DDR1-IN-1 is an effective and specific DDR1 receptor tyrosine kinase inhibitor (IC50: 105 nM), about 3-fold selectivity over DDR2.

DDR1:105 nM

在U2OS细胞中，DDR1-IN-1通过EC50为86 nM抑制DDR1的基础自磷酸化，并且在没有胶原蛋白刺激的情况下显示出更强的DDR1自磷酸化抑制作用。在一系列具有DDR1功能增益突变和/或过表达的不同癌症细胞株中，DDR1-IN-1在低于10 μM的浓度下不抑制增殖，而GSK2126458增强了DDR1-IN-1的抗增殖活性。[1]

General procedure for the EC50 test: DDR1 is induced by 2 Gg/ml doxycycline for 48 hrs prior to DDR1 activation by rat tail collagen I. The DDR1 over-expressed U2OS is pre-treated by media containing each concentration of the compound for 1 hr and treated by changing the media to the EC50 test media containing 10 Gg/ml collagen and each concentration of the compound for 2 hrs. Each cells is washed with cold PBS three times and lysed with the lysis buffer (50 mMTris, pH 7.5, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 100 mMNaF, 2 mM Na3VO4, 1 mM PMSF, 10 Gg/ml aprotinin, and 10 Gg/ml leupeptin). The activation of DDR1 is quantified by density using program ImageJ to determine EC50 following Western blot using anti-activated human DDR1b (Y513).

Cells are plated in triplicate at a density of 3000 cells per well in 96-well plates and 1500 cells per well in 384-well plates. Compounds of various concentrations are added into plates for 48 hours. Cell viability is determined using the CellTiter-Glo and CCK-8. Both assays are performed according to the manufacturer's instructions. For CellTiter-Glo assay, luminescence is determined in a multi-label reader. For CCK-8 assay, absorbance is measured in a microplate reader at 450 nM. Data are normalized to control group (DMSO) and represented by the mean of at least two independent measurement with standard error <20%. GI50 were calculated using Prism 5.0.(Only for Reference)