Presentation on theme: "Supplementary Figure S1. Determination of the binding mode of the FGFR inhibitor CH5183284/Debio 1347. Lineweaver-Burk plot of FGFR1 with different concentrations."— Presentation transcript:
Supplementary Figure S1. Determination of the binding mode of the FGFR inhibitor CH5183284/Debio 1347. Lineweaver-Burk plot of FGFR1 with different concentrations of ATP in presence of various concentrations of CH5183284/Debio 1347. Linear graphs intersected all with y-axis (i.e. V max unchanged) which does support an ATP-competitive mode-of-action of CH5183284/Debio 1347 for FGFR1. Supplementary Figure S1.
Supplementary Figure S2. Effects of CH5183284/Debio 1347 and cediranib on in vitro VEGF-induced tube formation (A) Inhibition of tube formation by CH5183284/Debio 1347 and cediranib in a HUVEC and ﬁbroblast co-culture system. Total vessel area was measured quantitatively for the area of capillary-like tube formation with Kurabo angiogenesis image analysis software. N=3. (B) Representative mages of tube formation of HUVEC. x4 objective. (a) DMSO treatment. (b) 0.1 µM of CH5183284/Debio 1347. (c) 1 µM of CH5183284/Debio 1347. (d) 0.01 µM of cediranib. (e) 0.1 µM of cediranib. A. (a) (b) (c) (d) (e) B. Supplementary Figure S2.
Supplementary Figure S3. Kinase activity of FGFR2 WT, V564F, V564I, and V564L mutants. The 293 cells were transiently transfected with pCXND3 FGFR2 WT, FGFR2 V564F, FGFR2 V564I, and FGFR2 V564L. The kinases were immunoprecipitated with anti-FGFR2 antibody. Then, kinase activity was evaluated by examining their ability to phosphorylate substrate peptide using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. (N=2) Relative kinase activity of FGFR2 to mock Supplementary Figure S3.
Supplementary Figure S4. Inhibition of cellular pY-FGFR2 WT, pY-FGFR2 V564F, pY- FGFR2 V564I, and pY-FGFR2 V564L. The HCT-116 cells were transiently transfected with pCXND3 FGFR2 WT, FGFR2 V564F, FGFR2 V564I, and FGFR2 V564L. A day after the transfection, the cells were treated with the indicated concentration of CH5183284/Debio 1347 or AZD4547 for 2 h. Cells were extracted and analyzed by Western blotting. WT V564F 0 V564L 0.010.030.10.31310 AZD4547 V564I 00.0010.0030.010.030.10.3100.010.030.10.3131000.0010.0030.010.030.10.31 CH5183284 (µM) AZD4547CH5183284 pY-FGFRFGFR2 Supplementary Figure S4.
pY-FGFR WT CH5183284AZD4547 NVP-BGJ398 DMSO TEL-FGFR2 V564F CH5183284AZD4547 NVP-BGJ398 DMSO Supplementary Figure S5. Susceptibility of CH5183284/Debio 1347 against gatekeeper mutants of FGFR2. (A) Anti-proliferative activity of PD173074 and cediranib against TEL-FGFR2 WT driven Ba/F3 cells and TEL-FGFR2 V564F-driven Ba/F3 cells. Three clones were treated with various concentrations of the indicated inhibitors for 4 days. The viable cells were counted with WST-8. (B) TEL-FGFR2 WT driven Ba/F3 and TEL-FGFR2 V564-driven Ba/F3 were treated with the indicated 1 μM of CH5183284/Debio 1347, AZD4547, and NVP-BGJ398 for 2 hours. Cells were extracted and analyzed by Western blotting. A. % inhibition Drug conc. μM ( log 10 ) B. PD173074cediranib Supplementary Figure S5.
