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Updated: May 17, 2026
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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
Implementation of a high-throughput screen for identifying small molecules to activate the Keap1-Nrf2-ARE pathway
Kai Connie Wu1, Peter R McDonald, Jie Jerry Liu
1Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, United States of America.
Plos One
|October 12, 2012
Summary
Researchers developed a high-throughput screen to find activators of the Keap1-Nrf2-ARE pathway. This screen identified potent Nrf2 activators, offering new chemical scaffolds for treating oxidative stress diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates antioxidant and electrophile-detoxifying genes.
- Nrf2 activation is crucial for combating oxidative and electrophilic stress-induced diseases.
- Identifying novel Nrf2 activators is a key therapeutic strategy.
Purpose of the Study:
- To develop and validate a high-throughput screening assay for identifying Nrf2 activators.
- To discover novel chemical compounds that activate the Keap1-Nrf2-ARE pathway.
- To analyze structure-activity relationships of identified Nrf2 activators.
Main Methods:
- Utilized AREc32 cells with a luciferase reporter gene under ARE promoter control for screening.
- Screened approximately 47,000 compounds to identify potential Nrf2 activators.
- Validated hits using concentration-response assays and measured Nqo1 mRNA induction in Hepa1c1c7 cells.
Main Results:
- Identified 238 top hits (0.5%) with >14.4-fold luminescence increase from 47,000 compounds.
- 231 compounds showed concentration-dependent luminescence increase; four scaffolds were enriched.
- 30 compounds were highly potent Nrf2 activators, and 17 increased Nqo1 mRNA levels.
Conclusions:
- Successfully developed and validated a high-throughput screen for Keap1-Nrf2-ARE pathway activators.
- Discovered novel Nrf2 activators with potential therapeutic applications.
- Identified promising chemical scaffolds for preventing oxidative stress-related toxicity and carcinogenesis.
