Identification of Adenylate Kinase 5 as a Protein Target of Ginsenosides in Brain Tissues Using Mass Spectrometry-Based Drug Affinity Responsive Target Stability (DARTS) and Cellular Thermal Shift Assay (CETSA) Techniques

Ginseng is a very famous Chinese
herbal medicine with various pharmacological
effects. Ginsenosides, the main effective compounds of ginseng, show
favorable biological activities in the central nervous system (CNS),
but the protein targets of ginsenosides in brain tissues have not
been clarified clearly. First, we screened proteins that interact
with ginsenosides by mass spectrometry-based drug affinity responsive
target stability (DARTS) and cellular thermal shift assay (CETSA).
Then, we identified and confirmed adenylate kinase 5 (AK5) as a target
protein of ginsenosides by biolayer interferometry (BLI), isothermal
titration calorimetry (ITC), and molecular docking. Finally, an enzyme
activity kit was used to determine the effect of 20­(S)-protopanaxadiol (PPD), a ginseng saponin metabolite, on AK5 activities in vivo and in vitro. We screened out seven
overlapping target proteins by proteomics of DARTS and CETSA. The
BLI direct action assays showed that the direct interaction of PPD
with AK5 was higher compared to the parental ginsenosides. Subsequently,
BLI kinetic analysis and ITC assay showed that PPD specifically bound
to AK5. Furthermore, key amino acid mutations predicted by molecular
docking decreased the affinity between PPD and AK5. Enzyme activity
assays showed that PPD increased AK5 activities in vivo and in vitro. The above-mentioned findings indicated
that AK5 is a protein target of ginsenoside in the brain and PPD is
considered to be a small-molecular activator of AK5, which can improve
comprehension of the molecular mechanisms of ginseng pharmacological
effects in the CNS and further develop AK5 activators based on the
dammarane-type triterpenoid structure.