Presentation on theme: "Supplementary Figure S1."— Presentation transcript:
1Supplementary Figure S1. Supplementary Figure S1. Determination of the binding mode of the FGFR inhibitor CH /Debio 1347.Lineweaver-Burk plot of FGFR1 with different concentrations of ATP in presence of various concentrations of CH /Debio Linear graphs intersected all with y-axis (i.e. Vmax unchanged) which does support an ATP-competitive mode-of-action of CH /Debio 1347 for FGFR1.
2Supplementary Figure S2. B.(a)(d)(b)(e)(c)Supplementary Figure S2. Effects of CH /Debio 1347 and cediranib on in vitro VEGF-induced tube formation(A) Inhibition of tube formation by CH /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 CH /Debio (c) 1 µM of CH /Debio (d) 0.01 µM of cediranib. (e) 0.1 µM of cediranib.
3Supplementary Figure S3. Relative kinase activity of FGFR2 to mockSupplementary 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)
4Supplementary Figure S4. CHAZD4547CHAZD45470.0010.0030.0010.003(µM)0.010.030.10.3100.010.030.010.030.010.03130.10.310.10.313100.10.31WTV564FV564IV564LpY-FGFRFGFR2Supplementary 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 CH /Debio 1347 or AZD4547 for 2 h. Cells were extracted and analyzed by Western blotting.
5Supplementary Figure S5. PD173074cediranib% inhibitionDrug conc. μM ( log10)Drug conc. μM ( log10)B.CHNVP-BGJ398CHNVP-BGJ398AZD4547AZD4547DMSODMSOpY-FGFRTEL-FGFR2WTV564FSupplementary Figure S5. Susceptibility of CH /Debio 1347 against gatekeeper mutants of FGFR2.(A) Anti-proliferative activity of PD 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 CH /Debio 1347, AZD4547, and NVP-BGJ398 for 2 hours. Cells were extracted and analyzed by Western blotting.
6Supplementary Figure S6. CHAZD4547Tumor volume (mm3)Days after the inoculationDays after the inoculationCH(mg/kg)AZD4547(mg/kg)B.Vehicle501002550pY-FGFRTEL-FGFR2 WTActinSupplementary Figure S6. Antitumor activity of CH /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 CH /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 CH /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)
7Supplementary Figure S7. CHdovitinibWT% inhibition% inhibitionN549HDrug conc. μM ( log10)Drug conc. μM ( log10)AZD4547NVP-BGJ398PD173074% inhibition% inhibition% inhibitionDrug conc. μM ( log10)Drug conc. μM ( log10)Drug conc. μM ( log10)Supplementary Figure S7. Kinase inhibition assay of FGFR2 WT or FGFR2 N549H mutantKinase inhibitory activity of FGFR inhibitors against FGFR2 WT or FGFR2 N549H were measured (n=3). CH , dovitinib, AZD4547, NVP-BGJ398, and PD showed 12, 5.2, 26, 19, 30 fold higher IC50 against FGFR2 N549H than FGFR2 WT, respectively.
