BML-277  (Synonyms: Chk2 Inhibitor II)

Activity of inhibitors of chk2 is determined by incubating inhibitory compounds with recombinant full-length chk2: 5 nM recombinant human Chk2, 50 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl₂, 25 μM synthetic peptide substrate (biotin-SGLYRSPSMPENLNRPR, 1 μM ATP, 50 μCi/mL [γ-³³P] ATP, and a protease inhibitor mixture. The reaction mixtures are incubated at 37°C for 3 h, and the peptide substrate is captured on streptavidin conjugated to agarose beads. The agarose beads are washed repeatedly with a 0.1% solution of Tween-20 in phosphate-buffered saline, pH 7.4. Enzyme activity at different BML-277 concentrations (6.25, 12.5, 25, 50, 100, and 200 nM) is determined by measuring the amount of radioactive phosphate bound to the substrate peptide by scintillation counting. In kinetic experiments ATP concentration is varied while the ratio between unlabeled and [γ-³³P] labeled ATP is kept constant. Reactions are stopped at different time points by addition of 50 mM cold ATP and samples are kept on ice during further processing^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

To determine the radioprotective effect of Chk2 inhibitors, purified T-cells are incubated at 100 000 cells per well in BML-277 (10^2.5 nM, 1 μM, 10^0.5 μM, 10 μM, and 10^1.5 μM) or vehicle (DMSO) at varying concentrations in 96-well stripwells for 1 h. Cells are then exposed to a dose of 0 or 10 Gy gamma irradiation from a ¹³⁷Cs source at a dose rate of 3.65 Gy/min and then returned to the incubator for a further 24 h. Cells are stained with Annexin V-FITC and propidium iodide, according to the manufacturers protocol. Apoptotic and surviving cells are quantitated with a FACSCalibur FACS machine. Data are reported as percent recovery-or the number of survivors from treatment groups minus the number of cells surviving in the irradiated control group divided by the number of surviving cells in the untreated control groups^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Arienti KL, et al. Checkpoint kinase inhibitors: SAR and radioprotective properties of a series of 2-arylbenzimidazoles. J Med Chem. 2005 Mar 24;48(6):1873-85.  [Content Brief]