AC480 (BMS-599626) 714971-09-2

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Molecular Weight:

567.01 AC480 (BMS-599626) is a selective and efficacious inhibitor of HER1 and HER2 with IC50 of 20 nM and 30 nM, ~8-fold less potent to HER4, >100-fold to VEGFR2, c-Kit, Lck, MET etc. Phase 1.


Biological Activity


BMS-599626 also inhibits the related receptor HER4, but with reduced
potency with IC50 of 190 nM. BMS-599626 is identified as an
ATP-Competitive Inhibitor for HER1 and as an ATP-noncompetitive
inhibitor for HER2 with Ki of 2 nM and 5 nM, respectively.
BMS-599626 inhibits the proliferation of tumor cells expressing high
levels of HER1 and/or HER2, including Sal2, BT474, N87, KPL-4, HCC202,
HCC1954, HCC1419, AU565, ZR-75-30, MDA-MB-175, GEO, and PC9 cells with
IC50 of 0.24 μM, 0.31 μM, 0.45 μM, 0.38μM, 0.94 μM, 0.34 μM, 0.75 μM,
0.63 μM, 0.51 μM, 0.84 μM, 0.90 μM and 0.34 μM, respectively.


While the
proliferation of the ovarian tumor cell line A2780 and MRC5 fibroblasts,
neither of which expresses HER1 or HER2, are not inhibited significant
by BMS-599626. A recent study shows that BMS-599626
significantly enhances the radiosensitivity of HN-5 cells expressing
both EGFR and Her2 cell, by promoting cycle redistribution and
inhibiting DNA repair.


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.






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 μL and contains 10 ng
of glutathione-S-transferase fusion protein or 150 ng of partially
purified HER2. The mixtures also contains 1.5 μM poly(Glu/Tyr) (4:1), 1
μM ATP, 0.15 μCi [γ-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 27°C for 1 hour and are terminated
by the addition of 10 μL of a stop buffer (2.5 mg/mL bovine serum
albumin and 0.3 M EDTA), followed by a 108-μL 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.






Method


All cell lines are maintained in RPMI 1640 supplemented with 10% fetal
bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells
are plated at 1,000 per well in 96-well plates and are cultured for 24
hours before BMS-599626 is added. BMS-599626 is diluted in culture
medium such that the final concentrations of DMSO are ≤ 1%. Following
the addition of BMS-599626, the cells are cultured for an additional 72
hours before cell viability is determined by measuring the conversion of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye with
the CellTiter96 kit.


For some cell lines, there is a lack of a
correlation between 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide dye Metabolism and cell number, and a thymidine uptake assay is
used to measure proliferation of these cell lines. Cells are plated in
96-well plates and treated with compounds as above. At the end of the
72-hour incubation, cells are pulsed with [3H]thymidine (0.4
μCi/well) for 3 hours before they are harvested.


Cells are digested with
2.5% trypsin for 10 minutes at 37 °C and are harvested by filtration
using a Packard Filtermate Harvester and GF/C Unifilter plates.
Incorporation of radioactive thymidine into nucleic acids is determined
by liquid scintillation counting.

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Product Categories : Protein Tyrosine Kinase > HER2 Inhibitor