In vitro activity: UNC2250 efficiently inhibits Mer phosphorylation in 697 B-ALL cells and Colo699 NSCLC cells. Moreover, UNC2250 also displays functional antitumor activity by reducing colony-forming potential in both rhabdoid tumor cells and NSCLC cells.

Kinase Assay: Activity assays are performed in a 384-well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, pH 7.4, containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% bovine serum albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions are terminated by addition of 20 μL of 70 mM EDTA. After an 180 min incubation, phosphorylated and unphosphorylated substrate peptides are separated in buffer supplemented with 1× CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data are analyzed using EZ Reader software.

Cell Assay: BT-12 rhabdoid tumor cells (10 000 cells) are cultured in 2.0 mL of 0.35% soft agar containing 0.5× RPMI medium, 7.5% FBS, and the indicated concentrations of 10 or DMSO vehicle only and overlaid with 0.5 mL of 1× RPMI medium containing 15% FBS and 10 or DMSO vehicle only. Medium and 10 or vehicle are refreshed 2 times per week. Colonies were stained with thiazolyl blue tetrazolium bromide and counted after 3 weeks. Colo699 NSCLC cells (15 000 cells) are cultured in 1.5 mL of 0.35% soft agar containing 1× RPMI medium and 10% FBS and overlaid with 2.0 mL of 1× RPMI medium containing 10% FBS and the indicated concentrations of 10 or DMSO vehicle only. Medium and 10 or vehicle are refreshed 3 times per week. Colonies are stained with nitrotetrazolium blue chloride and counted after 2 weeks.

J Med Chem. 2013 Dec 12;56(23):9683-92.