Journal of Ginseng Research (May 2020)

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

  • Feiyan Chen,
  • Kexuan Zhu,
  • Lin Chen,
  • Liufeng Ouyang,
  • Cuihua Chen,
  • Ling Gu,
  • Yucui Jiang,
  • Zhongli Wang,
  • Zixuan Lin,
  • Qiang Zhang,
  • Xiao Shao,
  • Jianguo Dai,
  • Yunan Zhao

Journal volume & issue
Vol. 44, no. 3
pp. 461 – 474

Abstract

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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. Keywords: Affinity chromatography, Creatine kinase, Fatigue, Ginseng, 20(S)-protopanaxadiol