DCC-2036, inhibitor

DCC-2036 (Rebastinib)

Cat. Num. AAA577676

• Purity: 99.5%
• Synonyms: DCC-2036; DCC-2036 (Rebastinib); inhibitor

• Purity/Purification: 99.5%
• Form/Format: POWDER

• CAS: 1020172-07-9
• Formula: C30H28FN7O3
• Solubility: DMSO: 103 mg/mL (186.1 mM); Ethanol: 13 mg/mL (23.5 mM); (< 1 mg/ml refers to the product slightly soluble or insoluble)
• Receptor (IC50): Abl1: 1.4nM; FLT3: 2nM; p-Abl1 (native): 2nM; p-Abl1 (T315I): 4nM; u-Abl1 (native): 0.75nM
• Kinase Assay: Assay of Abl1 kinase isoforms and determination of inhibitor potency: Activity of u-Abl1native is determined by following the production of ADP from the kinase reaction through coupling with the pyruvate kinase/lactate dehydrogenase system. In this assay, the oxidation of NADH (measured as a decreased A340nm) is continuously monitored spectrophotometrically. The final reaction mixture (100 uL, in a 384-well Corning plate) is prepared as follows: An Abl1 kinase/coupled assay components mixture is prepared containing u-Abl1 kinase (1 nM), Abltide (EAIYAAPFAKKK, 0.2 mM), MgCl2 (9 mM), pyruvate kinase (~ 4 units), lactate dehydrogenase (~ 0.7 units), phosphoenol pyruvate (1 mM), and NADH (0.28 mM) in 90 mM Tris containing 0.1 % octyl-glucoside and 1 % DMSO, pH 7.5. Separately, an inhibitor mixture is prepared containing DCC-2036 serially diluted 3-fold in DMSO followed by dilution into buffer composed of 180 mM Tris, pH 7.5, containing MgCl2 (18 mM) and 0.2 % octylglucoside. Fifty ?L of the inhibitor mixture is mixed with 50 uL of the above Abl1 kinase/coupled assay components mixture, which is then incubated at 30 °C for 2 hours before 2 uL of 25 mM ATP (500 ?M, final) is added to start the reaction. The reaction is recorded every 2 minutes for 2.5 hours at 30 °C on a Polarstar Optima or Synergy2 plate reader. Reaction rate (slope) is calculated using the 1 to 2 hour time frame with reader's software. Percent inhibition is obtained by comparison of reaction rate with that of a DMSO control. IC50 values are calculated from a series of percent inhibition values determined at a range of inhibitor concentrations using GraphPad Prism. The kinase assay for Abl1T315I, p-Abl1native or Abl1H396P is assayed the same as above except that 2.2 nM Abl1T315I, 1 nM p-Abl1 native or 1.3 nM Abl1H396P is used. The above assay format is also used for kinases other than Abl1 with the exception of TIE2, for which a fluorescence polarization/Transcreener format is used. The assay conditions are the same as described above except that PolyE4Y (final 1 mg/mL) is used as the substrate and one hour preincubation is used.
• Smiles: CNC(=O)c1nccc(Oc2cc(F)c(NC(=O)Nc3cc(nn3c3cc4c(cc3)nccc4)C(C)(C)C)cc2)c1
• Cell Assay: viable cells are quantified by Resazurin or MTT assay. Cells are diluted in medium to be added to each well of a 96-well tissue culture-treated plate. All cells are incubated overnight and maintained in a humidified atmosphere at 37°C and 5% CO2. Cells are treated the following day. Serum-free medium is used during treatment with DCC-2036. MTT is used to assess the viability of cells following treatment. Aliquots of 20 mL of stock MTT solution are added to each well containing 200 mL of medium (10% final solution) and incubated with the cells for 2 hours. Following incubation the medium is removed and 200 mL of dimethylsulfoxide added to solubilize the formazan crystals. The absorbance is read on the plate reader at 550 and 690 nm. A subtraction analysis of the dual wavelength is performed (D550 to D690) to increase accuracy of the measurement (Only for Reference); ; Cell line: Ba/F3 cells and primary Ph+ leukemia cells
• Animal Experiment: Animal Model: Ba/F3 cells transformed to interleukin-3 independence by transduction with either Bcr-Abl1native or Bcr-Abl1T315I retrovirus are injected intravenously into syngeneic Balb/c mice.
• Preparation and Storage: 2 years -80°C in solvent; 3 years -20°C powder.

Structure

Related Product Information for DCC-2036, inhibitor

DCC-2036 (Rebastinib) is a conformational control Bcr-Abl inhibitor for Abl1(WT, IC50: 0.8 nM) and Abl1(T315I, IC50: 4 nM), also inhibits LYN, SRC, HCK, FGR, FLT3, KDR, and Tie-2, and low activity to c-Kit.