Screening and identification of covalent Nrf2 activators with α,β-unsaturated imide structures

Abstract

The Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is a central regulator of cellular defense mechanisms against oxidative stress. The pharmacological activation of this pathway has emerged as a promising strategy for the treatment of diseases such as neurodegeneration and inflammation. In this study, we performed a high-throughput luciferase reporter assay to screen the original compound library comprising small molecules synthesized at Kobe Pharmaceutical University. Initial screening identified KU-0016, KU-0017, and KU-0171 as moderate activators of the Nrf2 pathway. Furthermore, structural comparison revealed a common α,β-unsaturated imide motif, which led to the synthesis of KU-0017-based analogs for structure–activity relationship analysis. Among them, KU-0479 exhibited potent Nrf2 activation and low cytotoxicity. Further assays demonstrated that KU-0479 increased the intracellular glutathione (GSH) levels and protected cells from hydrogen peroxide–induced oxidative damage in a GSH-dependent manner. Moreover, LC–MS analysis confirmed the formation of mono-GSH adducts, and covalent docking simulations suggested covalent binding at the Cys151 residue of Keap1. These findings highlight KU-0479 as a synthetically accessible covalent Nrf2 activator with favorable redox-modulating properties and therapeutic potential.