8Supplementary Table S1. Supplementary Table S1 Crystallographic data collection and refinement statistics for FGFR1 in complex with CH /Debio 1347.Data collectionX-ray sourcePXII/X10SA (SLS)Wavelength [Å]1.000DetectorPILATUS 6MTemperature [K]100Resolution [Å]Observed reflections16371823073Unique reflections484447015Completeness [%]99.399.1Rsym [%]3.369.6Rmeas [%]4.493.2I/sigma(I)14.91.5Space groupC 1 2 1Unit cell (a, b, c) [Å]211.21, 56.75, 65.45, 90, , 90RefinementResolutionNumber of reflections(working/test)39318 / 1954Rcryst [%]22.3Rfree [%]25.7Number of atoms:Protein4460Ligand54Water75Phosphate ion5Deviation from ideal geometry:Bond lengths [Å]0.010Bond angles [ °]1.03Ramachandran plot:Most favoured regions92.2Additional allowed regions7.0Generously allowed regions0.8
9Supplementary Table S2. Supplementary Table S2 Kinase selectivity profile of CH /Debio The values of % competition at 0.1 or 1 μM CH /Debio 1347 in DiscoveRx’s KINOMEscan for 442 kinases including mutated kinases.Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanAAK1<65ABL1(E255K)-phosphorylatedABL1(F317I)-nonphosphorylatedABL1(F317I)-phosphorylatedABL1(F317L)-nonphosphorylatedABL1(F317L)-phosphorylatedABL1(H396P)-nonphosphorylated74ABL1(H396P)-phosphorylatedABL1(M351T)-phosphorylatedABL1(Q252H)-nonphosphorylated65ABL1(Q252H)-phosphorylatedABL1(T315I)-nonphosphorylatedABL1(T315I)-phosphorylatedABL1(Y253F)-phosphorylatedABL1-nonphosphorylatedABL1-phosphorylatedABL2ACVR1ACVR1BACVR2AACVR2B72ACVRL1ADCK3ADCK4AKT1AKT2AKT3ALKAMPK-alpha1AMPK-alpha2ANKK1ARK5ASK1ASK2AURKAAURKBAURKCAXLBIKEBLK85BMPR1ABMPR1BBMPR2BMXBRAFBRAF(V600E)BRKBRSK1BRSK2
10Supplementary Table S2 (cont). Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanBTK<65CAMK1CAMK1DCAMK1GCAMK2ACAMK2BCAMK2DCAMK2GCAMK4CAMKK1CAMKK2CASKCDC2L1CDC2L2CDC2L5CDK11CDK2CDK3CDK4-cyclinD1CDK4-cyclinD3CDK5CDK7CDK8CDK9CDKL1CDKL2CDKL3CDKL5CHEK1CHEK2CITCLK1CLK2CLK3CLK4CSF1R7699CSKCSNK1A1CSNK1A1LCSNK1DCSNK1ECSNK1G1CSNK1G2CSNK1G3CSNK2A1CSNK2A2CTKDAPK1DAPK2
11Supplementary Table S2 (cont). Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanDAPK3<65DCAMKL1DCAMKL2DCAMKL3DDR172DDR2DLKDMPKDMPK2DRAK1DRAK2DYRK1ADYRK1BDYRK2EGFREGFR(E746-A750del)EGFR(G719C)EGFR(G719S)EGFR(L747-E749del, A750P)EGFR(L747-S752del, P753S)EGFR(L747-T751del,Sins)EGFR(L858R)EGFR(L858R,T790M)EGFR(L861Q)EGFR(S752-I759del)EGFR(T790M)EIF2AK1EPHA1EPHA2EPHA3EPHA4EPHA5EPHA6EPHA7EPHA8EPHB1EPHB2EPHB3EPHB4EPHB692ERBB2ERBB3ERBB4ERK1ERK2ERK3ERK4ERK5ERK8
