| Assay Method Information | |
| | HER2-Phosphorylating Activity |
| Description: | For setting the conditions for the method for measuring the in vitro inhibitory activity of a compound against HER2-phosphorylating activity, ProfilerPro Peptide 22 from PerkinElmer Inc. was used as a substrate on the basis of the report (PLoS One, 6 (7), e21487, 2011) on HER2 kinase reaction using, as a substrate, a peptide having the same sequence (5-FAM-EEPLYWSFPAKKK-CONH2) as that of ProfilerPro Peptide 22. The purified recombinant human HER2 protein used in the test was purchased from Carna Biosciences, Inc. Also, staurosporine (Eur. J. Biochem., 234, p. 317-322, 1995; and Nat. Biotechnol., 26 (1), p. 127-132, 2008), which is a multikinase inhibitor having Her2 inhibitory activity, was purchased from Enzo Life Sciences, Inc. (item No.: ALX-380-014) and used as a positive control in this test.For the inhibitory activity measurement of each compound, the compound of the present invention or staurosporine was first serially diluted with dimethyl sulfoxide (DMSO). Next, the HER2 protein, the substrate peptide (final concentration: 0.5 uM), manganese chloride (final concentration: 10 mM), ATP (final concentration: 6 uM), and the solution of the compound of the present invention in DMSO (final concentration of DMSO: 5%) were added into a buffer solution for kinase reaction (15 mM Tris (pH 7.5), 2 mM dithiothreitol, and 0.01% Tween 20), and the mixture was incubated at 25° C. for 40 minutes for kinase reaction. The reaction was terminated by adding EDTA (final concentration: 30 mM) thereto. Finally, the unphosphorylated substrate peptide (S) and the phosphorylated peptide (P) were separated and detected by microcapillary electrophoresis using LabChip EZ Reader II (PerkinElmer Inc.). The amount of phosphorylation reaction was determined from the respective peak heights of S and P. The compound concentration which can suppress the phosphorylation reaction by 50% was defined as an IC50 value (nM). |
| Affinity data for this assay | |
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