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 ℃ 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 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ 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.

BML-277 is novel, potent and highly selective inhibitor of the chk2 with the IC50 value of 15±6.9nM [1].

BML-277 has shown the ATP-competitive inhibition of chk2 with the Ki value of 37nM. BML-277 has also been exhibited to be a chk2 inhibitor with the IC50 value of 15±6.9nM and efficiently rescue T-cell populations from the apoptosis of radiation-induced in a concentration-dependent fashion with the EC50 of 3~7.6μM. The Km of ATP for chk2 is 99μM and assuming that intracellular ATP concentration is 10mM. Apart from these, BML-277 has shown the inhibition through docking into a homology model of chk2 ATP binging site [1].

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.