Supplemental Materials & Methods
Antibodies and kinases
Following primary antibodies were used for this study: Phospho-FGF receptor (Tyr653/654) antibody (#3471) for pY-FGFR, phospho-PDGF receptor α (Tyr849)/PDGF receptor β (Tyr857) antibody (#3170) for pY-PDGFR, PDGF receptor α antibody (#3164) for PDGFRα, phospho-c-Kit (Tyr719) antibody (#3391) for pY-c-KIT, c-Kit antibody (#3392) for c-KIT, phospho-VEGF receptor 2 (Tyr1059) (D5A6) antibody (#2474) for pY-KDR, KDR antibody (#2479) for KDR from Cell Signaling Technology. FGFR2 antibody (F0300) for FGFR2 from SIGMA-ALDRICH. FGFR1 antibody (sc-7945) for FGFR1, FGFR4 antibody (sc-124) for FGFR4, and FGFR3 antibody (sc-13121) for FGFR3 from Santa Cruz Biotechnology.
Following secondary antibodies were used for this study: Anti-rabbit IgG, HRP-linked Antibody (NA934V) and Anti-mouse IgG, HRP-linked Antibody (NA931V) from GE Healthcare Life Sciences.
Following primary recombinant proteins were used for kinase assay: FGFR1, FGFR2, FGFR3, FGFR4, FLT1, KDR, ABL, EPHA2, KIT, SRC, FLT3, TIE2, EGFR, MET, INSR, ALK, JAK2, HER2, EPHB2, RON, AXL, LTK, FAK, PKA, AKT1, PKACα, PKACβ1, PKACβ2, p38α, AurA, CDC2/CycB1, CHK1, CHK2, TBK1, and FGFR2 N549H from Carna Biosciences. PDGFR beta, MEK1, Raf-1 from Millipore Corporation.
Determination of the binding mode
The inhibitory activity of CH5183284/Debio 1347 against FGFR1 was evaluated using a radiometric filter assay (Millipore) by measuring the incorporation of 33Pi with a microplate scintillation counter (Microbeta, Perkin Elmer) (Proqinase, Germany). A dose response assay with increasing amounts of ATP (1-200 μM) was performed in the absence or presence of the inhibitor CH5183284/Debio 1347 at seven concentrations corresponding to its IC10 to IC75 for FGFR1, with each ATP-concentration measured in duplicates.
Kinase autophosphorylation, substrate and ATP background were determined as controls in absence of compound. Enzymatic parameters Km[ATP] and Vmax values of FGFR1 under the influence of the different concentrations of CH5183284/Debio 1347 were calculated based on non-linear regression analysis and used to plot the results by the method described by Lineweaver and Burk. The linear graphs intersect with the x-axis at -1/Km and with the y-axis at 1/Vmax.
Tube formation assay
The Angiogenesis Kit (Kurabo Industries Ltd.) was charged with the test compound at final concentration of 0.1 or 1 µM of CH5183284/Debio 1347 or 0.01 or 0.1 µM of cediranib and incubated in a CO2 incubator (37 C, 5%) in the 10 ng/ml VEGF containing medium. After 11 days of incubation, capillary-like tubes formed were fixed with 70% ethanol and visualized with a CD31 Staining Kit (also produced by Kurabo Industries Ltd.). Under a microscope, stain images of the wells were photographed (x4 objective) stored as an image file, and measured quantitatively for the area of capillary-like tube formation with Kurabo angiogenesis image analysis software (1).
Crystallization and structural determination of FGFR1-CH5183284/Debio 1347 complex
Protein crystallography was performed by Roche pharmaceutical (Nutley, US). The kinase domain of human FGFR1 (residues 462–763) was expressed in E.coli with a GST-fusion tag, which was removed by protease cleavage during purification; the kinase domain was then purified using affinity, size exclusion, and ion exchange chromatography.
Crystals were obtained at 4°C from sitting drops using a reservoir solution (5–20% PEG 550 MME and, 0.1 M maleic acid [pH 5.5]) by vapor diffusion. The crystals were shock-frozen in liquid nitrogen after the addition of 22% ethyleneglycol. Diffraction data were collected at 90 K at the PXII beamline in SLS using a PILATUS 6M detector (DECTRIS Ltd.). The data set was processed with XDS (2) and scaled with SCALA (3) in the space group C121. The structure of the FGFR1 and CH5183284/Debio 1347 complex was determined by molecular replacement by Phaser (4) with an insulin receptor kinase (PDB ID 1GAG). The crystals contain two monomers of the protein in the asymmetric unit. The model was rebuilt manually in COOT (5), and refined with REFMAC5 (6) to a final resolution of 2.20 Å. B-factors were refined isotropically. The final model consisted of residues 464-763 and 462-762 with some breaks in disordered regions. The resulting electron density revealed an unambiguous binding mode of CH5183284/Debio 1347.
Immunoprecipitation of FGFR2
The 293 cells were transiently transfected with pCXND3 FGFR2 WT, FGFR2 V564F, FGFR2 V564I, and FGFR2 V564L. Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology) supplemented with cOomplete, PhosSTOP, cOmplete ULTRA (Roche). For immunoprecipitation of FGFR2 proteins, anti-FGFR2 antibody (Sigma F0300)-conjugated Dynabeads Protein G (Invitrogen) was added to cell lysate and incubated for 1 hour. Immunoprecipitations were washed 2 times in the wash buffer and 2 times in the enzyme dilution buffer subjected to kinase assay.
Transient expression and compound treatment
The V564F, V564I, and V564L were generated from the FGFR2 wild type (WT) by PCR-based site-directed mutagenesis. Then, the FGFR2 WT, V564F, V564I, and V564L were inserted into pCXND3 vector (Kaketsuken). The HCT116 cells transfected with 1 µg FGFR2 WT, V564F, V564I, and V564L plasmid DNA were treated with 0.1% DMSO or CH5183284/Debio 1347 for 2 hours.
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