Implementation of a High-Throughput Screen for Identifying Small Molecules to Activate the Keap1-Nrf2-ARE Pathway

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01–30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.

The Impact of miRNA-Based Molecular Diagnostics and Treatment of NRF2-Stabilized Tumors

“…therapy. Indeed, several previous studies have described the exploration and identification of small molecules abrogating the pathway (36,37). However, it has also been shown that carcinogenesis, especially in the earliest stages, can be suppressed by the activation of NRF2, and many drugs enhance NRF2 activity (38,39).…”

Astroglia as a cellular target for neuroprotection and treatment of neuro‐psychiatric disorders

“…Numerous cell‐based and in silico screens have identified Nrf2‐activating compounds (Schaap, Hancock, Wilderspin, & Wells, 2013; Wang et al, 2013; Williamson et al, 2012; Wu, McDonald, Liu, Chaguturu, & Klaassen, 2012), including triterpenoid 2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oate‐methylamide (CDDO‐MA), puerarin, sulforaphane, CDDO‐ethyl amide and others. Nrf2 activators demonstrated activity in in vitro and in vivo in different neurodegenerative mouse models, protecting neurons, decreasing the accumulation of aberrant proteins and increasing life span (Buendia et al, 2016; Joshi and Johnson 2012).…”

Polyhydroxylated fullerene attenuates oxidative stress-induced apoptosis via a fortifying Nrf2-regulated cellular antioxidant defence system

“…Under unstressed physiological conditions, Nrf2 is bound to the repressor Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and easily degraded by ubiquitin-proteasome. 7 Upon activation, the Nrf2/Keap1 complex is dissociated, and Nrf2 is released from Keap1 and translocates into the nucleus, where it forms a heterodimer with its obligatory partner Maf and binds to the ARE sequence to activate transcription of a battery of antioxidant and cytoprotective genes, including heme oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase 1 (NQO1), and γ-glutamate cysteine ligase. 8 Therefore, agents that modulate the Nrf2/ARE pathway would be expected to have beneficial effects in ameliorating oxidative stress-related diseases by upregulation of phase II antioxidant enzymes.…”

“…8 Therefore, agents that modulate the Nrf2/ARE pathway would be expected to have beneficial effects in ameliorating oxidative stress-related diseases by upregulation of phase II antioxidant enzymes. 7,8 The use of nanoparticles in biomedicine is currently an active area of research because nanoparticles hold great promise as novel and effective antioxidants for oxidative stress-related diseases. 9,10 The advantages of using nanoparticles as antioxidants over currently available antioxidants are due to the possibility that they have a versatile surface that can be decorated with active agents, 11 can penetrate the cell membrane, 12 can quench radicals without the need for assistance from other detoxifying molecules, 13 and possess higher physical stability in biological media, 14 thus extending their applications to the treatment of oxidative damage.…”