Abstract
Transcription factor Nrf2 and its negative regulator Keap1 orchestrate a comprehensive cytoprotective response against oxidative, electrophilic, metabolic, and inflammatory stress. Keap1 is a drug target, with several small molecules currently in drug development. Here, we show that the isoquinoline PRL-295 increased Keap1 thermostability in lysates from cells stably expressing fluorescently-tagged Keap1. The thermostability of endogenous Keap1 also increased in intact cells and in the murine liver following PRL-295 treatment. Fluorescence Lifetime Imaging - Forster Resonance Energy Transfer (FLIM-FRET) experiments in cells co-expressing sfGFP-Nrf2 and Keap1-mCherry further showed that PRL-295 prolonged the fluorescence lifetime of the donor, indicating disruption of the Keap1-Nrf2 protein complex. Orally-administered PRL-295 to mice activated the Nrf2-transcriptional target NAD(P)H:oxidoreductase 1 in liver and decreased plasma alanine aminotransferase upon acetaminophen-induced hepatic injury. Thus, PRL-295 engages the Keap1 protein target in cells and in vivo, disrupting its interaction with Nrf2, leading to activation of Nrf2-dependent transcription, and hepatocellular protection.
Highlights
The transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2, encoded by NFE2L2) and its main negative regulator, the Kelch-like ECH associated protein 1 (Keap1), orchestrate the coordinated expression of large networks of proteins, allowing cellular and organismal adaptation to oxidative, electrophilic, metabolic, and inflammatory stress (Hayes and Dinkova-Kostova, 2014; Yamamoto et al, 2018)
Transcription factor Nrf2 and its negative regulator Keap1 orchestrate a cytoprotective response against oxidative, metabolic, and inflammatory stress
We show that PRL-295 binds to Keap1 in the cellular environment, disrupting its interaction with Nrf2, which leads to enhanced gene expression of the classical Nrf2 target NAD(P)H:quinone oxidoreductase 1 (NQO1) in cells and in vivo in the mouse liver
Summary
The transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf, encoded by NFE2L2) and its main negative regulator, the Kelch-like ECH associated protein 1 (Keap1), orchestrate the coordinated expression of large networks of proteins, allowing cellular and organismal adaptation to oxidative, electrophilic, metabolic, and inflammatory stress (Hayes and Dinkova-Kostova, 2014; Yamamoto et al, 2018). Keap serves as a substrate adaptor for a Cullin3-based RING E3 ubiquitin ligase, which continuously targets Nrf for ubiquitination and proteasomal degradation. This process can be disrupted by numerous small molecules, which target Keap either by covalently modifying its sensor cysteines or by binding to it non-covalently and inhibiting its protein–protein interactions with Nrf (Cuadrado et al, 2019). Clinical trials with the electrophilic Nrf inducers sulforaphane, dimethyl fumarate, bardoxolone methyl, and oltipraz have demonstrated beneficial effects in humans, and some are promising protective agents against unavoidable exposures to environmental pollutants (Yagishita et al, 2019, 2020). The Keap1/Nrf protein complex is a recognized drug target, several Nrf activators are in advanced clinical trials, including in patients with COVID-19, and dimethyl fumarate is used in clinical practice for the treatment of psoriasis (under the brand name Skilarence) and relapsing forms of multiple sclerosis (brand name Tecfidera) (Atanasov et al, 2021; Cuadrado et al, 2019, 2020)
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