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Biological Activity
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Description
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PHA-665752 is a potent, selective and ATP-competitive c-Met inhibitor with IC50 of 9 nM. |
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Targets
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c-Met |
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IC50 |
9 nM [1] |
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In Vitro
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PHA-665752 significantly inhibits c-Met kinase activity with Ki of 4 nM, and exhibits >50-fold selectivity for c-Met compared with various tyrosine and serine-threonine kinases. PHA-665752 potently inhibits the HGF-stimulated c-Met autophosphorylation with IC50 of 25-50 nM. PHA-665752 also significantly blocks HGF- and c-Met-dependent functions such as cell motility and cell proliferation with IC50 of 40-50 nM and 18-42 nM, respectively. In addition, PHA-665752 potently inhibits HGF-stimulated or constitutive phosphorylation of mediators of downstream of c-Met such as Gab-1, ERK, Akt, STAT3, PLC-γ, and FAK in multiple tumor cell lines. [1] PHA-665752 inhibits cell growth in TPR-MET-transformed BaF3 cells with IC50 of <60 nm,="" and="" inhibits="" constitutive="" cell="" motility="" and="" migration="" by="" 92.5%="" at="" 0.2="" μm.="" inhibition="" of="" c-met="" by="" pha665752="" (0.2="" μm)="" also="" induces="" cell="" apoptosis="" of="" 33.1%="" and="" g1="" cell="" cycle="" arrest="" with="" cells="" in="" g1="" phase="" increasing="" from="" 42.4%="" to="" 77.0%.="" pha665752="" can="" cooperate="" with="" rapamycin="" to="" inhibit="" cell="" growth="" of="" tpr-met-transformed="" baf3="" cells="" and="" non-small="" cell="" lung="" cancer="" h441="" cells.="">[2] |
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In Vivo
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Administration of PHA-665752 induces a dose-dependent tumor growth inhibition of S114 xenografts by 20 %, 39% and 68%, at dose of 7.5, 15, and 30 mg/kg/day, respectively. [1] PHA665752 treatment significantly reduces the tumor growth of NCI-H69, NCI-H441 and A549 in mouse xenografts by 99%, 75%, and 59%, respectively. PHA665752 also significantly inhibits angiogenesis by >85%, due to decreasing the production of vascular endothelial growth factor and increasing the production of the angiogenesis inhibitor thrombospondin-1. [3] |
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Clinical Trials
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Features
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Protocol
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Kinase Assay
[1]
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| In vitro enzyme assay |
The c-Met kinase domain GST-fusion protein is used for the c-Met assay. The IC50 value of PHA-665752 for the inhibition of c-Met is based on phosphorylation of kinase peptide substrates or poly-glu-tyr in the presence of ATP and divalent cation (MgCl2 or MnCl2 10-20 mM). The linear range (i.e., the time period over which the rate remains equivalent to the initial rate) is determined for c-Met, and the kinetic measurement and IC50 determination are performed within this range. |
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Cell Assay
[1]
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| Cell Lines |
S114, GTL-16, NCI-H441, and BxPC-3 |
| Concentrations |
Dissolved in DMSO, final concentrations ~10 μM |
| Incubation Time |
18, or 72 hours |
| Methods |
For proliferation assays, cells are grown in medium with 0.1% FBS for 48 hours after which they are treated with various concentrations of PHA-665752 in HGF (50 ng/mL) in a medium containing 2% FBS. After 18 hours, cells are incubated with BrdUrd for 1 hour, fixed, and stained with anti-BrdUrd peroxidase-conjugated antibody, and plates are read at 630 nm. For apoptosis assays, cells are grown in medium with 2% FBS in presence and absence of HGF (50 ng/mL) and various concentrations of PHA-665752 for 72 hours. After 72 hours, a mixture containing ethidium bromide and acridine orange is added, and apoptotic cells (bright orange cells or cell fragments) are counted by fluorescence microscopy. |
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Animal Study
[1]
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| Animal Models |
Female athymic mice (nu/nu) bearing S114 or GTL-16 tumor xenografts |
| Formulation |
Formulated in vehicle (L-lactate (pH 4.8) and 10% polyethylene glycol) |
| Doses |
~30 mg/kg/day |
| Administration |
Injection via bolus i.v. |
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References |
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[1] Christensen JG, et al. Cancer Res, 2003, 63(21), 7345-7355.
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[2] Ma PC, et al. Clin Cancer Res, 2005, 11(6), 2312-2319.
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[3] Puri N, et al. Cancer Res, 2007, 67(8), 3529-3534.
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