| Assay Method Information | |
| | Inhibitory Effects of Compounds on Kinase Activity Assay |
| Description: | 1) IRAK4 kinase was dissolved in kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT and 0.01% Brij-35) to a final concentration of 6 nM.[0542]2) The substrate peptide FAM-P8 and ATP were dissolved in the above kinase buffer. The final concentrations of the IRAK4 substrate peptide FAM-P8 and ATP were 3 μM and 10 μM respectively.[0543]3) Dilution of the compounds: the compound was first diluted to 50 μM, and then diluted down with a 4-fold gradient of DMSO. The solution without the compound and kinase is used as a blank control, corresponding to the “minimum value” shown below; and the solution without the compound but with kinase, adenosine 5′-triphosphate disodium salt hydrate, DMSO and buffer is used as a positive control, corresponding to the “maximum value” shown below. 4) Kinase reaction and termination: 10 μL of kinase buffer was added to a 384-well plate containing 5 μL of the compound to be tested, and incubated at room temperature for 10 minutes. Another 10 μL of buffer containing the substrate peptide and adenosine 5′-triphosphate disodium salt hydrate was added to the 384-well plate. After incubation at 28° C. for one hour, 25 μL stop solution (100 mM HEPES pH 7.5, 50 mM EDTA, 0.2% Coating Reagent #3 and 0.015% Brij-35) was added to each well to terminate the reaction.[0544]5) Data reading: the CaliperEZ Reader II instrument was used to read the conversion rate data. Conditions: downstream voltage −500V, upstream voltage −2250V, base pressure −0.5 PSI, and screening pressure −1.2 PSI.[0545]6) Data calculation: the conversion rate data was copied from CaliperEZ Reader II and converted into inhibition rate data. The calculation formula is as follows:Inhibitionpercentage(%)=(maximumvalue-conversionrate)/(maximumvalue-minimumvalue)*100%IC50 values were fitted using XLFit excel add-in version 5.4.0.8,Y=Bottom+(Top−Bottom)/(1+(IC50 /X){circumflex over ( )}HillSlope) Fitting formula |
| Affinity data for this assay | |
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