In vitro activity: Ceritinib (formerly known as LDK378; trade name: Zykadia) is novel, potent and selective inhibitor against ALK (anaplastic lymphoma kinase positive) with IC50 of 0.2 nM in cell-free assays, it shows 40- and 35-fold selectivity against IGF-1R and InsR, respectively. Ceritinib was approved by FDA in April 2014 for the treatment non-small cell lung cancer (NSCLC). In Phase I trials, Ceritinib showed a marked clinical response in 78 patients with anaplastic ALK+ metastatic non-small cell lung cancer who had progressed during or after crizotinib therapy or had not been previously treated with crizotinib. LDK378 shows great anti-proliferative activity in Ba/F3-NPM-ALK and Karpas290 cells with IC50 of 26.0 nM and 22.8 nM, compared with IC50 of 319.5 nM and 2477 nM in Ba/F3-Tel-InsR and Ba/F3-WT cells.

Kinase Assay: All kinases are expressed as either Histidine- or GST-tagged fusion proteins using the baculovirus expression technology except for the untagged ERK2 which is produced in E. coli. The kinase activity is measured in the LabChip mobility shift assay. The assay is performed at 30°C for 60 min. The effect of LDK378 on the enzymatic activity is obtained from the linear progress curves in the absence and presence of LDK378 and routinely determines from one reading (end point measurement).

Cell Assay: Luciferase-expressing cells are incubated with serial dilutions of LDK378 or DMSO for 2-3 days. Luciferase expression is used as a measure of cell proliferation/survival and is evaluated with the Bright-Glo Luciferase Assay System. IC50 values are generated by using XLFit software.