Fgfr2

CH5451098 (N-cyclopropyl-2-[2-[8-[(2R)-2,3-dihydroxypropoxy]-6,6-dimethyl-11-oxo-naphtho[2,3-b]benzofuran-3-yl]ethynyl]pyridine-4-carboxamide), a selective inhibitor of AXL, was synthesized at Chugai Pharmaceutical Co. Ltd. (Fig 1). The following recombinant proteins were used for the kinase assay: AXL, Mer, Tyro3, Flt3, JAK2, EGFR, HER2, KIT, PDGFRβ, FGFR2, KDR, MET, ALK, EPHA2, EPHB2, FAK, SRC, ABL, ERK1, PKA, AKT1, PKACα, PKACβ1, AurA, CHK1, CHK2, MNK1, CDK1, and p38α from Carna Biosciences (Kobe, Japan), and Raf1 from Thermo Fisher Scientific (Waltham, MA). Recombinant mouse TGFβ1 (mTGFβ1) and recombinant mouse Gas6 (mGas6) were purchased from R&D systems. All other chemicals were of the highest purity available. The following primary antibodies were used for this study: anti-beta catenin (8480), anti-zinc finger E-box-binding homeobox 1 (Zeb1) (3396), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (8884) antibody from Cell Signaling Technology (Danvers, MA). As a secondary antibody, this study used Anti-rabbit IgG (7074) from Cell Signaling Technology.

The surface plasmon resonance experiments were performed using a Biacore S200 system (Cytiva, Marlborough, MA, USA) equipped with a CM5 sensor chip. The ligands FGFR1, FGFR2, and FGFR3 (Carna Biosciences) at a concentration of 20 μg/mL in 10 mM sodium acetate, pH 4.5, were immobilized using amine-coupling chemistry at densities of 17,050 RU (300 s), 17,150 RU (400 s), and 12,950 (550 s) on flow cells 2, 3, 4, respectively, with flow cell 1 left blank for reference. Kinetics analysis was performed in running buffer (10 mM HEPES, 150 mM NaCl, and 0.01% Tween20, pH 7.4), and data were collected at a rate of 1 Hz. Triplicate injections of CPL304110 at concentrations of 50 nM, 16.7 nM, 5.6 nM, 1.85 nM, 0.61 nM, and 0.2 nM and a triple buffer blank were injected in single-cycle mode over all four surfaces simultaneously at a flow rate of 30 μL/min and a temperature of 25°C. The complex was allowed to associate for 60 s and dissociate for 300 s, with no regeneration steps. The data were fit to a simple 1:1 interaction model using the global data analysis option available within Biacore S200 Evaluation Software 1.1.1 (Cytiva).

The inhibitory activity of CPL304110 against tested proteins was evaluated with ADP-Glo assay (Promega, Madison, WI, USA) following the manufacturer’s protocol. CPL304110 was dissolved in 100% DMSO and used as a stock solution, and serial dilutions were prepared using dilution buffer (20 mM Tris pH 7.5, 10 mM MgCl₂, 0.1 mM Na₃VO₄, 0.01% Triton X-100, and 2.5 mM DTT). Kinases FGFR1, FGFR2, and FGFR3 (Carna Biosciences, Kobe, Japan) were diluted in buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.05% Triton X-100, and 1 mM DTT) to final concentrations of 0.5 ng/μL, 0.4 ng/μL, and 1 ng/μL, respectively. Luminescence intensity was measured using a Victor Light luminometer (PerkinElmer, Inc.). IC₅₀ values were determined in GraphPad Prism 8 software by fitting to individual points of the curve by non-linear regression (log(inhibitor) vs. normalized response - Variable slope). Each compound was tested in at least six technical replicates in two separate experiments.