Identification and Characterization of a Novel and Specific Inhibitor of the Ataxia-Telangiectasia Mutated Kinase ATM

Abstract

The serine/threonine protein kinase ATM signals to cell cycle and DNA repair components by phosphorylating downstream targets such as p53, CHK2, NBS1, and BRCA1. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia, which is characterized by hypersensitivity to ionizing radiation and a failure of cells to arrest the cell cycle after the induction of DNA double-strand breaks. It has thus been proposed that ATM inhibition would cause cellular radio-and chemosensitization. Through screening a small molecule compound library developed for the phosphatidylinositol 3-kinase-like kinase family, we identified an ATP-competitive inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), that inhibits ATM with an IC 50 of 13 nmol/L and a Ki of 2.2 nmol/L. KU-55933 shows specificity with respect to inhibition of other phosphatidylinositol 3-kinase-like kinases. Cellular inhibition of ATM by KU-55933 was demonstrated by the ablation of ionizing radiation-dependent phosphorylation of a range of ATM targets, including p53, ␥H2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase.

ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks

“…The ATM kinase inhibitor KU55933 (Hickson et al, 2004) inhibited ATM activation and this protein was not detected at the site of cleavage, but as expected Nbs1 still localized to the break, since this is ATM-independent. Furthermore, while ectopically expressed ATM localized to I-Ppo1 DSB in A-T cells, neither a kinase dead nor an S1981A mutant form of ATM bound to the DSB.…”

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors

“…Since ATM showed the relatively early activation following thymidine treatment in the presence or absence of Chk1 inhibitor, the effect of ATM inhibition on immortalized normal urothelial cells during DNA replication stress was then examined. hTERT-NHU cells were firstly treated with 10 M of the highly specific ATM inhibitor KU55933 (Hickson et al, 2004) for 2 hr and then incubated in the presence or absence of thymidine or gemcitabine for 24hr.…”

ATR-dependent radiation-induced γH2AX foci in bystander primary human astrocytes and glioma cells

“…The following agents were used to potentially abrogate bystander signalling pathways inducing gH2AX foci in nontargeted cells: 1% DMSO as a ROS scavenger, 0.5 mg/ml Filipin for abrogation of signalling through glycosphingolipidenriched membrane microdomains or lipid rafts, 10 mg/ml anti-TGF-beta 1 (all Sigma, Poole, UK), 5 mM ATM inhibitor KU-55933 (Hickson et al, 2004) and 5 mM DNA-PK inhibitor NU 7026 (Veuger et al, 2003) (both kindly provided by G Smith, KuDos, Cambridge, UK). Cells were incubated with the inhibitors 10 min before irradiation and the inhibitors were present during irradiation and subsequent incubation time.…”

“…We found that both cell types were capable of generating signals inducing gH2AX foci in bystander cells for a prolonged time up to 24-48 h after irradiation. The ATM inhibitor KU-55933 (Hickson et al, 2004) and the DNA-PK inhibitor NU 7026 (Veuger et al, 2003) could not suppress the induction of gH2AX foci in bystander cells suggesting the involvement of ATR in H2AX phosphorylation in these cells. This could be confirmed by the finding that ATR-mutated cells did not show the induction of gH2AX foci in bystander cells.…”