Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy

Sci Rep. 2017 Jun 21;7(1):3945. doi: 10.1038/s41598-017-04233-3.


The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 μM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antioxidant Response Elements
  • Drug Evaluation, Preclinical
  • Drug Repositioning*
  • Hep G2 Cells
  • Humans
  • Kelch-Like ECH-Associated Protein 1 / metabolism*
  • NF-E2-Related Factor 2 / metabolism*
  • Oxidative Stress
  • Protein Binding
  • Spectrometry, Fluorescence
  • Up-Regulation


  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human