CHEMBL5739385

Enzymologic Experiment of FGFR4: In this experiment, the inhibitory effect of the compounds on FGFR4 kinase activity was tested by a fluorescence resonance energy transfer (TR-FRET) method, and the half maximal inhibitory concentration (IC50) of the compounds on the FGFR4 kinase activity was determined. 1) 1˜5 μL of FGFR4 enzyme solution was added to a 384-well plate, and the final concentration of the enzyme was 0.1˜5 nM. 2) 1˜5 μL of diluted solution in gradient of the compound was added. 3) 1˜5 μL of a substrate mixture containing substrate polypeptide with a final concentration of 5˜50 nM and ATP with a final concentration of 10˜200 μM was added. 4) The mixture was incubated for 0.5˜3 hours at room temperature. 5) 10 μL of EDTA and a test solution comprising labeled antibody were added, and the plate was incubated for 1 hour at room temperature. 6) The fluorescence signal values of each plate were determined by a microplate reader at 665 nm. 7) The inhibition rates were calculated according to the fluorescence signal values.