Rebastinib (DCC-2036)

BCR

553, 59

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.

DCC-2036 is currently in Phase I/II clinical trials in patients with chronic myeloid leukemia.

<=100 mg/kg

0.8 nM, 0.8 nM, 0.8 nM, 0.8 nM, 0.8 nM, 0.8 nM

In a mouse allograft model bearing Ba/F3-Bcr-Abl1T315I leukemia cells, DCC-2036 treatment by oral gavage at 100 mg/kg once daily, effectively inhibits Bcr-Abl1 signaling and significantly prolongs mouse survival. [1]

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 (ca. 4 units), lactate dehydrogenase (ca. 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 % octyl-glucoside. Fifty uL of the inhibitor mixture is mixed with 50 uL of the above Abl1 kinase/coupled assay components mixture, which is then incubated at 30C for 2 hours before 2 uL of 25 mM ATP (500 uM, final) is added to start the reaction. The reaction is recorded every 2 minutes for 2.5 hours at 30C 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.

DMSO 111 mg/mL, Water <1 mg/mL, Ethanol 16 mg/mL

1-(3-tert-butyl-1-(quinolin-6-yl)-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea