizes with a variety of transcriptional regulatory proteins, including members of the activator protein-1 family (Jun and Fos), and the small Maf family of transcription factors [6]. These protein complexes bind to antioxidant response elements (ARE) located in the upstream promoter region of a battery of genes, and drives their transcription [7]
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