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

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.