AC480 (BMS-599626)

SAL2 murine salivary gland tumor, N87 human gastric carcinoma, BT474 human breast tumor, A549 human non–small-cell lung tumor, and GEO human colon tumor are maintained and passaged in athymic female nude mice.

(S)-morpholin-3-ylmethyl 4-(1-(3-fluorobenzyl)-1H-indazol-5-ylamino)-5-methylpyrrolo[1, 2-f][1, 2, 4]triazin-6-ylcarbamate

0-10 uM

<=240 mg/kg

20 nM, 20 nM, 20 nM, 20 nM, 20 nM, 20 nM

In vivo, oral administration of BMS-599626 results in a dose-dependent inhibition of Sal2 tumor growth at doses ranging from 60 mg/kg to 240 mg/kg, yielding a potent antitumor activity in a human breast tumor KPL-4 xenograft at its maximum tolerated dose of 180 mg/kg, and also has similar antitumor activity in other HER2 amplified xenograft models, as well as other HER1-overexpressing xenograft models. [1]

Protein kinase assays, The entire cytoplasmic sequences of HER1, HER2, and HER4 are expressed as recombinant proteins in Sf9 insect cells. HER1 and HER4 are expressed as fusion proteins with glutathione-S-transferase and are purified by affinity chromatography on glutathione-S-Sepharose. HER2 is subcloned into the pBlueBac4 vector and expressed as an untagged protein using an internal methionine codon (M687) for translation initiation. The truncated HER2 protein is isolated by chromatography on a column of DEAE-Sepharose equilibrated in a buffer that contains 0.1 M NaCl, and the recombinant protein is eluted with a buffer containing 0.3 M NaCl. For the HER kinase assays, reaction volumes are 50 uL and contains 10 ng of glutathione-S-transferase fusion protein or 150 ng of partially purified HER2. The mixtures also contains 1.5 uM poly(Glu/Tyr) (4:1), 1 uM ATP, 0.15 uCi [gamma-33P]ATP, 50 mM Tris-HCl (pH 7.7), 2 mM DTT, 0.1 mg/mL bovine serum albumin, and 10 mM MnCl2. Reactions are allowed to proceed at 27C for 1 hour and are terminated by the addition of 10 uL of a stop buffer (2.5 mg/mL bovine serum albumin and 0.3 M EDTA), followed by a 108-uL mixture of 3.5 mM ATP and 5% trichloroacetic acid. Acid-insoluble proteins are recovered on GF/C Unifilter plates with a Filtermate harvester. Incorporation of radioactive phosphate into the poly(Glu/Tyr) substrate is determined by liquid scintillation counting. Percent inhibition of kinase activity is determined by nonlinear regression analyses and data are reported as the inhibitory concentration required to achieve 50% inhibition relative to control reactions (IC50). Data are the averages of triplicate determinations. All other tyrosine kinases are also assayed using poly(Glu/Tyr) as a substrate. Kinetics of HER1 and HER2 inhibition are determined in reaction mixtures that contains varying concentrations of ATP and BMS-599626.

567, 01

, , Dr. Zhang of Tianjin Medical University, BMS-599626 (AC480)purchased from Selleck, Breast cancer cells were pretreated with 100ng/ml EGF for 15 min and then treated with the indicated concentrations of BMS-599626.

2 years -20CPowder, 2 weeks4Cin DMSO, 2 months-80Cin DMSO