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. upplementary Figure S1.
Supplementary Figure S1. Supplementary Figure S1. Determination of the binding mode of the FGFR inhibitor CH5183284/Debio 1347. - PowerPoint PPT Presentation
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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. Vmax 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.
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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)
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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.
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Supplementary Figure S4.
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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.
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-8 -6 -4 -2 0 2-20
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WT clone3 V564F clone1
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WT clone1 WT clone2
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Supplementary Figure S5.
Actin
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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)
Tum
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Days after the inoculation Days after the inoculation
Unit cell (a, b, c) [Å] 211.21, 56.75, 65.45, 90, 107.43, 90
Refinement
Resolution 43.35-2.15 Number of reflections (working/test)
39318 / 1954
Rcryst [%] 22.3 Rfree [%] 25.7 Number of atoms:
Protein 4460Ligand 54Water 75Phosphate ion 5
Deviation from ideal geometry: Bond lengths [Å] 0.010Bond angles [ °] 1.03
Ramachandran plot: Most favoured regions 92.2 Additional allowed regions 7.0 Generously allowed regions 0.8
Supplementary Table S1.
Supplementary Table S2Kinase 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.
Supplementary Table S3Selective antiproliferative activity of CH5183284/Debio 1347 against cancer cell lines harboring genetic alterations in FGFR
Cell line Tumor typeCH5183284 IC50 (µM)
FGFR genetic alterations
SNU-16 Gastric cancer 0.017 FGFR2 gene amplificationKato-III Gastric cancer 0.018 FGFR2 gene amplificationHSC-39 Gastric cancer 0.050 FGFR2 gene amplification
AGS Gastric cancer 2.9HGC-27 Gastric cancer 3.0 MKN-1 Gastric cancer 3.0 MKN-45 Gastric cancer 5.0 SNU-1 Gastric cancer 5.0 JR-St Gastric cancer 7.1
NCI-N87 Gastric cancer 10NUGC-4 Gastric cancer >10MKN-74 Gastric cancer >10NUGC-3 Gastric cancer >10
SCH Gastric cancer >10MKN-28 Gastric cancer >10SNU-5 Gastric cancer >10
MFE-296 Endometrial cancer 0.042 FGFR2 N549K mutationAN3 CA Endometrial cancer 0.054 FGFR2 K310R/N549K mutationMFE-280 Endometrial cancer 0.41 FGFR2 S252W mutationHEC-59 Endometrial cancer 2.8HEC-1-B Endometrial cancer 5.7HEC-1-A Endometrial cancer 9.2
KLE Endometrial cancer 10HEC-151 Endometrial cancer 10RL95-2 Endometrial cancer >10
HEC-50B Endometrial cancer >10HEC-108 Endometrial cancer >10RT112/84 Bladder cancer 0.018 FGFR3-TACC3 fusionUM-UC-14 Bladder cancer 0.11 FGFR3 S249C mutation
SW780 Bladder cancer 0.12 FGFR3-BAIAP2L1 fusionRT4 Bladder cancer 0.35 FGFR3-TACC3 fusionT24 Bladder cancer 2.65637 Bladder cancer 3.5J82 Bladder cancer 4.1
SCaBER Bladder cancer 5.8 JMSU1 Bladder cancer 7.1
UM-UC-3 Bladder cancer 7.2HT-1376 Bladder cancer 10HT-1197 Bladder cancer >10TCCSUP Bladder cancer >10BFTC-905 Bladder cancer >10
DMS 114 Small cell lung cancer 0.18 FGFR1 gene amplificationNCI-H2227 Small cell lung cancer 1.4 NCI-H82 Small cell lung cancer 2.6SHP-77 Small cell lung cancer 5.6
NCI-H526 Small cell lung cancer 8.7NCI-H1930 Small cell lung cancer >10
DMS 53 Small cell lung cancer >10SUM-52PE Breast cancer 0.018 FGFR2 gene amplificationMFM-223 Breast cancer 0.058 FGFR2 gene amplificationHCC2218 Breast cancer 1.2
MCF10DCIS.com Breast cancer 1.6MDA-MB-157 Breast cancer 2.3
DU-4475 Breast cancer 3.1Hs 578.T Breast cancer 3.7MCF10A Breast normal 6.2HCC38 Breast cancer 6.4
SUM-44PE Breast cancer 6.9 FGFR1 gene amplificationSUM-229PE Breast cancer 7.4MDA-MB-453 Breast cancer 7.7MDA-MB-468 Breast cancer 8.0
MCF7 Breast cancer 8.4HCC1500 Breast cancer 8.6HCC1187 Breast cancer 9.2
MDA-MB-175-VII Breast cancer >10CAL-120 Breast cancer >10 FGFR1 gene amplificationHCC1569 Breast cancer >10HCC1599 Breast cancer >10ZR-75-1 Breast cancer >10 FGFR1 gene amplification
MDA-MB-231 Breast cancer >10JIMT-1 Breast cancer >10 FGFR1 gene amplification
CAMA-1 Breast cancer >10 FGFR1 gene amplificationHCC1395 Breast cancer >10 FGFR1 S125L mutation
BT-474 Breast cancer >10HCC1806 Breast cancer >10HCC70 Breast cancer >10
HCC1954 Breast cancer >10T47D Breast cancer >10
HCC1419 Breast cancer >10
Supplementary Table S3 (cont).
Cell line Tumor typeCH5183284 IC50 (µM)
FGFR genetic alterations
ZR-75-30 Breast cancer >10HCC1937 Breast cancer >10SK-BR-3 Breast cancer >10HCC1428 Breast cancer >10COLO-824 Breast cancer >10UACC-812 Breast cancer >10HCC1143 Breast cancer >10
MDA-MB-361 Breast cancer >10BT-20 Breast cancer >10
MDA-MB-134-VI Breast cancer >10 FGFR1 gene amplificationBT-483 Breast cancer >10
NCI-H716 Colorectal cancer 0.013 FGFR2 gene amplificationCOLO-205 Colorectal cancer 2.9
HCT-8 Colorectal cancer 3.2CaR-1 Colorectal cancer 5.8SW48 Colorectal cancer 5.9
LS174T Colorectal cancer 7.5LS513 Colorectal cancer 7.9HT-29 Colorectal cancer 7.9
COLO-201 Colorectal cancer 7.9NCI-H508 Colorectal cancer 8.3
SW620 Colorectal cancer 8.3RKO Colorectal cancer 8.7
SW480 Colorectal cancer 8.8LS411N Colorectal cancer 9.0 HCT-116 Colorectal cancer 10LS1034 Colorectal cancer >10HCT-15 Colorectal cancer >10
WiDr Colorectal cancer >10CW-2 Colorectal cancer >10T84 Colorectal cancer >10
SW948 Colorectal cancer >10SW1417 Colorectal cancer >10DLD-1 Colorectal cancer >10
SW1463 Colorectal cancer >10LoVo Colorectal cancer >10
SW403 Colorectal cancer >10Caco-2 Colorectal cancer >10
COLO-320DM Colorectal cancer >10SW1116 Colorectal cancer >10