Kinase inhibition is investigated using one of three assay formats: [³³P]phosphoryl transfer, luciferase-coupled chemiluminescence, or AlphaScreen tyrosine kinase technology. IC₅₀s are calculated by nonlinear regression analysis using XLFit.³³P -Phosphoryl Transfer Kinase Assay Reactions are performed in 384-well white, clear bottom, high-binding microtiter plates. Plates are coated with 2 μg/well of protein or peptide substrate in a 50 μL volume of coating buffer contained 40 μg/mL substrate (poly(Glu, Tyr) 4:1, 22.5 mM Na₂CO₃, 27.5 mM NaHCO₃, 50 mM NaCl and 3 mM NaN₃. Coated plates are washed once with 50 μL of assay buffer following overnight incubation at room temperature (RT). Test compounds and enzymes are combined with ³³P-γ-ATP (3.3 μCi/nmol) in a total volume of 20 μL. The reaction mixture is incubated at RT for 2 hours and terminated by aspiration. The microtiter plates are subsequently washed 6 times with 0.05% Tween-PBS buffer (PBST). Scintillation fluid (50 μL/well) is added and incorporated ³³P is measured by liquid scintillation spectrometry using a MicroBeta scintillation counter.Luciferase-Coupled Chemiluminescence Assay Reactions are conducted in 384-well white, medium binding microtiter plates. In a first step enzyme and compound are combined and incubated for 60 minutes; reactions are initiated by addition of ATP and peptide substrate (poly(Glu, Tyr) 4:1) in a final voume of 20 μL, and incubated at RT for 2-4 hours. Following the kinase reaction, a 20 μL aliquot of Kinase Glo is added and luminescence signal is measured using a Victor plate reader. Total ATP consumption is limited to 50%. AlphaScreenTM Tyrosine Kinase Assay Donor beads coated with streptavidin and acceptor beads coated with PY100 anti-phosphotyrosine antibody are used. Biotinylated poly(Glu,Tyr) 4:1 is used as the substrate. Substrate phosphorylation is measured by addition of donor/acceptor beads by luminescence following donor-acceptor bead complex formation. Kinase and test compounds are combined and preincubated for 60 minutes, followed by addition of ATP, and biotinylated poly(Glu, Tyr) in a total volume of 20 μL in 384-well white, medium binding microtiter plates. Reaction mixtures are incubated for 1 hour at room temperature. Reactions are quenched by addition of 10 μL of 15-30 μg/mL AlphaScreen bead suspension containing 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20. After 2-16 hours incubation at room temperature plates are read using an AlphaQuest reader.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

B16F10, A549, and HT29 cells (1.2×10³ per well) are mixed with soft agar and seeded in a 96-well plate containing 10% FBS and EXEL-2880 over a base agar layer. For normoxic conditions, the plates are incubated (37°C) for 12 to 14 days in 21% oxygen, 5% CO₂, and 74% nitrogen, whereas incubation (37°C) under hypoxic conditions is done in a hypoxia chamber in 1% oxygen, 5% CO₂, and 94% nitrogen. The number of colonies is evaluated under each condition following addition of 50% Alamar Blue and fluorescence detection.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

In vivo target modulation studies are done in naive mice or mice bearing B16F10 tumors. Foretinib or vehicle (0.9% normal saline) is administered at 10 mL/kg via oral gavage. For examination of Met phosphorylation in liver, HGF (10 μg/mouse) is administered i.v. 10 min before harvest. For examination of Flk-1/KDR phosphorylation in lung, VEGF (10 μg/mouse) is administered i.v. 30 min before harvest 0.5 h later. Receptor phosphorylation analysis is determined by immunoblot analysis.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Qian F, et al. Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res, 2009, 69(20), 8009-8016.  [Content Brief]

  [2]. Kataoka Y, et al. Foretinib (GSK1363089), a multi-kinase inhibitor of MET and VEGFRs, inhibits growth of gastric cancer cell lines by blocking inter-receptor tyrosine kinase networks. Invest New Drugs, 2011.  [Content Brief]

  [3]. Eder JP, et al. A phase I study of foretinib, a multi-targeted inhibitor of c-Met and vascular endothelial growth factor receptor 2. Clin Cancer Res, 2010, 16(13), 3507-3516.  [Content Brief]