Abstract
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.
Highlights
Oxidative stress occurs as a result of increased reactive oxygen species (ROS) and/or depressed capacity of the antioxidant system[1]
We focused on identified chemical compounds that efficiently disrupted the binding of the ETGE motif in Nrf[2] to the DGR domain in Keap[1]
Most of these drugs are approved for animal or human use, and their pharmacological and toxicological profiles have been defined and published
Summary
Oxidative stress occurs as a result of increased reactive oxygen species (ROS) and/or depressed capacity of the antioxidant system[1]. In terms of preventive medicine, activation of Nrf[2] signaling pathway is considered as a promising strategy for cancer chemoprevention, leading to the expression of cytoprotective genes against cancer. These reports support the idea that the activation of Nrf[2] could contribute to prevention of numerous diseases. Recent studies have shown that several natural products, including sulforaphane, Glycyrrhiza glabra, resveratrol, curcumin, and epigallocatechin-3-gallate have the potential as an Nrf[2] activator with cytoprotective functions[14,15,16,17,18]. We focused on “drug repositioning”, i.e., the application of known drugs to treat new indications, in an attempt to reduce costs and risks
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