| Assay Method Information | |
| | Kinase Activity Test Assay |
| Description: | The effect of the disclosed compounds as protein kinase inhibitors can be evaluated by the following experiments. The AXL (h) kinase activity of the compounds of the invention is tested by the following methods:Reagents used in the experiment were AXL (Carna Bioscience, Cat No.: 08-107) and FLPeptide30 (PerkinElmer, Cat No.: 760430). The instruments involved were thermotank (Thermo Scientific), oscillator (QILINBEIER), EZ Reader (PerkinElmer, Cat No.: 122919), Non-contact stage pipetting system (Labcyte Inc., Cat No.: Echo 550), and non-contact nanoliter pipetting system (TECAN, Cat No.: EVO200).Experimental Method[0298]Compound dilution: 1) Dissolve the compound in DMSO to the appropriate concentration; 2) Using TECAN EVO200 to dilute 10 concentrations in 384 microplates by three times gradient, the highest concentration is 1 mM; Echo550 was used to transfer 20 nL solution from the dilution plate to the test plate.Experimental Method of Kinase 1) Prepare solution 1, as shown in Table C below:TABLE CReagent Solution 1MgCl2 10mMBrij-35 0.050%DTT 2mMBSA 0.05%EGTA 1mMHEPE (pH 7.5)50mM AXL 1.333nM 2) Add 15 μL solution 1 to each well of the test plate and incubate at room temperature for 30 minutes.[0301]3) Prepare solution 2, as shown in Table D below:TABLE DReagent Solution 2MgCl2 10mMBrij-35 0.050%DTT 2mMBSA 0.05%EGTA 1mMHEPE (pH 7.5) 50mMFLPeptide 6 μMATP 400μM[0302]4) Add 5μL solution 2 to start the reaction, and the final volume of each well is 20 μL. The composition of the system is shown in Table E below:TABLE E Reagent Final concentration MgCl2 10mMBrij-35 0.050%DTT 2mMBSA 0.05%EGTA 1mMHEPE (pH 7.5) 50mMFLPeptide 1.5μMATP 100 μMAXL 1nM[0303]5) Incubated at 25° C. for 90 min, and then added 75 μL stop solution (containing 0.5 M EDTA) to terminate the reaction.[0304]6) The sample of each well was analyzed using EZ reader. |
| Affinity data for this assay | |
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