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Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy
Yuki Yoshizaki1, Takayasu Mori1, Mari Ishigami-Yuasa2
1Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Scientific Reports
|June 23, 2017
Summary
Researchers screened 1,633 drugs to find Keap1-Nrf2 inhibitors. Two drugs successfully disrupted the Keap1-Nrf2 interaction, upregulating the Nrf2-ARE pathway and its protective stress response enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is crucial for cellular defense against oxidative and electrophilic stress.
- The Cul3/Keap1 E3 ubiquitin ligase complex targets Nrf2 for ubiquitination and degradation, thus regulating its expression.
Purpose of the Study:
- To identify inhibitors of the Keap1-Nrf2 protein-protein interaction through drug repositioning.
- To upregulate Nrf2 expression and subsequent transcription of antioxidant response element (ARE)-controlled genes, such as HO-1.
Main Methods:
- Development and utilization of a fluorescence correlation spectroscopy (FCS) screening system to detect Keap1-Nrf2 binding.
- Screening of 1,633 existing drugs for their ability to inhibit Keap1-Nrf2 interaction.
- Validation of drug efficacy in HepG2 cells by measuring Nrf2 protein levels, ARE gene promoter activity, and HO-1 mRNA expression.
Main Results:
- The FCS system successfully detected the Keap1-Nrf2 binding with a KD of 2.6 μM.
- Initial screening identified 12 candidate drugs, with 2 demonstrating significant Nrf2 protein upregulation in HepG2 cells.
- These two drugs also enhanced ARE gene promoter activity and HO-1 mRNA expression, confirming Keap1-Nrf2 dissociation and Nrf2-ARE pathway activation.
Conclusions:
- Drug repositioning screening using FCS is an effective method for identifying Keap1-Nrf2 binding inhibitors.
- Two known drugs were identified as promising activators of the Nrf2-ARE pathway, offering potential therapeutic applications for oxidative stress-related conditions.
