Sphaeropsidin A covalently binds to Cys 151 of Keap1 to attenuate LPS-induced acute pneumonia in mice

Redox Biol. 2025 May:82:103621. doi: 10.1016/j.redox.2025.103621. Epub 2025 Mar 27.

Abstract

Introduction: Kelch ECH-associating protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2) axis is crucial for regulating oxidative stress and inflammatory responses in acute pneumonia. Sphaeropsidin A (SA) is a antioxidant diterpenoid isolated from Sphaeropsis sapinea f. sp. cupressi, discovered as a novel Nrf2 agonist by our research group previously. However, the accurate function and mechanism of SA in treating acute pneumonia are still unknown.

Methods: The therapeutic effect of SA was evaluated in LPS-induced acute pneumonia in mice. The underlying mechanism of action was then analyzed by transcriptomics. The direct target of SA was identified through the synthesis of SA-biotin probe, and the binding amino acid residues were found and verified by LC-MS/MS analysis and site-specific mutation. Finally, knockout mice were employed to verify the mechanism of SA.

Results: Our data indicated that SA significantly inhibited LPS-induced acute pneumonia in mice via up-regulating Nrf2, inhibiting NLRP3 inflammasome and NF-κB activation, and identified Keap1 as the direct target of SA. Specifically, the effective dose of SA in mice was only 2 mg/kg. SA selectively covalent bound to Keap1 in cysteine 151 residue (Cys151). SA mediated the activation of Nrf2 and reduced the level of ROS, thereby inhibiting the NF-κB and NLRP3 inflammasome. Besides, SA formed hydrogen bond with ASP48 of ASC, blocking its oligomerization and inhibiting the activation of NLRP3 inflammasome.

Conclusion: This study indicates that SA might be a new covalent molecule of Keap1 to activate Nrf2, and is a promising drug candidate or lead molecule for the therapy of acute pneumonia through regulating Nrf2/NF-κB/NLRP3 inflammasome axis.

Keywords: Acute pneumonia; Keap1; NF-κB; NLRP3 inflammasome; Nrf2; Sphaeropsidin A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cysteine* / chemistry
  • Cysteine* / metabolism
  • Disease Models, Animal
  • Diterpenes* / chemistry
  • Diterpenes* / pharmacology
  • Inflammasomes / metabolism
  • Kelch-Like ECH-Associated Protein 1* / chemistry
  • Kelch-Like ECH-Associated Protein 1* / genetics
  • Kelch-Like ECH-Associated Protein 1* / metabolism
  • Lipopolysaccharides / adverse effects
  • Male
  • Mice
  • Mice, Knockout
  • NF-E2-Related Factor 2 / metabolism
  • NF-kappa B / metabolism
  • NLR Family, Pyrin Domain-Containing 3 Protein / metabolism
  • Pneumonia* / chemically induced
  • Pneumonia* / drug therapy
  • Pneumonia* / etiology
  • Pneumonia* / metabolism
  • Pneumonia* / pathology
  • Protein Binding

Substances

  • Kelch-Like ECH-Associated Protein 1
  • Lipopolysaccharides
  • NF-E2-Related Factor 2
  • Keap1 protein, mouse
  • Diterpenes
  • Cysteine
  • Inflammasomes
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • NF-kappa B
  • Nfe2l2 protein, mouse