DDR1-IN-2 (Synonyms: 7rh)

Kinase experiment:

The effects of compounds (including DDR1-IN-2) on the kinases DDR1 and DDR2 are assessed by using a LanthaScreen Eu kinase activity assay technology. Kinase reactions are performed in a 10 µL solution in low-volume 384-well plates. The kinase reaction buffer consists of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA; the concentration of Fluorescein-Poly GAT substrate in the assay is 100 nM. Kinase reactions are initiated by the addition of 100 nM ATP in the presence of serially diluted compounds (DDR1-IN-2). The reactions are allowed to proceed for 1 h at room temperature before a 10 µL preparation of EDTA (20 mM) and Eu-labeled antibody (4 nM) in TR-FRET dilution buffer are added. The final concentration of antibody in the assay well is 2 nM, and the final concentration of EDTA is 10 mM. The plate is allowed to incubate at room temperature for one more hour before the TR-FRET emission ratios of 665 nm/340 nm are acquired on a multilabel reader. Data analysis and curve fitting are performed using GraphPad Prism4 software[1].

References:

[1]. Wang Z, et al. Tetrahydroisoquinoline-7-carboxamide Derivatives as New Selective Discoidin Domain Receptor 1 (DDR1) Inhibitors. ACS Med Chem Lett. 2017 Feb 9;8(3):327-332.