Phytomedicine : international journal of phytotherapy and phytopharmacology · 2025
Covalent targeting GAPDH by Pudilan formula and baicalein suppresses macrophage Warburg effect to alleviate acute lung injury.
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Jiang Mengyao, Yang Yang, Zhang Ying, Zhang Panpan, Zhang Qianyu, Yang Tong, Zhang Guoxin, Zhou Jie, Chu Zheng, Li Shuyu, Wang Yaxu, Zhang Junzhe, Shen Shengnan, Song Ping, Wang Jigang, Tang Huan
Аннотация
BACKGROUND: Acute lung injury (ALI) is a severe inflammatory syndrome characterized by dysregulated macrophage activation and cytokine release, leading to high mortality rates. The traditional Chinese medicine (TCM) formula Pudilan (PDL) has demonstrated clinical efficacy against inflammatory diseases including ALI. However, its multi-component complexity has obscured the precise molecular targets and mechanisms underlying its anti-inflammatory action.
PURPOSE: This study aimed to systematically identify the direct protein targets of PDL and elucidate the mechanistic basis for its anti-inflammatory effects in ALI.
METHODS: We employed a cysteine-reactive activity-based protein profiling (ABPP) chemoproteomic strategy to map covalent targets of PDL in macrophages. Target engagement and functional impact were validated through competitive ABPP, cellular thermal shift assay (CETSA), enzymatic activity assays, bio-layer interferometry (BLI), and molecular docking. Functional consequences were assessed using metabolic flux analysis (Seahorse), cytokine profiling, and models of LPS-induced inflammation both in vitro and in vivo.
RESULTS: ABPP identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the primary covalent target of PDL, specifically modified at its catalytic cysteine residue (Cys150). Baicalein (BC) was pinpointed as the principal bioactive component responsible for GAPDH engagement. Both PDL and BC covalently bound to GAPDH Cys150, inhibiting its enzymatic activity. Functionally, this inhibition suppressed glycolytic flux (the Warburg effect), promoted a metabolic shift toward oxidative phosphorylation (OXPHOS) in macrophages, and reversed pro-inflammatory M1 polarization. This metabolic reprogramming was functionally linked to the suppression of NF-κB p65 nuclear translocation and inflammatory mediator production. Critically, both PDL and BC alleviated LPS-induced lung injury in a murine ALI model, with direct evidence of GAPDH engagement and inhibition confirmed in lung tissues.
CONCLUSION: Our study establishes GAPDH as a direct covalent target of PDL and its active component BC, defining a novel "metabolism-immunity" axis through which PDL ameliorates ALI. The ABPP-driven target deconvolution strategy overcomes traditional limitations in TCM mechanism research. These findings provide a mechanistic foundation for the anti-inflammatory efficacy of PDL and offer a targeted therapeutic strategy for inflammatory lung injury.
PURPOSE: This study aimed to systematically identify the direct protein targets of PDL and elucidate the mechanistic basis for its anti-inflammatory effects in ALI.
METHODS: We employed a cysteine-reactive activity-based protein profiling (ABPP) chemoproteomic strategy to map covalent targets of PDL in macrophages. Target engagement and functional impact were validated through competitive ABPP, cellular thermal shift assay (CETSA), enzymatic activity assays, bio-layer interferometry (BLI), and molecular docking. Functional consequences were assessed using metabolic flux analysis (Seahorse), cytokine profiling, and models of LPS-induced inflammation both in vitro and in vivo.
RESULTS: ABPP identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the primary covalent target of PDL, specifically modified at its catalytic cysteine residue (Cys150). Baicalein (BC) was pinpointed as the principal bioactive component responsible for GAPDH engagement. Both PDL and BC covalently bound to GAPDH Cys150, inhibiting its enzymatic activity. Functionally, this inhibition suppressed glycolytic flux (the Warburg effect), promoted a metabolic shift toward oxidative phosphorylation (OXPHOS) in macrophages, and reversed pro-inflammatory M1 polarization. This metabolic reprogramming was functionally linked to the suppression of NF-κB p65 nuclear translocation and inflammatory mediator production. Critically, both PDL and BC alleviated LPS-induced lung injury in a murine ALI model, with direct evidence of GAPDH engagement and inhibition confirmed in lung tissues.
CONCLUSION: Our study establishes GAPDH as a direct covalent target of PDL and its active component BC, defining a novel "metabolism-immunity" axis through which PDL ameliorates ALI. The ABPP-driven target deconvolution strategy overcomes traditional limitations in TCM mechanism research. These findings provide a mechanistic foundation for the anti-inflammatory efficacy of PDL and offer a targeted therapeutic strategy for inflammatory lung injury.