Orantinib (NSC 702827) , a excellent effective against PDGFR autophosphorylation with Ki of 8 nM, also highly inhibits Flk-1 and FGFR1 trans-phosphorylation. It shows little effect against IGF-1R, Met, Src, Lck, Zap70, Abl and CDK2 and does not suppresses EGFR.

PDGFRβ:8 nM(Ki)

在HT29人类结肠癌肿瘤模型中,TSU-68（200 mg/kg）降低肿瘤边缘的平均血管通透性和肿瘤中心的平均血浆容积分数.在携带多种移植瘤的无胸腺小鼠,包括A375,Colo205,H460,Calu-6,C6,SF763T,和SKOV3TP5细胞等,TSU-68（75-200 mg/kg）抑制细胞生长.兔VX2肝脏肿瘤模型中, TSU-68（200 mg/kg）增加注射化学治疗的效果.在C6神经胶细胞移植瘤中, TSU-68（75 mg/kg）也阻断肿瘤血管生成.

TSU-68是ATP竞争性抑制剂, 作用于Flk-1/KDR磷酸转移, FGFR1磷酸转移,和PDGFRβ激酶时, Ki分别为2.1 μM, 1.2 μM,和8 nM。在人类骨髓性白血病MO7E细胞中,TSU-68（IC50=0.1-1 μM）抑制肝细胞因子受体c-kit的酪氨酸自磷酸化, 也抑制ERK1/2磷酸化。在SCF刺激的MO7E细胞中,TSU-68（IC50=0.29 μM）也抑制细胞增殖,并诱导凋亡。过量表达PDGFRβ的NIH-3T3细胞中,TSU-68（0.03-0.1 μM）抑制PDGF刺激的PDGFRβ 酪氨酸磷酸化。在血管内皮生长因子刺激的HUVECs中,TSU-68（0.03-10 μM)抑制KDR酪氨酸磷酸化。在过量表达EGFR 的NIH-3T3细胞中, TSU-68(100 μM) 不抑制EGF刺激的EGFR酪氨酸磷酸化。TSU-68抑制血管内皮生长因子驱动的和FGF驱动的HUVECs分裂,平均IC50分别为0.34和 9.6 μM。

trans-Phosphorylation Reactions: Tyrosine kinase assays to quantitate the trans-phosphorylation activity of Flk-1 and FGFR1 are performed in 96-well microtiter plates precoated (20 μg/well in PBS; incubated overnight at 4 °C) with the peptide substrate poly-Glu,Tyr (4:1). Excess protein binding sites are blocked with 1-5% (w/v) BSA in PBS. Purified GST-FGFR1 (kinase domain) or GST-Flk-1 (cytoplasmic domain) fusion proteins are then added to the microtiter wells in 2 × concentration kinase dilution buffer consisting of 100 mM HEPES, 50 mM NaCl, 40 μM NaVO4, and 0.02% (w/v) BSA. The final enzyme concentration for GST-Flk-1 and GST-FGFR1 is 50 ng/mL. SU6668 is dissolved in DMSO at 100× the final required concentration and diluted 1:25 in Water. Twenty-five μL of diluted SU6668 are subsequently added to each reaction well. The kinase reaction is initiated by the addition of different concentrations of ATP in a solution of MnCl2 so that the final ATP concentrations spanned the Km for the enzyme, and the final concentration of MnCl2 is 10 mM. The plates are incubated for 5-15 min at room temperature before stopping the reaction with the addition of EDTA. The plates are then washed three times with TBST. Rabbit polyclonal antiphosphotyrosine antisera are added to the wells at a 1: 10000 dilution in TBST containing 0.5% (w/v) BSA, 0.025% (w/v) nonfat dry milk, and 100 μM NaVO4 and incubated for 1 hour at 37 °C. The plates are then washed three times with TBST, followed by the addition of goat anti-rabbit antisera conjugated with HRP. The plates are incubated for 1 hour at 37 °C and then washed three times with TBST. The amount of phosphotyrosine in each well is quantitated after the addition of 2,2

Cells are seeded (3 × 105 cells/35-mm well) in DMEM containing 10% (v/v) FBS and grow to confluence and then quiesced in DMEM containing 0.1% serum for 2 hours before drug treatment. HUVECs (seeded at 2 × 106 cells/10-cm plate) are grown to confluence in endothelial cell growth media and then quiesced in endothelial cell basal media containing 0.5% FBS for 24 hours before drug treatment. All cell lines are incubated with SU6668 for 1 hour before ligand stimulation (100 ng/mL) for 10 min. Western blotting is perfor (Only for Reference)