Orantinib (SU6668)

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 H₂O. 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

HUVECs, and NIH-3T3 cells overexpressing PDGFRβ or EGFR

0.03-10 μM , dissolved in DMSO as 10 mM stock solution

1 hour (before ligand stimulation)

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

Mouse (Female, BALB/c, nu/nu) xenograft models of A375, Colo205, H460, Calu-6, C6, SF763T, and SKOV3TP5 tumor cells

75-200 mg/kg

Via i.p. injection or oral gavage once daily.