OSI-930 728033-96-3

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Molecular Weight:

443.44 OSI-930 is a Potent Inhibitor of Kit, KDR and CSF-1R with IC50 of 80 nM, 9 nM and 15 nM, respectively; also potent to Flt-1, c-Raf and Lck and low activity against PDGFRα/β, Flt-3 and Abl. Phase 1.


Biological Activity


OSI-930 inhibits the cell proliferation in the HMC-1 cell line with IC50
of 14 nM without significant effect on growth of the COLO-205 cell line
that does not express a constitutively active mutant receptor tyrosine
kinase. Moreover, OSI-930 also induces Apoptosis in HMC-1 cell line with
EC50 of 34 nM. A recent study shows that OSI-930 inactivates purified, recombinant cytochrome P450 (P450) 3A4 with a Ki of 24 μM in a time- and concentration-dependent mode.


OSI-930, administrated at the maximally efficacious dose of 200 mg/kg by
oral gavage, exhibits potent antitumor activity in a broad range of
preclinical xenograft models including HMC-1, NCI-SNU-5, COLO-205 and
U251 xenograft models.


Protein kinase assays


Protein kinase assays are either done in-house by ELISA-based assay
methods (Kit, KDR, PDGFRα, and PDGFRβ) or by a radiometric method.
In-house ELISA assays used poly(Glu:Tyr) as the substrate bound to the
surface of 96-well assay plates; phosphorylation is then detected using
an antiphosphotyrosine antibody conjugated to HRP. The bound antibody is
then quantitated using ABTS as the peroxidase substrate by measuring
the absorbance at 405/490 nm. All assays uses purified recombinant
kinase catalytic domains that are either expressed in insect cells or in
bacteria.


The Kit and EGFR protein used for in-house assays are
prepared internally; other enzymes are obtained. Recombinant Kit protein
is expressed as an NH2-terminal glutathione S-transferase
fusion protein in insect cells and is initially purified as a
nonphosphorylated (nonactivated) enzyme with a relatively high Km for ATP (400 μM). In some assays, an activated (tyrosine
phosphorylated) form of the enzyme is prepared by incubation with 1 mM
ATP for 1 hour at 30 °C. The phosphorylated protein is then passed
through a desalting column to remove the majority of the ATP and stored
at −80 °C in buffer containing 50% glycerol.


The resultant preparation
has a considerably higher specific activity and a lower Km for ATP (25
μM) than the initial nonphosphorylated preparation. The inhibition of
Kit autophosphorylation by OSI-930 is assayed by incubation of the
nonphosphorylated enzyme at 30 °C in the presence of 200 μM ATP and
various concentrations of OSI-930. The reaction is stopped by removal of
aliquots into SDS-PAGE sample buffer followed by heating to 100 °C for 5
minutes. The degree of phosphorylation of Kit is then determined by
immunoblotting for both total Kit and phosphorylated Kit.






Method


For assays of cell proliferation and apoptosis, cells are seeded into
96-well plates and incubated for 2 to 3 days in the presence of OSI-930
at various concentrations. Inhibition of cell growth is determined by
luminescent quantitation of the intracellular ATP content using
CellTiterGlo. Induction of caspase-dependent apoptosis by OSI-930 is
quantitated by an enzymatic caspase 3/7 assay. Inhibition of
Angiogenesis by OSI-930 is monitored using the rat aortic ring
endothelial sprout outgrowth assay.


Sections of aorta are prepared from
CO2-euthanized male rats and cultured in vitro in a collagen
matrix in the presence or absence of OSI-930. The collagen matrix is
prepared from type 1 rat tail collagen solubilized in 0.1% acetic acid
at 3 mg/mL, which is combined with 0.125 volume collagen buffer (0.05 N
NaOH, 200 mM HEPES, 260 mM NaHCO3), 0.125 volume of medium
199, 0.0125 volume of 1 M NaOH, and 1% GlutaMax.


Aortic rings are
embedded in 0.4 mL of this matrix in six-well plates, to which 0.5 mL
endothelial basal medium and the appropriate amount of OSI-930 is added;
the rings are then incubated for 10 days and the resultant angiogenic
sprout outgrowth is digitally quantitated from images by measurement of
the sprout-containing area within a series of concentric rings around
the aortic tissue area.

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Product Categories : Protein Tyrosine Kinase > c-Kit Inhibitor