| Assay Method Information | |
| | Inhibitory Activity Against KHK Kinase In Vitro |
| Description: | 1) The present compounds and the control drug were formulated in DMSO to 10 mM as stock solutions for test.2) The stock solutions of the compounds of the present invention and the control drug were diluted by 10-fold to 1 mM, and then further diluted in a 3-fold gradient to 11 concentrations, with the highest concentration being 1 mM.3) 0.1 μL of the compounds of the present invention and the control drug as diluted was transferred with Echo550 to a 384-well plate, in duplicate per concentration, and centrifuged at 1000 rpm for 1 min.2. Enzyme Reaction Test1) 5 μL of KHK-C kinase working solution was added to the 384-well plate, centrifuged at 1000 rpm for 1 min, and incubated at room temperature of 25° C. for 15 min.2) 5 μL of the substrate working solution was added to the 384-well plate to initiate the kinase reaction, centrifuged at 1000 rpm for 1 min, and incubated at room temperature of 25° C. for 60 min.3) The final concentrations of the KHK-C kinase reaction were 1 nM for KHK-C, 100 μM for ATP, 200 μM for D-Fructose, 50 mM for HEPES, 10 mM for MgCl2, and 0.01% for Brij35, and the final concentration of DMSO was 1%.4) The final concentrations of the test compounds and the control drug were 10000 nM, 3333.33 nM, 1111.11 nM, 370.37 nM, 123.457 nM, 41.15 nM, 13.71 nM, 4.572 nM, 1.524 nM, 0.508 nM, and 0.169 nM, respectively.3. Reaction Termination and Detection1) The 384-well plate was added with 10 μL of the ADP Glo reagent, centrifuged at 1000 rpm for 1 min, and then incubated at room temperature of 25° C. for 40 min.2) The 384-well plate was added with 20 μL of the kinase detection reagent, centrifuged at 1000 rpm for 1 min, and then incubated at room temperature of 25° C. for 40 min.3) After the reaction was completed, the fluorescence value LUM was read on Envision.4. Data AnalysisThe following equation was used to calculate the inhibition (% inh):inhibition(%)=100%×LumHC-LumCPDLumHC-LumLCwherein, |
| Affinity data for this assay | |
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