12Supplementary Table S2 (cont). Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanERN1<65FAKFERFESFGFR190100FGFR28797FGFR38099FGFR3(G697C)85FGFR4FGR82FLT1FLT3FLT3(D835H)FLT3(D835Y)FLT3(ITD)FLT3(K663Q)FLT3(N841I)FLT3(R834Q)FLT4FRKFYNGAKGCN2(Kin.Dom.2,S808G)GRK1GRK4GRK7GSK3AGSK3BHCKHIPK1HIPK2HIPK3HIPK4HPK1HUNKICKIGF1RIKK-alphaIKK-betaIKK-epsilonINSRINSRRIRAK1IRAK3IRAK4ITKJAK1(JH1domain-catalytic)JAK1(JH2domain-pseudokinase)JAK2(JH1domain-catalytic)
13Supplementary Table S2 (cont). Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanJAK3(JH1domain-catalytic)<65JNK1JNK2JNK3KIT92100KIT(A829P)KIT(D816H)KIT(D816V)KIT(L576P)8799KIT(V559D)95KIT(V559D,T670I)KIT(V559D,V654A)83LATS1LATS2LCKLIMK1LIMK2LKB1LOKLRRK2LRRK2(G2019S)LTKLYN65LZKMAKMAP3K1MAP3K15MAP3K2MAP3K3MAP3K4MAP4K2MAP4K3MAP4K4MAP4K5MAPKAPK2MAPKAPK5MARK1MARK2MARK3MARK4MAST1MEK1MEK2MEK3MEK4MEK570MEK6MELKMERTK
14Supplementary Table S2 (cont). Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanMET<65MET(M1250T)MET(Y1235D)MINKMKK7MKNK1MKNK2MLCKMLK1MLK2MLK3MRCKAMRCKBMST1MST1RMST2MST3MST4MTORMUSKMYLKMYLK2MYLK4MYO3AMYO3BNDR1NDR2NEK1NEK11NEK2NEK3NEK4NEK5NEK6NEK7NEK9NIM1NLKOSR1p38-alphap38-betap38-deltap38-gammaPAK1PAK2PAK3PAK4PAK6PAK7
15Supplementary Table S2 (cont). Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanPCTK1<65PCTK2PCTK3PDGFRA7699PDGFRB92100PDPK1PFCDPK1(P.falciparum)PFPK5(P.falciparum)PFTAIRE2PFTK1PHKG1PHKG2PIK3C2BPIK3C2GPIK3CAPIK3CA(C420R)PIK3CA(E542K)PIK3CA(E545A)PIK3CA(E545K)PIK3CA(H1047L)PIK3CA(H1047Y)PIK3CA(I800L)PIK3CA(M1043I)PIK3CA(Q546K)PIK3CBPIK3CDPIK3CGPIK4CBPIM1PIM2PIM3PIP5K1APIP5K1CPIP5K2BPIP5K2CPKAC-alphaPKAC-betaPKMYT1PKN1PKN2PKNB(M.tuberculosis)PLK1PLK2PLK3PLK4PRKCDPRKCEPRKCHPRKCI
16Supplementary Table S2 (cont). Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanPRKCQ<65PRKD1PRKD2PRKD3PRKG1PRKG2PRKRPRKXPRP4PYK2QSKRAF1RET91RET(M918T)94RET(V804L)RET(V804M)RIOK1RIOK2RIOK3RIPK1RIPK2RIPK4RIPK5ROCK1ROCK2ROS1RPS6KA4(Kin.Dom.1-N-terminal)RPS6KA4(Kin.Dom.2-C-terminal)RPS6KA5(Kin.Dom.1-N-terminal)RPS6KA5(Kin.Dom.2-C-terminal)RSK1(Kin.Dom.1-N-terminal)RSK1(Kin.Dom.2-C-terminal)RSK2(Kin.Dom.1-N-terminal)RSK3(Kin.Dom.1-N-terminal)RSK3(Kin.Dom.2-C-terminal)RSK4(Kin.Dom.1-N-terminal)RSK4(Kin.Dom.2-C-terminal)S6K1SBK1SgK110SGK3SIKSIK2SLKSNARKSNRKSRCSRMSSRPK1
17Supplementary Table S2 (cont). Kinase% competition at 100 nM in KINOMEscan% competition at 1000 nM in KINOMEscanSRPK2<65SRPK3STK16STK33STK35STK36STK39SYKTAK1TAOK1TAOK2TAOK3TBK1TECTESK1TGFBR1TGFBR2TIE1TIE2TLK1TLK2TNIKTNK1TNK2TNNI3KTRKATRKBTRKCTRPM6TSSK1BTTKTXKTYK2(JH1domain-catalytic)TYK2(JH2domain-pseudokinase)TYRO3ULK1ULK2ULK3VEGFR2VRK2WEE1WEE2YANK1YANK2YANK3YESYSK1YSK4ZAKZAP70