| キナーゼ試験 |
The ability of ZD6474 to inhibit the kinase activity associated with the VEGFRs KDR, Flt-1, and Flt-4 was determined using a previously described ELISA. Briefly, ZD6474 was incubated with enzyme, 10 mm MnCl2, and 2 μm ATP in 96-well plates coated with a poly(Glu, Ala, Tyr) 6:3:1 random copolymer substrate. Phosphorylated tyrosine was then detected by sequential incubation with a mouse IgG anti-phosphotyrosine 4G10 antibody, a horseradish peroxidase-linked sheep anti-mouse immunoglobulin antibody, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Microcal Origin software was used to interpolate IC50 values by nonlinear regression. This methodology was adapted to examine selectivity versus tyrosine kinases associated with EGFR, PDGFRβ, Tie-2, FGFR1, c-kit, erbB2, IGF-IR, and FAK. All enzyme assays (tyrosine or serine-threonine) used appropriate ATP concentrations at or just below the respective Km (0.2–14 μm). Selectivity versus serine-threonine kinases (CDK2, AKT, and PDK1) was examined using a relevant scintillation proximity assay (SPA) in 96-well plates. CDK2 assays contained 10 mm MnCl2, 4.5 μm ATP, 0.15 μCi of [γ-33P]ATP/reaction, 50 mm HEPES (pH 7.5), 1 mm DTT, 0.1 mm sodium orthovanadate, 0.1 mm sodium fluoride, 10 mm sodium glycerophosphate, 1 mg/ml BSA fraction V, and a retinoblastoma substrate (part of the retinoblastoma gene, 792–928, expressed in a glutathione S-transferase expression system; 0.22 μm final concentration). Reactions were allowed to proceed at room temperature for 60 min before quenching for 2 h with 150 μl of a solution containing EDTA (62 mm final concentration), 3 μg of a rabbit immunoglobulin anti-glutathione S-transferase antibody and protein A SPA-polyvinyltoluene beads (0.8 mg/reaction). Plates were then sealed, centrifuged (1200 × g for 5 min), and counted on a Topcount NXT Microplate scintillation counter for 30 s [1]. |
参考文献
投与量変換