Actin TEL-FGFR2 WT pY-FGFR Vehicle501005025 CH5183284 (mg/kg) AZD4547 (mg/kg) Supplementary Figure S6. Antitumor activity of CH5183284/Debio 1347 and AZD4547 in Ba/F3 TEL-FGFR2 WT-driven mouse model. (A) Mice bearing Ba/F3 TEL-FGFR2 WT cells were treated with CH5183284/Debio 1347 or AZD4547 orally once daily for 11 days at the indicated doses. Tumor volume and body weight change for each dose group were measured. Data are shown as mean ± SD (n = 5). (B) Suppression of phospho-FGFR inhibition in xenograft tissue. Mice bearing Ba/F3 TEL- FGFR2 WT cells were treated for 11 days at 50 and 100 mg/kg of CH5183284/Debio 1347 or 25 and 50 mg/kg of AZD4547, and xenograft tumors were extracted at 4 hours post- dosing and analyzed by Western blotting. (n = 3) Tumor volume (mm3) Days after the inoculation A. CH5183284AZD4547 B. Supplementary Figure S6.
Supplementary Figure S7. Kinase inhibition assay of FGFR2 WT or FGFR2 N549H mutant Kinase inhibitory activity of FGFR inhibitors against FGFR2 WT or FGFR2 N549H were measured (n=3). CH5183284, dovitinib, AZD4547, NVP-BGJ398, and PD173074 showed 12, 5.2, 26, 19, 30 fold higher IC 50 against FGFR2 N549H than FGFR2 WT, respectively. CH5183284 AZD4547NVP-BGJ398PD173074 dovitinib % inhibition Drug conc. μM ( log 10 ) % inhibition Drug conc. μM ( log 10 ) % inhibition Drug conc. μM ( log 10 ) % inhibition Drug conc. μM ( log 10 ) WT N549H Supplementary Figure S7.
Supplementary Table S1 Crystallographic data collection and refinement statistics for FGFR1 in complex with CH5183284/Debio 1347. Data collection X-ray sourcePXII/X10SA (SLS) Wavelength [Å]1.000 DetectorPILATUS 6M Temperature [K]100 Resolution [Å]47.1-2.022.13-2.02 Observed reflections16371823073 Unique reflections484447015 Completeness [%]99.399.1 R sym [%]3.369.6 R meas [%]4.493.2 I/sigma(I)14.91.5 Space groupC 1 2 1 Unit cell (a, b, c) [Å]211.21, 56.75, 65.45, 90, 107.43, 90 Refinement Resolution43.35-2.15 Number of reflections (working/test) 39318 / 1954 R cryst [%]22.3 R free [%]25.7 Number of atoms: Protein4460 Ligand54 Water75 Phosphate ion5 Deviation from ideal geometry: Bond lengths [Å]0.010 Bond angles [ °]1.03 Ramachandran plot: Most favoured regions92.2 Additional allowed regions7.0 Generously allowed regions0.8 Supplementary Table S1.
Supplementary Table S2 Kinase selectivity profile of CH5183284/Debio 1347. The values of % competition at 0.1 or 1 μM CH5183284/Debio 1347 in DiscoveRx’s KINOMEscan for 442 kinases including mutated kinases. Kinase % competition at 100 nM in KINOMEscan % competition at 1000 nM in KINOMEscan AAK1<65 ABL1(E255K)-phosphorylated<65 ABL1(F317I)-nonphosphorylated<65 ABL1(F317I)-phosphorylated<65 ABL1(F317L)-nonphosphorylated<65 ABL1(F317L)-phosphorylated<65 ABL1(H396P)-nonphosphorylated<6574 ABL1(H396P)-phosphorylated<65 ABL1(M351T)-phosphorylated<65 ABL1(Q252H)-nonphosphorylated<6565 ABL1(Q252H)-phosphorylated<65 ABL1(T315I)-nonphosphorylated<65 ABL1(T315I)-phosphorylated<65 ABL1(Y253F)-phosphorylated<65 ABL1-nonphosphorylated<65 ABL1-phosphorylated<65 ABL2<65 ACVR1<65 ACVR1B<65 ACVR2A<65 ACVR2B<6572 ACVRL1<65 ADCK3<65 ADCK4<65 AKT1<65 AKT2<65 AKT3<65 ALK<65 AMPK-alpha1<65 AMPK-alpha2<65 ANKK1<65 ARK5<65 ASK1<65 ASK2<65 AURKA<65 AURKB<65 AURKC<65 AXL<65 BIKE<65 BLK<6585 BMPR1A<65 BMPR1B<65 BMPR2<65 BMX<65 BRAF<65 BRAF(V600E)<65 BRK<65 BRSK1<65 BRSK2<65 Supplementary Table S2.