Implementation of a high-throughput screen for identifying small molecules to activate the Keap1-Nrf2-ARE pathway.
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.
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, United States of America.
Kai Connie
KC
Wu
Peter R
PR
McDonald
Jie Jerry
JJ
Liu
Rathnam
R
Chaguturu
Curtis D
CD
Klaassen
D000818:Animals; D061745:Antioxidant Response Elements / Q000235:genetics*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D051379:Mice; D015394:Molecular Structure; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D009930:Organic Chemicals / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011728:Pyridones / Q000737:chemistry / Q000494:pharmacology; D011744:Pyrimidinones / Q000737:chemistry / Q000494:pharmacology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D054852:Small Molecule Libraries
D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D009930:Organic Chemicals; D011728:Pyridones; D011744:Pyrimidinones; D054852:Small Molecule Libraries; D008156:Luciferases; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human