Protein target identifi cation of ginsenosides in skeletal muscle tissues: discovery of natural smallmolecule activators of muscle-type creatine kinase

Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, themechanism 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 potentialtarget 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 thestudy, was selected as a representative to confirm direct binding and its biological importance. Biolayerinterferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPDspecifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by moleculardocking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activityin vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the functionof the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delayingexercise-induced lactate accumulation, and improving exercise performance in mice. Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginsengreduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can helpin further developing better CK-MM activators based on the dammarane-type triterpenoid structure. KCI Citation Count: 14