| Assay Method Information | |
| | Chk1 and Chk2 Inhibitory Activity Assay |
| Description: | The compounds of Examples 1 to 66 were diluted at various concentrations and transferred to a 384-well plate by 5 μl, and 10 μl of a mixed solution (2×) of Chk1 kinase (Thermo, P3040) or Chk2 kinase (Thermo, PV3367) and a substrate (Thermo, PV3508) was added to wells treated with the compounds. 5 μl of an ATP solution at a 4× concentration was added to the test well and reacted at room temperature for 1 hour. 20 μl of LanthaScreen Terbium-CREB p-Ser 133 antibody (Thermo, PV3542) was added to the reaction well and incubated at room temperature for 30 minutes. Conversion data was measured by an EnVision 2014 multi label reader (PerkinElmer). The activity for each concentration was determined by the ratio (FRET ratio=FR) of the value obtained by dividing the value at 520 nm by the value at 490 nm among the emission wavelength values for each wavelength (Em520/Em490). The results obtained for each concentration of the compounds were the average values obtained in two wells, and the IC50 values of the compounds were calculated using PRIZM software for analysis of the results |
| Affinity data for this assay | |
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