Kinase Assay Panel

The KINOMEscan screening platform (Discoverx Corporation, San Diego, CA, DiscoverX scanMAX Kinase Assay Panel) employing an active site-directed competition binding assay was used to quantitatively measure interactions between the two Roche DDR1 inhibitors on and 468 kinases and disease relevant mutant variants. In brief, T7 kinase-tagged phage strains were grown in parallel in 24-well blocks or 96-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection = 0.4) and incubated with shaking at 32 °C until lysis (90–150 min). The lysates were centrifuged (6000g) and filtered (0.2 μm) to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 min at RT to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and the two test compounds in 1× binding buffer (20% SeaBlock, 0.17× PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 40× stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.02 mL. The assay plates were incubated at RT with shaking for 1 h and the affinity beads were washed with wash buffer (1× PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1× PBS, 0.05% Tween 20, 0.5 μM nonbiotinylated affinity ligand) and incubated at RT with shaking for 30 min. The kinase concentration in the eluates was measured by qPCR. The selectivity score (S-score) was calculated for both compounds. The compounds were screened at the concentrations requested, and results for primary screen binding interactions were reported as percent competition (% competition).