In vitro activity: ZM 306416 (also known as CB 676475) is a potent inhibitor or antagonist for VEGFR1 (vascular endothelial growth factor receptor 1) tyrosine kinase with IC50 of 0.33 μM, but it was also found to be an inhibitor of EGFR with IC50 of<10 nM. Using A549-EGFRB cells for EGFRB assay, it was revealed that ZM 306416 exhibited inhibitory activity on VEGFR with IC50 value of 670 nM. When tested with human thyroid follicular cells. ZM 306416 treatment reducing VEGFR2 phosphorylation and inhibited endogenous, steady-state levels of p42/44 MAPK phosphorylation. Taken together, these results demonstrate ZM 306416's potential use in the clinic.

Kinase Assay: The activity of confirmed positives was assessed in a panel of kinases consisting of EGFR, VEGFR1, SRC and ABL kinase using a luminescence ADP production kinase assay as previously described. The potency of each compound was measured in dose response studies in 384-well format using 12 doubling dilutions in duplicate with 10 μM and 1 μM compound concentration as the upper limit. All reagents transfers were performed using the PP-384-M Personal Pipettor. Tested compounds or controls were added to the wells at a volume of 1 μL to white 384 well microtiter plates (Corning #3570, Corning, NY). Controls consisted of 1% DMSO (v/v) (high control) and 30 μM staurosporine in 1% DMSO (v/v) (low controls). The assay buffer was 25 mM Hepes/NaOH, pH 7.5 and contained 10 mM MgCl2, 2 mM TCEP, 20 mM β-Glycerol Phosphate, and 100 μM Na3VO4; for each kinase on the panel 4 μL of kinase dilution in assay buffer were added to the wells to reach a final concentration of 50 nM enzyme. Of note, the specific activity of the kinases of the panel is unknown and therefore the concentration of active enzyme in the preparation is unknown. After enzyme addition, kinase and compound were pre-incubated for 10 minutes at room temperature. Then 5 μL of a mix containing ATP and Poly(Glu, Tyr) substrates in solution in assay buffer were added to the wells both to reach a final concentration of 200 μM. After 45 minutes reaction at room temperature, 10 μL of ADP-Glo Reagent were added to each well. After 40 minutes incubation, 20 μL Kinase Detection Reagent were added followed by 60 minute incubation. The luminescence signal output was measured on the LEADseeker(TM). Dose response curves were plotted using SigmaPlot and represent the mean data from duplicates, the error bars correspond to the standard error of the regression. The low limit for calculating compound IC50 in the assay conditions was 10 nM.

Cell Assay: The anti-proliferative effect of confirmed hits was assessed against a panel of established cell lines that includes those harboring wild type EGFR (A549-EGFRB, A549 and H2030 cell lines) and those harboring the activating L858R EGFR mutation (H3255 and HCC4011 cell lines). A549-EGFRB cells were cultured as previously described. H2030, H3255 and HCC4011 cells were cultured as previously described. A549 cells were cultured in F12K media (Invitrogen Cat.# 21127-022) supplemented with 10% FBS (PAA Cat.# A15-201). The anti-proliferative effect of each compound was assessed in dose response studies in 384-well format using 12 doubling dilutions in duplicate with 10 μM and 1 μM compound concentration as the upper limit and using the Alamar Blue viability assay as previously described. Control wells consisted of 1% DMSO (v/v) (high control) and 1 μM “killer mix” in 1% DMSO (v/v) (low control). Final compound incubation time with cells was 120 hours. In dose response curves plotted using SigmaPlot 9.0 (Systat Software, San Jose, CA), the mean data from duplicates is presented and the error bars correspond to the standard error of the regression.