AG-490 (Tyrphostin B42)

 

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Biological Activity

Description	AG-490 (Tyrphostin B42) is an inhibitor of EGFR with IC50 of 0.1 μM, 135-fold more selective for EGFR versus ErbB2, also inhibits JAK2 with no activity to Lck, Lyn, Btk, Syk and Src.
Targets	EGFR	ErbB2	JAK2
IC50	0.1 μM	13.5 μM [1]	~10 μM [2]
In vitro	AG-490 inhibits HER-2 driven cell proliferation with IC50 of 3.5 μM. [1] Corresponding to the specific dose-dependent inhibition of constitutively activated JAK2 in pre-B acute leukemia (ALL) cells, AG-490 (5 μM) almost completely blocks the growth of all ALL cells by inducing programmed cell death, with no deleterious effect on normal hematopoiesis. AG-490 does not inhibit the activities of Lck, Lyn, Btk, Syk, and Src. [2] AG-490 (60-100 μM) blocks the constitutive activation of Stat3sm, and inhibits spontaneous as well as interleukin 2-induced growth of mycosis fungoides (MF) tumor cells with IC50 values of 75 μM and 20 μM, respectively. [3] AG-490 potently inhibits IL-2-mediated human T cell growth with an IC50 of 25 μM by blocking the activities of JAK3 and STAT5a/b. [4] Although AG-490 alone has no effect on proliferation of FDrv210H cells at a concentration of 5 μM, AG-490 can synergize with STI571 to enhance its inhibitory effect on p210bcr-abl driven proliferation. [5] AG-490 significantly inhibits the constitutive activation of Stat3 in MOPC, MPC11, and S194 cells, leading to dramatic dose-dependent apoptosis. [6] AG-490 (100 μM) inhibits Akt phosphorylation, inhibits the activation of nuclear factor-κB, and causes the activation of GSK-3β, leading to the reduction of c-Myc. AG-490 (50 μM) can induce apoptosis of imatinib-resistant BaF3 cells expressing T315I and E255K mutants of Bcr-Abl. [7] AG-490 at 30 μM inhibits not only Epo-induced phosphorylation of wild-type JAK2 but also constitutive phosphorylation of the JAK2 V617F mutant. AG-490 also potently inhibits cytokine-independent cell growth induced by the JAK2 V617F mutant in BaF3 cells. [8]
In vivo	Administration of AG-490 drastically reduces the numbers of CD45+ and HLA-DR+ cells from 48 % and 46% in bone marrow of untreated mice, as well as 38% and 22% in the spleen of untreated mice to undetectale levels.[2] In vivo administration of AG-490 causes murine myeloma tumor cell apoptosis but does not inhibit IL-12-mediated macrophage activation and IFN-γ production by lymphocytes. [6] Consistent with the in vitro blocking of JAK2 V617F mutant activity, AG-490 treatment at 0.5 mg/day for 10 days effectively inhibits JAK2 V617F mutant-induced tumorigenesis and tumor cell invasion in nude mice. [8] Combined therapy with AG-490 and IL-12 induces greater antitumor effects than either agent alone in a murine myeloma tumor model. [6]
Features

Protocol(Only for Reference)

Kinase Assay: 

^[1]

In vitro kinase autophosphorylation

AG-490 is dissolved in DMSO 10%-H2O-ethanol 45%. Crude membrane extracts (0.125 μg/mL) are preactivated with EGF (20 nM) in 50 mM HEPES buffer, pH 7.6, and 125 mM NaCl, for 15 minutes at 4 °C. Autophosphorylation activity of EGFR or ErbB2 kinase is assayed at 4 °C for 30 seconds in V-shaped 96-well plates. Membrane extracts (8 μL) are added to each well containing reaction mixture (12 μL, 50mM, HEPES, pH 7.4,125 mM NaCl, 12 mM M8Ac2, 2 mM MnCl2, 1 mM NaVO3, 1 μM ATP, and 1 μCi[γ-32P]ATP, final concentrations) and increasing concentrations of AG-490 (4 μL). After termination by addition of hot sample buffer, the samples are run on a 6% SDS-polyacrylamide gel electrophoresis minigel, the gels dried, and autoradiography performed during the linear exposure time period. The receptor bands are scanned densitrometrically, and the results analyzed by the Ez-Fit program. For the analysis of autophosphorylation of JAK2, JAK2 is immunoprecipitated by using anti-JAK2 antibody from lysates of G2 cells pretreated for 16 hours with increasing concentrations of AG-490 (0-50 μM). Immune complexes are then immunoblotted with anti-phosphotyrosine antibody. A dose-dependent inhibition of in vitro kinase activity is demonstrated by assessing JAK2 autophosphorylation.

Cell Assay: 

^[2]

Cell lines	Pre-B ALL
Concentrations	Dissolved in DMSO, final concentrations ~ 50 μM
Incubation Time	16 hours
Method	Cells are exposed to different concentrations of AG-490 for 16 hours. For the determination of cell proliferation, [3H]tymidine (1 μCi) is added 6 hours or more before the cultures are terminated. Cells are then collected and samples counted in a liquid scintillation counter.

Animal Study: 

^[2]

Animal Models	SCID mice intravenously injected with ALL cells
Dosages	0.85 mg + 0.5 mg daily
Administration	Continuous pump infusion supplemented with daily intraperitoneal injections
Solubility	1% DMSO/30% polyethylene glycol/1% Tween 80, 30 mg/mL

1

References

Chemical Information

Download AG-490 (Tyrphostin B42) SDF

Chemical Name	(E)-N-benzyl-2-cyano-3-(3,4-dihydroxyphenyl)acrylamide

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.2943	2.943	5.886	8.829

Customer Reviews (2)

Click to enlarge	Rating
	Source	Carcinogenesis. 2013 Sep;34(9):1994-9 . AG-490 (Tyrphostin B42) purchased from Selleck
	Method	Western blot
	Cell Lines	MDA231 cells
	Concentrations	5, 10, 20, 50 uM
	Incubation Time	4 h
	Results	IL6 normally induces WASF3 expression in MDA231 cells, but co-treatments with AG490 (a pan-JAK inhibitor) show a dose-dependent reduction of both WASF3 levels and activated STAT3 levels.

Product Citations (4)

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  IL-17A Stimulates the Progression of Giant Cell Tumors of Bone. [Xu M, et al. Clin Cancer Res 2013;19(17):4697-705]

  
  PubMed: 23857601


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  The Activity of the Androgen Receptor Variant AR-V7 Is Regulated by FOXO1 in a PTEN-PI3K-AKT-Dependent Way [Sanjay N, et al. Prostate 2012;73(3):267-77]

  
  PubMed: 22821817


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  RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis. [Li C, et al. Biochem Biophys Res Commun 2013;429(3-4):156-62]

  
  PubMed: 23142594