摘要：SummaryTranscription factor Nrf2 and its negative regulator Keap1 orchestrate a cytoprotective response against oxidative, metabolic, and inflammatory stress. Keap1 is a drug target, with several small molecules in drug development. Here, we show that the isoquinoline PRL-295 increased Keap1 thermostability in lysates from cells expressing fluorescently tagged Keap1. The thermostability of endogenous Keap1 also increased in intact cells and murine liver following PRL-295 treatment. Fluorescence Lifetime Imaging–Förster Resonance Energy Transfer (FLIM-FRET) experiments in cells co-expressing sfGFP-Nrf2 and Keap1-mCherry further showed that PRL-295 prolonged the donor fluorescence lifetime, indicating disruption of the Keap1-Nrf2 protein complex. Orally administered PRL-295 to mice activated the Nrf2transcriptional target NAD(P)H:quinone oxidoreductase 1 (NQO1) in liver and decreased the levels of plasma alanine aminotransferase and aspartate aminotransferase upon acetaminophen-induced hepatic injury. Thus, PRL-295 engages the Keap1 protein target in cells andin vivo, disrupting its interaction with Nrf2, leading to activation of Nrf2-dependent transcription and hepatocellular protection.Graphical abstractDisplay OmittedHighlights•PRL-295 increases the thermostability of Keap1 in cells and in the murine liver•PRL-295 disrupts the Keap1-Nrf2 protein complex in single live cells•Orally administered PRL-295 activates the Nrf2 transcriptional target NQO1 in murine liver•Orally administered PRL-295 to mice decreases acetaminophen-induced hepatic injuryBiological sciences; Biochemistry; Molecular interaction
