Development of an enzyme-linked immunosorbent assay for Keap1-Nrf2 interaction inhibitors identification
Yan Wang,
Chu-Ying Xiao,
Huang-Quan Lin,
Jian-Shu Hu,
Tsz-Ming Ip,
David Chi-Cheong Wan
Affiliations
Yan Wang
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; Center for Translation Medicine Research and Development, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, The Chinese Academy of Sciences, Shenzhen, 518055, China; Corresponding author. 1068 Xueyuan Avenue, Shenzhen University Town, Nanshan, Shenzhen, PR China.
Chu-Ying Xiao
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
Huang-Quan Lin
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, 518057, China
Jian-Shu Hu
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
Tsz-Ming Ip
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
David Chi-Cheong Wan
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; Corresponding author. 326A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area39, The Chinese University of Hong Kong, Shatin, N.T., SAR Hong Kong, PR China.
Development of Keap1–Nrf2 interaction inhibitors is a promising strategy for the discovery of therapeutic agents against oxidative stress-mediated diseases. Two motifs of Nrf2, ETGE and DLG motif, are responsible for Keap1-Nrf2 binding. Previously, ETGE peptide or ETGE-derived peptide-based approaches were used to detect Keap1-Nrf2 interaction; however, these approaches are not able to monitor Keap1-DLG motif binding. We first report here a novel Enzyme-linked Immunosorbent Assay (ELISA) approach to detect the protein-protein interaction of full length Keap1 and Nrf2. In our assay, the test compounds can target either ETGE or DLG binding site, therefore facilitating the exploration of diverse Keap1-Nrf2 inhibitors. Three FDA-approved drugs, zafirlukast, dutasteride and ketoconazole, were found to inhibit the Keap1-Nrf2 interaction with IC50 of 5.87, 2.81 and 1.67 μM, respectively. Additionally, these three drugs also activated Nrf2 pathway in neuroblasts and lipopolysaccharide (LPS)-challenged mice. The results presented here indicate that the ELISA approach has the capacity to identify Keap1-Nrf2 inhibitors.
