Fig 2: CHMFL-VEGFR2-002 inhibits VEGFR2 activity. (A) IC50 determination of CHMFL-VEGFR2-002 with purified VEGFR2 kinase protein using the ADP-Glo assay. (B) GI50 determination of CHMFL-VEGFR2-002 with TEL-VEGFR2-BaF3 cells using the CCK8 assay. (C) EC50 determination of CHMFL-VEGFR2-002 on auto-phosphorylation of VEGFR2 in TEL-VEGFR2-BaF3 cells. (D) The cellular on-target effect of CHMFL-VEGFR2-002 using the Cellular Thermal Shift Assay. (E) Docking of CHMFL-VEGFR2-002 into VEGFR2 kinase X-ray crystal structure (PDB ID: 4AG8).

Fig 3: Anti-angiogenesis effect of CHMFL-VEGFR2-002 in vitro. (A) CHMFL-VEGFR2-002 inhibited phosphorylation of VEGFR2 in HUVEC cells. (B) CHMFL-VEGFR2-002 impaired capillary tube formation of HUVEC cells. HUVECs were plated on Matrigel with DMSO or various concentrations of compounds. After 24 h, tubes were photographed and measured. (C) CHMFL-VEGFR2-002 inhibited HUVEC invasion in transwell assay. 5 × 104 HUVECs were seeded in the top chamber with various concentrations of compounds. After 24 h, the invaded HUVEC cells were photographed and counted. (D) CHMFL-VEGFR2-002 inhibited HUVEC cell migration in wound healing assay. CHMFL-VEGFR2-002 group is compared with DMSO group, and significance of difference is indicated. The distance of migration is measured by ImageJ. Data are mean±SD (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control treatment.

Fig 4: XQ-2d-His-SH2 CM-(Arg)9 influenced multiple signaling cascades of PANC-1 cells. (A) Inhibition of EGFR, VEGFR2, IGF-1R, Src, AKT, and ERK1/2 phosphorylation by XQ-2d-His-SH2 CM-(Arg)9 at different incubation time or concentrations was examined in PANC-1 cells. (B-D) The panels showed reciprocal immunoprecipitation of EGFR and GRB2, IGF-1R and IRS1, VEGFR2, and SHC in PANC-1 cells treated as indicated above. IP, immunoprecipitation; IB, immunobloting. Data shown are representative of three independent experiments

Fig 5: CHMFL-VEGFR2-002 inhibited tumor growth. (A) Anti-tumor efficacy of CHMFL-VEGFR2-002 in the peritoneal dissemination model. MKN45 cells were inoculated into nude mice and treated with DMSO, CHMFL-VEGFR2-002, and sunitinib. After 17 days, tumors in CHMFL-VEGFR2-002 group were significantly less than DMSO group (tumors are marked with arrows), *P < 0.05 versus control treatment. (B) Body weight monitoring of CHMFL-VEGFR2-002 in mouse xenograft model. (C) CHMFL-VEGFR2-002 increased the survival rate of C57 mice bearing B16-F10 compared with DMSO. Data are mean±SD (n=5).