LANCE Ultra TR-FRET assays

The ability of all target compounds to inhibit the activation of FGFR1 kinase domain was assessed using LANCE Ultra TR-FRET assays, and the inhibitor TKI258 was used as the control. The assays were carried out in a final volume of 50 µL per well in a white Packard OptiPlate-384 (PerkinElmer, Waltham, MA, USA). Each tested compound was diluted as 40, 4, and 0.4 µM in 1× kinase base buffer containing 50 mM (4-(2-hydroxyethyl)-1-piperazinethaneesulfonic acid) (pH 7.5), 10 mM MgCl₂, 1 mM ethylenebis(oxyethylenenitrilo) tetraacetic acid, 2 mM dl-dithiothreitol, and 0.01% Tween-20. Then, 2.5 µL of the compounds was transferred to an assay plate. A standard enzymatic reaction, initiated by the addition of 5 µL of 2.5× peptide solution to 2.5 µL of 4× enzyme, contained 4 nM FGFR1 kinase, 11.9 µM ULight-JAK-1 (Tyr1023) peptide (PerkinElmer), 47 µM ATP, and 1× kinase base buffer. After 90-minute incubation at room temperature, the reaction was stopped by the addition of 5 µL termination buffer (40 mM ethylenediaminetetraacetic acid). Afterwards, 5 µL of 4× antibody (Eu-anti-phospho-tyrosine antibody [PT66] at a final concentration of 2 nM) was added to each well of the assay plate for 1 hour. The product and substrate in each independent reaction were separated using a 12-sipper microfluidic chip (Caliper Life Sciences) run on a Caliper LC3000 (Caliper Life Sciences). The resulting data were collected from the EnVision^® Multilabel Reader (PerkinElmer). Then, the original values were converted into the inhibition ratio. All experiments were repeated independently three times.

In addition, some compounds were screened for their selective kinase inhibition. The inhibitory concentration or IC₅₀ values on RTKs were determined by the same method.