Inhibition of purified chk2

Activity of chk2 inhibitor was determined by incubating BML-277 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 were incubated at 37 °C for 3 hrs, and the peptide substrate was captured on streptavidin conjugated to agarose beads. The agarose beads were washed repeatedly with a 0.1% solution of Tween-20 in phosphate-buffered saline, pH 7.4. Enzyme activity at different BML-277 concentrations was determined by measuring the amount of radioactive phosphate bound to the substrate peptide by scintillation counting. In the kinetic experiment, ATP concentration was varied while the ratio between unlabeled and [γ-33P] labeled ATP was kept constant. Reactions were stopped at different time points by addition of 50 mM cold ATP and samples were kept on ice during further processing.

Cell lines

Human T-cells

Preparation method

The solubility of this compound in DMSO is > 18.2 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 °C for several months.

Reacting condition

25 hrs

Applications

BML-277 efficiently prevented T-cell populations from radiation-induced apoptosis in a concentration-dependent manner, with the EC50 value ranging from 3 to 7.6 μM.

References:

[1]. Arienti KL1, Brunmark A, Axe FU, McClure K, Lee A, Blevitt J, Neff DK, Huang L, Crawford S, Pandit CR, Karlsson L, Breitenbucher JG. Checkpoint kinase inhibitors: SAR and radioprotective properties of a series of 2-arylbenzimidazoles. J Med Chem. 2005 Mar 24;48(6):1873-85.