2.11. Inhibition of GSK-3β

The inhibitory activity of farrerol on GSK-3β was evaluated using the Kinase-Glo assay which was performed in assay buffer (50 mmol/L HEPES pH 7.5, 1 mmol/L EDTA, 1 mmol/L EGTA, and 15 mmol/L magnesium acetate) using the black 96-well plate as previously reported [26]. In brief, the reaction system contained a series of different concentrations of the test compound, 20 ng of enzyme, 25 μmol/L substrate, and 1 μmol/L ATP in a total volume of 40 μL. Then, the reaction mixture was incubated at 30°C for 30 min and reaction was stopped by the addition of 40 μL Kinase-Glo reagent. Glow-type luminescence was recorded after 10 min using a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale, CA, USA). SB 415286, a GSK-3β inhibitor, was used as positive control to validate the method in our laboratory. The inhibitory activity was measured indirectly by quantifying the amount of ATP remaining in solution following a kinase reaction. In specific, the luminescent signal is proportional to the amount of ATP remaining in the reaction and the inhibitory activity of the test compound. Maximal kinase (average positive) and luciferase (average negative) activities were used to calculate the inhibitory activity. The former was measured in the absence of inhibitor and the latter in the presence of reference compound inhibitor SB415286 (IC₅₀ = 77.5 nmol/L) at total inhibition concentration (5 μmol/L) [27]. The percentage inhibition was calculated by the following equation: inhibition rate (%) = (luminescence of test inhibitor − luminescence of average positive)/(luminescence of average negative − luminescence of average positive) × 100. IC₅₀ values were determined by nonlinear regression analysis of GSK-3β inhibition versus different concentrations of the test compound using GraphPad Prism 5.0 software (San Diego, CA, USA).