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Protein target identification of ginsenosides in skeletal muscle tissues: discovery of natural small-molecule activators of muscle-type creatine kinase

Journal of Ginseng Research 2020년 44권 3호 p.461 ~ 474
 ( Chen Feiyan ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Pathology and Pathophysiology

 ( Zhu Kexuan ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Pathology and Pathophysiology
 ( Chen Lin ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Physiology
 ( Ouyang Liufeng ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Pathology and Pathophysiology
 ( Chen Cuihua ) - Nanjing University of Chinese Medicine Basic Medical College Research Center
 ( Gu Ling ) - Nanjing University of Chinese Medicine Basic Medical College Research Center
 ( Jiang Yucui ) - Nanjing University of Chinese Medicine Basic Medical College Research Center
 ( Wang Zhongli ) - Jiujiang University School of Nursing
 ( Lin Zixuan ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Pathology and Pathophysiology
 ( Zhang Qiang ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Pathology and Pathophysiology
 ( Shao Xiao ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Pathology and Pathophysiology
 ( Dai Jianguo ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Pathology and Pathophysiology
 ( Zhao Yunan ) - Nanjing University of Chinese Medicine School of Medicine and Life Science Department of Pathology and Pathophysiology

Abstract


Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, the mechanism of action is not completely understood, and its molecular targets remain largely unknown.

Methods: By screening for proteins that interact with the primary components of ginseng (ginsenosides) in an affinity chromatography assay, we have identified muscle-type creatine kinase (CK-MM) as a potential target in skeletal muscle tissues.

Results: Biolayer interferometry analysis showed that ginsenoside metabolites, instead of parent ginsenosides, had direct interaction with recombinant human CK-MM. Subsequently, 20(S)-protopanaxadiol (PPD), which is a ginsenoside metabolite and displayed the strongest interaction with CK-MM in the study, was selected as a representative to confirm direct binding and its biological importance. Biolayer interferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPD specifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by molecular docking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activity in vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the function of the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delaying exercise-induced lactate accumulation, and improving exercise performance in mice.

Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginseng reduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can help in further developing better CK-MM activators based on the dammarane-type triterpenoid structure.

키워드

Affinity chromatography; Creatine kinase; Fatigue; Ginseng; 20(S)-protopanaxadiol
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