Abstract
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.
Highlights
Oxidative stress has been demonstrated involving into many pathophysiological processes that would lead to diseases including cancer, diabetes, cardiovascular and neurodegenerative diseases [1]
When the cell is challenged with oxidative stress, there is a conformation change of Kelch-like ECH-associated protein 1 (Keap1) through dissociation of nuclear factor erythroid 2-related factor 2 (Nrf2), followed by Nrf2 nuclear translocation to trigger the transcriptional activation of NADPH: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) [2]
ETGE motif and DLG motif locating on Neh2 domain are responsible for Keap1-Nrf2 binding (Fig. 1B and C) [4]
Summary
Oxidative stress has been demonstrated involving into many pathophysiological processes that would lead to diseases including cancer, diabetes, cardiovascular and neurodegenerative diseases [1]. An alternative assay should be available to validate the bioactivity of hits that are filtered by FP or FRET screening Both of these two approaches employed Nrf peptide or Nrf2-derived peptide to monitor Keap1-Nrf ETGE peptide disruption (Fig. 1C). FP and FRET assays are two common approaches for evaluating Keap1-Nrf PPI inhibitors [5]. Both FP and FRET assays utilize fluorescent label strategy which can be applied to HTS assay development. Either FITC or CFP is relatively sensitive to autofluorescence of test compounds These peptides in fluorescence-based assays mimic ETGE motif that docks with Keap1-DC (Fig. 1C), indicating that these assays monitor Keap1-Nrf ETGE peptide disruption [6,7]. The ELISA approach was selected for our assay development to avoid autofluorescence interference produced by test compounds
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