| Assay Method Information | |
| | Inhibitory Activity on EGFR Kinase |
| Description: | The compound of the present invention had a good inhibitory activity on an EGFR kinase, and the IC50 values were all less than 10 μM. The specific experiment was as follows:1. Preparation of 1× concentration kinase buffer1 volume of 5 times concentration of an enzyme buffer diluted with 4 volumes of distilled water; 5 mM magnesium chloride; 1 mM DTT; and 1 mM MnCl2.2. Test method of activity of compounda) a compound diluent was transferred to each well of an assay plate (784075, Greiner) using Echo 550;b) the assay plate was sealed and centrifuged at 1,000 g for 1 min;c) 2×EGFR (D770-N771 ins NPG T790M) was prepared in a 1× kinase buffer;d) 5 μl of the 2×EGFR (D770-N771 ins NPG T790M) was added to a 384-well assay plate (784075, Greiner);e) the assay plate was centrifuged at 1,000 g for 30 s and incubated at room temperature for 10 min;f) 2× TK-substrate-biotin (2 μM) and ATP were added to the 1× kinase buffer and mixed;g) the reaction was started after adding 5 μl of the TK-substrate-biotin (2 μM) and ATP mixture;h) the assay plate was centrifuged at 1,000 g for 30 s; and the assay plate was sealed and incubated at room temperature for 40 min;i) 4× Sa-XL 665 was prepared in an HTRF detection buffer;j) 5 μl of the Sa-XL 665 and 5 μl of a TK-antibody-cryptate were added to each well of the assay plate;k) the assay plate was centrifuged at 1,000 g for 30 s and incubated at room temperature for 1 h; andl) fluorescence signals at 615 nm (cryptate) and 665 nm (XL665) were read on an Envision 2104 reader.3. Data analysis3.1 Calculation of ratio (665/615 nm) of each well3.2 Calculation of inhibition rateInhibitionrate=100-(signalmpd-SignalAve_PC)/(SignalAve_VC-SignalAve_PC)×100.3.3 Calculation of IC50 value of compound and drawing dose-effect curve of compoundIC50 values were calculated using GraphPad 6.0 by applying a non-linear regression curve (dose-effect relationship-variable slope) of the logarithm of the compound concentration versus the inhibition rate. |
| Affinity data for this assay | |
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