Figure 5. CP466722 can be used as a molecular switch to regulate cellular ATM kinase activity.

(a) CP466722 potently inhibits ATM for at least 8h in culture. HeLa cells were preincubated with DMSO, 6μM CP466722 or 10μM KU55933 to reach the experimental start point (i.e. 0h). After preincubation, cells were exposed to mock-IR (control) or IR (2Gy) at the indicated time points (0–8h). Following irradiation cells were incubated at 37°C for 30min before being harvested (the times displayed in the figure represent the time of IR-exposure and do not include the 30min recovery time). To determine whether these compounds displayed a limited half-life with respect to inhibition of ATM kinase activity, ATM-intermolecular autophosphorylation at Serine 1981 and phosphorylation of Chk2 (Thr68) were determined by western blotting analysis (representative of several repeat experiments). (b) CP466722 and KU55933 display rapid and complete reversibility of ATM kinase inhibition in culture. HeLa cells were preincubated with DMSO, 6μM CP466722 or 10μM KU55933 to reach the experimental start point (i.e. 0h). After preincubation, the compounds were either left on the cells (+DMSO, +CP466722 & + KU55933) or removed (−DMSO, −CP466722 & − KU55933) and fresh media added. Following wash off, the cells were exposed to mock-IR (control) or IR (2Gy) at the indicated times (0–4h). Irradiated cells were incubated at 37°C for 30min before being harvested (the times displayed in the figure represent the time of IR-exposure and do not include the 30min recovery time). To determine whether the inhibition of ATM kinase activity had been reversed by removal of the compounds, ATM-intermolecular autophosphorylation at Serine1981 and phosphorylation of downstream ATM-targets were determined by western blotting analysis (representative of several repeat experiments).