PHA-665752

 

Distributor Tel: +886-2-2827-1197jimmy@new.abscience.com.tw

Biological Activity

Description	PHA-665752 is a potent, selective and ATP-competitive c-Met inhibitor with IC50 of 9 nM, >50-fold selectivity for c-Met than RTKs or STKs.
Targets	c-Met
IC50	9 nM [1]
In vitro	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]
In vivo	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]
Features

Protocol(Only for Reference)

Kinase Assay: ^[1]

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.

Cell Assay: ^[1]

Cell lines	S114, GTL-16, NCI-H441, and BxPC-3
Concentrations	Dissolved in DMSO, final concentrations ~10 μM
Incubation Time	18, or 72 hours
Method	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.

Animal Study: ^[1]

Animal Models	Female athymic mice (nu/nu) bearing S114 or GTL-16 tumor xenografts
Dosages	~30 mg/kg/day
Administration	Injection via bolus i.v.
Solubility	30% PEG400/0.5% Tween80/5% propylene glycol, 30 mg/mL

1

References

Chemical Information

Download PHA-665752 SDF

Chemical Name	(R,Z)-5-(2,6-dichlorobenzylsulfonyl)-3-((3,5-dimethyl-4-(2-(pyrrolidin-1-ylmethyl)pyrrolidine-1-carbonyl)-1H-pyrrol-2-yl)methylene)indolin-2-one

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.64161	6.4161	12.8322	19.2483

Customer Reviews (1)

Click to enlarge	Rating
	Source	Dr. Zhang of Tianjin Medical University. PHA-665752 purchased from Selleck
	Method	Western blot
	Cell Lines
	Concentrations	0-100 μM
	Incubation Time
	Results	PHA-665752 treatment resulted in a reduction of c-Met phosphorylation in a concentration-dependent manner.

Product Citations (9)

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  Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. [Zhang Z, et al. Nat Genet 2012;44(8):852-60]

  
  PubMed: 22751098


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  Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. [Walter AO, et al. Cancer-Discov 2013;3(12):1404-15]

  
  PubMed: 24065731


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  Proliferation State and Polo‐Like Kinase1 Dependence of Tumorigenic Colon Cancer Cells. [Francescangeli F, et al. Stem Cells 2012;30(9):1819-30]

  
  PubMed: 22753241