| Assay Method Information | |
| | Test for Binding to ATP Binding Site of MYT1 |
| Description: | Table 3-1 to 3-5: The binding capacity of each compound to the ATP binding site of MYT1 Kinase protein was evaluated. 5× Kinase Buffer A (manufactured by Thermo Fisher Scientific Inc., PV3189) was diluted to 5 times with Milli-Q water, thereby preparing 1× Kinase Buffer. Using 1× Kinase Buffer, MYT1 protein (manufactured by Carna Biosciences, Inc.) was diluted to a concentration of 0.005 μM and Eu-Anti-GST Antibody was diluted to a concentration of 1 μM. In addition, Kinase Tracer 178 (manufactured by Thermo Fisher Scientific Inc., PV5593) was diluted to a concentration of 0.1 μM using 1× Kinase Buffer. To a 96 well-plate, 2.5 μL of a compound diluted with DMSO was added. Thereafter, 5 μL of a solution in which MYT1 protein and Eu-GST-Antibody diluted above were mixed in a ratio of 1:1 was added, and 2.5 μL of Kinase Tracer 178 diluted above was further added thereto. The mixture was well mixed and then allowed to stand at room temperature for 30 minutes. Thereafter, fluorescence wavelengths of 665 nm and 615 nm generated by irradiation with excitation light of 340 nm were detected using Envision (manufactured by PerkinElmer). The proportion of Tracer bound to MYT1 protein was calculated for every condition by dividing the intensity at the fluorescence wavelength of 665 nm by the intensity at the fluorescence wavelength of 615 nm. The inhibition rate of each compound added at each concentration was calculated by setting the signal when the compound was not added as 100% and the signal when MYT1 protein was not added as 0%, and the calculation results of IC50 were shown in Tables 3-1 to 3-5. When the compound exhibits IC50 of 10 μM or less, the compound can be determined to have binding activity to the ATP binding site of MYT1 protein. |
| Affinity data for this assay | |
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