>>Signaling Pathways>> Tyrosine Kinase>> Src>>A 419259 trihydrochloride

A 419259 trihydrochloride (Synonyms: RK-20449)

Catalog No.GC17789

419259 삼염산염은 Src, Lck 및 Lyn에 대해 각각 9nM, 3nM 및 3nM의 IC50을 갖는 Src 계열 키나제 억제제입니다.

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A 419259 trihydrochloride Chemical Structure

Cas No.: 1435934-25-0

Size 가격 재고 수량
1mg
US$42.00
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5mg
US$98.00
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10mg
US$138.00
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25mg
US$265.00
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Sample solution is provided at 25 µL, 10mM.

Product Documents

Quality Control & SDS

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Protocol

Kinase experiment:

In vitro kinase assays are performed on His(6)-tagged Lck (residues 62-509) and full-length c-Abl purified from Sf-9 cells, and commercial sources of Lyn, Src and PKC. Lck, Lyn, Src and Abl activities are measured with an ELISA-based assay. Flat bottom 96-well ELISA plates are incubated with a 200 μg/mL solution of Poly(Glu,Tyr) 4 : 1 substrate in phosphate buffered saline (PBS) for 1 h at 37°C and then washed with PBS containing 0.1% Tween-20 (PBS-T). Inhibitor dilutions are added to the washed plates already containing the appropriate enzyme in kinase assay buffer (250 mM Mopso, pH 6.75, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.02% BSA, 2 mM Na3VO4, 5% DMSO, 5 μM ATP). After incubation at room temperature for 20 min, plates are washed three times with PBS-T and plate-bound phosphotyrosine is detected with an anti-phosphotyrosine-HRP antibody conjugate and subsequent color development with K-Blue reagents. All assays are optimized to use the least amount of enzyme necessary for a reproducible signal-to-noise ratio[1].

Cell experiment:

K-562 cells are grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS), and 50 g/mL Gentamycin. Meg-01 cells are cultured in Vitacell modified RPMI 1640 (ATCC), supplemented with 10% FCS and 50 μg/mL Gentamycin. The human GM-CSF-dependent myeloid leukemia cell line TF-1 is grown in RPMI 1640 supplemented with 10% FCS, 50 μg/mL Gentamycin, and 1 ng/mL of recombinant human GM-CSF. DAGM murine myeloid leukemia cells are cultured in RPMI 1640 supplemented with 10% FCS, 50 μg/mL Gentamycin, and 0.5 ng/mL recombinant IL-3. Concentrated stock solutions of PP2 (5 mM) and A-419259 (2 mM) are prepared in DMSO and stored at -20°C. Cellular proliferation is measured using the Live/Dead growth assay. This assay employs calcein-AM, a fluorogenic esterase substrate that is taken up by viable cells and hydrolyzed intracellularly, releasing a green fluorescent product. Briefly, 104 cells are plated per well in 96-well plates for each day of a 4-day time course. Various concentrations of PP2, A-419259 or vehicle control are added to the wells (five wells per concentration per day) and the plates are incubated at 37°C. At each time point, one plate is centrifuged at 1500 g for 10 min to pellet the cells. Cells are washed with phosphate buffered saline (PBS), and calcein-AM is added to each well to a final concentration of 1 μM. Plates are incubated in the dark at room temperature for 1 h. The plates are then read at 485/530 nm (excitation/emission) using a SpectraMax Gemini XS fluorescent plate reader and data are analysed with SoftMax Pro software[1].

References:

[1]. Wilson MB, et al. Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr-Abl signal transduction and oncogenesis. Oncogene. 2002 Nov 21;21(53):8075-88.
[2]. Pene-Dumitrescu T, et al. An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259.Oncogene (2008) 27, 7055-7069

Background

A 419259 trihydrochloride is an orally available inhibitor of lck with IC50 value of 15 nM [1].

Lck is a src family tyrosine kinase. Inhibition of this enzyme results in a prevention of T-cell activation and provides an application for treating T-cell dependent processes such as autoimmune and inflammatory diseases. A 419259 trihydrochloride is screened out by its potent inhibitory activity against the recombinant lck (64–509) with IC50 value of 15nM. It is also a selective inhibitor. It shows inactive against a panel of other kinases including cMet, IGFR, PKC and PDGFR. Besides that, A 419259 trihydrochloride is found to inhibit the production of IL-2 with IC50 value of 25nM in Jurkat cells. Moreover, administration of A 419259 trihydrochloride reduces IL-2 production in mice with ED50 value of 2.5 mg/kg. A 419259 trihydrochloride also has efficacy of the antigen specific T-Cell immune response. Administration of this compound inhibits IFN-γ production during the in vivo priming phase [1].

Reference:
[1] Calderwood D J, Johnston D N, Munschauer R, et al.  Pyrrolo [2, 3-d] pyrimidines containing diverse N-7 substituents as potent inhibitors of Lck. Bioorganic & medicinal chemistry letters, 2002, 12(12): 1683-1686.

Chemical Properties

Cas No. 1435934-25-0 SDF
Synonyms RK-20449
Chemical Name 7-((1r,4r)-4-(4-methylpiperazin-1-yl)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine trihydrochloride
Canonical SMILES CN1CCN([C@@]2([H])CC[C@@](N3C=C(C(C3=NC=N4)=C4N)C5=CC=C(OC6=CC=CC=C6)C=C5)([H])CC2)CC1.Cl.Cl.Cl
Formula C29H37Cl3N6O M.Wt 592
Solubility 55 mg/mL in water Storage Store at -20°C
General tips Please select the appropriate solvent to prepare the stock solution according to the solubility of the product in different solvents; once the solution is prepared, please store it in separate packages to avoid product failure caused by repeated freezing and thawing.Storage method and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored at -20°C, please use it within 1 month.
To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time.
Shipping Condition Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request.

Complete Stock Solution Preparation Table

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1 mg 5 mg 10 mg
1 mM 1.6892 mL 8.4459 mL 16.8919 mL
5 mM 0.3378 mL 1.6892 mL 3.3784 mL
10 mM 0.1689 mL 0.8446 mL 1.6892 mL
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Average Rating: 5 ★★★★★ (Based on Reviews and 24 reference(s) in Google Scholar.)

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