|Size||Stock||Price||Quantity||Add to Cart||Quotation Online|
|Cat. No. :||CS-0877|
|CAS No. :||915385-81-8|
|M. Wt. :||380.9104|
|Solubility:||DMSO: ≥ 3.7 mg/mL|
ABC294640 is a selective, competitive sphingosine kinase 2 (SK2) inhibitor with Ki of 9.8 μM. IC50 & Target: Ki: 9.8 μM (SK2) In Vitro: Using recombinant human SK1 and SK2, ABC294640 demonstrates dose-dependent inhibition of SK2 with an IC50 of approximately 60 μM without affecting the activity of SK1 at concentrations up to at least 100 μM. In contrast, N,N-dimethylsphingosine (DMS) inhibits both SK1 and SK2 with IC50 values of approximately 60 and 20 μM, respectively. Kinetic analyses of varying concentrations of ABC294640 in the presence of 2.5 to 25 μM sphingosine indicated a Ki of 9.8±1.4 μM for the inhibition of SK2. ABC294640 decreases [3H]S1P formation in a dose-dependent fashion with an IC50 value of 26 μM. IC50 values for ABC294640 are approximately 50 and 60 μM for A-498 and Bxpc-3 cells, respectively; whereas the IC50 values for ABC294735 are approximately 20 and 40 μM for these cells. In Vivo: ABC294640 induces a transient minor decrease in the hematocrit of rats. Hematology studies indicate decreases in red blood cell number and hematocrit of approximately 20% in animals given either 100 or 250 mg/kg/day; and a slight increase in neutrophils and decrease in basophils in the treated rats. Mice are gavaged with ABC294640 (50 mg/kg), a selective inhibitor of sphingosine kinase-2 (SK2), 1 h before surgery and subjected to 1 h-warm ischemia to ~70% of the liver followed by reperfusion. ABC294640-treatment largely prevented the increase of sphingosine-1-phosphate (S1P) after ischemia-reperfusion (IR) in vivo.
Protocol:Kinase Assay: The IC50 values for ABC294640 and DMS are determined by a newly developed HPLC-based SK activity assay. In brief, the test compounds are incubated with recombinant SK1 or SK2 and NBD-Sph in the isozyme-selective assay buffers detailed below with 1 mg/mL fatty acid-free bovine serum albumin, 100 μM ATP, and 400 μM MgCl2. The product, i.e., NBD-S1P, is separated from NBD-Sph by HPLC as follows: Waters 2795 HPLC system with a Waters 2495 fluorescence detector, C8 Chromolith RP-8e column (100×4.6 mm), 1 mL/min mobile phase (acetonitrile/20 mM sodium phosphate buffer, pH2.5, at 45:55). Fluorescence is monitored with excitation at 465 nm and emission at 531 nm. The ratio of NBD-S1P/(NBD-Sph+NBD-S1P) is used as a measure of SK activity. SK-isozyme selective assay buffers each contain 20 mM Tris, pH7.4, 5 mM EDTA, 5 mM EGTA, 3 mM β-mercaptoethanol, 5% glycerol, 1× protease inhibitors and 1× phosphatase inhibitors. For the SK1 assay buffer, 0.25% (final) Triton X-100 is added; and for the SK2 buffer, 1 M (final) KCl is added. Assays are run for 2 h at room temperature, and then a 1.5 volume of methanol is added to terminate the kinase reaction. After 10 min, the samples are centrifuged at 20,000g to pellet the precipitated protein, and the supernatants are analyzed by HPLC. In experiments to determine the Ki for inhibition of SK2 by ABC294640, the ADP Quest assay system is used to measure kinase activity in the presence of varying concentrations of sphingosine and ABC294640. Cell Assay: ABC294640 is prepared in DMSO and stored, and then diluted with appropriate media before use.To determine the effects of the test compounds (e.g., ABC294640) on proliferation, cells are plated into 96-well microtiter plates and allowed to attach for 24 h. Varying concentrations of ABC294640 are added to individual wells and the cells are incubated for an additional 72 h. At the end of this period, the number of viable cells is determined by use of the sulforhodamine-binding assay. The percentage of cells killed is calculated as the percentage decrease in sulforhodamine-binding compare with control cultures. Regression analyses of inhibition curves are performed by use of GraphPad Prism. Animal Administration: ABC294640•HCl is prepared by dissolving 9.6 g of ABC294640 in 50 mL of CH2Cl2, and slowly adding an equimolar amount of 1M HCl in ether. Then ABC294640•HCl is prepared in 0.375% Polysorbate-80 in PBS (Rat).
ABC294640 is prepared in 0.375% Tween 80 in PBS, pH 7.1 (Mice).Rat
Sprague-Dawley male rats (7-8 weeks old) are orally dosed with 0, 100, or 250 mg of ABC294640•HCl/kg in 0.375% Polysorbate-80 in PBS daily for 7 days. The animals are observed daily for viability, signs of gross toxicity, and behavioral changes, and a battery of detailed observations are performed on study days 1 and 7. Blood is sampled from all animals on day 8 of the study for hematology, clinical biochemistry, and serology assessments, and the animals are sacrificed. Gross necropsies are performed on all study rats, and selected organs and tissues are evaluated in the control and high-dose level groups.
Male C57BL/6 (8-9 weeks) mice are gavaged with 50 mg/kg of ABC294640, or an equivalent volume of vehicle (0.375% Tween 80 in phosphate buffered saline, pH 7.1) 1 h before surgery. Under ether anesthesia, ischemia to 70% of the total liver is induced for 1 h. After opening the vascular clamp, the non-ischemic liver lobes are removed, and mice are observed 7 days for survival. Sham operation included equivalent anesthesia and laparotomy without ischemia.
Shi Y, et al. Sphingosine kinase-2 inhibition improves mitochondrial function and survival after hepatic ischemia-reperfusion. J Hepatol. 2012 Jan;56(1):137-45.
Beljanski V, et al. Combined anticancer effects of sphingosine kinase inhibitors and sorafenib. Invest New Drugs. 2011 Dec;29(6):1132-42.
Liu Q, et al. Inhibition of sphingosine kinase-2 suppresses inflammation and attenuates graft injury after liver transplantation in rats. PLoS One. 2012;7(7):e41834.
French KJ, et al. Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2. J Pharmacol Exp Ther. 2010 Apr;333(1):129-39.
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