2. Academic Validation
  3. The electrophilic metabolite of kynurenine, kynurenine-CKA, targets C151 in Keap1 to derepress Nrf2

The electrophilic metabolite of kynurenine, kynurenine-CKA, targets C151 in Keap1 to derepress Nrf2

  • bioRxiv. 2025 Nov 18:2025.11.18.689077. doi: 10.1101/2025.11.18.689077.
Jialin Feng 1,  Mara Carreño 2,  Hannah Jung 3,  Sharadha Dayalan Naidu 1,  Nicole Arroyo Diaz 2,  Abel D Ang 1,  Bhargavi Kulkarni 2,  Dorothy Kisielewski 1,  Takafumi Suzuki 4,  Masayuki Yamamoto 4,  John D Hayes 1,  Tadashi Honda 5,  Beatriz Leon-Ruiz 6,  Aimee L Eggler 3,  Dario A Vitturi 2,  Albena T Dinkova-Kostova 1 7
Affiliations

Affiliations

  • 1 Jacqui Wood Cancer Centre, Division of Cancer Research, School of Medicine, University of Dundee, Dundee, UK.
  • 2 Department of Pathology, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL, USA.
  • 3 Department of Chemistry and Biochemistry, Villanova University, Villanova, PA, USA.
  • 4 Department of Biochemistry and Molecular Biology, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
  • 5 Department of Chemistry and Institute of Chemical Biology & Drug Discovery, Stony Brook University, Stony Brook, NY, USA.
  • 6 Innate Cells and Th2 Immunity Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
  • 7 Department of Physiology, Pharmacology and Therapeutics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
PMID: 41332654 DOI: 10.1101/2025.11.18.689077
Abstract

The Kelch-like ECH-associated protein 1 / Nuclear factor-erythroid 2 p45-related factor 2 (Keap1/Nrf2) system responds to a wide array of structurally diverse small molecules, of both exogenous and endogenous origin, by inducing a robust cytoprotective program that allows adaptation during oxidative, metabolic and inflammatory stress. Here, we report that exposure to the tryptophan metabolite kynurenine and its electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA) leads to an increase in the abundance of transcription factor Nrf2 and induction of Nrf2-target genes, including NAD(P)H:quinone oxidoreductase 1 (NQO1), in murine and human cells. Additionally, both kynurenine and Kyn-CKA activate the Aryl Hydrocarbon Receptor (AhR). Using cellular thermal shift assays, we found that Kyn-CKA increases the thermal stability of Keap1-mCherry fusion protein, but not free mCherry, indicating target engagement of Keap1, the principal repressor of Nrf2. The use of purified recombinant BTB domain of Keap1 and its C151S mutant counterpart revealed that Kyn-CKA reacts with wild-type, but not C151S mutant, Keap1-BTB, and at a much faster rate than with the small molecule thiol N-acetyl cysteine, demonstrating Kyn-CKA is targeted to react with C151 by the surrounding protein environment. In close agreement, Kyn-CKA increased the abundance of Nrf2 and expression of NQO1 in mouse embryonic fibroblast (MEF) cells expressing wild-type Keap1, but its inducer potency was greatly diminished in C151S-Keap1 mutant MEFs. Experiments in WT, AhR-knockout, and Nrf2- knockout primary murine bone marrow-derived macrophages showed that Nrf2 is required for the acute anti-inflammatory activity of Kyn-CKA, whereas AhR is dispensable. Together, these findings demonstrate that Kyn-CKA targets C151 in Keap1 to derepress Nrf2 and reveal that Nrf2, but not AhR, is a main contributor to the anti-inflammatory activity of Kyn-CKA in macrophages.

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