CHEMBL5734840

QSS Assist FP assay: Rsk1: Inhibitory activity of the compound according to the present invention on Rsk1 kinase activity in vitro was assayed using the QSS Assist FP assay kit (Carna Biosciences, Inc.).In the inhibition assay of inhibitory activity of the compound, the test compound was diluted by serial dilution with dimethyl sulfoxide (DMSO). Subsequently, an Rsk1 protein, a substrate peptide (final concentration: 100 nM), magnesium chloride (final concentration: 10 mM), ATP (final concentration: 30 μM), and the solution of the test compound in DMSO (DMSO final concentration: 5%) were added to the kinase reaction buffer (20 mM HEPES (pH 7.4), 2 mM dithiothreitol, 0.01% Tween-20), and the mixture was incubated at 25° C. for 40 minutes to perform the kinase reaction. The IMAP Progressive Binding Reagent diluted to 400-fold with the IMAP Progressive Binding Buffer A (Molecular Devices, LLC) was added to terminate the kinase reaction. The reaction product was allowed to stand at room temperature in the dark for 120 minutes and assayed with the use of PHERAstar (BMG LABTECH; excitation wavelength: 485 nm; detection wavelength: 520 nm). The phosphorylation reaction level was determined based on the degree of fluorescence polarization, and the concentration of the compound at which phosphorylation could be inhibited by 50% was defined as the IC50 level (nM)