CCT241533 is a potent and selective inhibitor of CHK2 that potentiates the cytotoxicity of PARP inhibitors

Abstract

CHK2 is a checkpoint kinase involved in the ATM-mediated response to double-strand DNA breaks. Its potential as a drug target is still unclear, but inhibitors of CHK2 may increase the efficacy of genotoxic cancer therapies in a p53 mutant background by eliminating one of the checkpoints or DNA repair pathways contributing to cellular resistance. We report here the identification and characterization of a novel CHK2 kinase inhibitor, CCT241533. X-ray crystallography confirmed that CCT241533 bound to CHK2 in the ATP pocket. This compound inhibits CHK2 with an IC(50) of 3 nmol/L and shows minimal cross-reactivity against a panel of kinases at 1 μmol/L. CCT241533 blocked CHK2 activity in human tumor cell lines in response to DNA damage, as shown by inhibition of CHK2 autophosphorylation at S516, band shift mobility changes, and HDMX degradation. CCT241533 did not potentiate the cytotoxicity of a selection of genotoxic agents in several cell lines. However, this compound significantly potentiates the cytotoxicity of two structurally distinct PARP inhibitors. Clear induction of the pS516 CHK2 signal was seen with a PARP inhibitor alone, and this activation was abolished by CCT241533, implying that the potentiation of PARP inhibitor cell killing by CCT241533 was due to inhibition of CHK2. Consequently, our findings imply that CHK2 inhibitors may exert therapeutic activity in combination with PARP inhibitors.

Figure 1. (A) Chemical structure of CCT241533 B) CCT241533 is an ATP competitive inhibitor of CHK2.

CHK2 enzyme activity was measured at different concentrations of ATP. (C) X-ray crystal structure of CCT241533 bound to CHK2. Key interacting residues surrounding the bound compound are show as ‘ball and stick’ representation. The carbon atoms of CCT241533 are colored in cyan, nitrogens in blue and oxygens in red. The electron density corresponding to an mF_o-DF_c omit map contoured at 2.5 σ (brown mesh) is shown. The relative positions of the αC helix, glycine-rich loop, DFG motif (magenta) and hinge region are also indicated. (D) Schematic diagram highlighting the key interactions of CCT241533 with the active site residues of CHK2. Dotted lines represent hydrogen bonds whilst solid curved lines represent hydrophobic interactions.

Figure 2. (A) Characterization of the CHK2 activation pathway and p53 status in a panel of human tumor cell lines.

Cells were irradiated (5 Gy) and samples taken 4 hours post treatment. Similar results were obtained in a repeat experiment. (B) Effect of CCT241533 on etoposide induced biomarker changes. HT-29 or HeLa cells were pre-incubated with different concentrations of CCT241533 for 1 hour, followed by the addition of 50 μM etoposide or DMSO control and incubated for a further 5 hours. Similar results were obtained in repeat experiments.

Figure 3. Effect of Bleomycin treatments on CHK2 activation and cell cycle distribution.

(A) HT-29 cells were treated with 600 IU/ml bleomycin and samples taken at indicated time. (B) HT-29 cells were treated for 4 hours with various concentration of bleomycin. Similar results were obtained in repeat experiments. (C) A549 cells (wild type p53) were treated with either vehicle (control), bleomycin alone, CCT241533 alone or a combincation of bleomycin and CCT241533 (D) HeLa cells (p53 defective) were treated as (C). Cell cycle distribution at 24 h was determined by PI/FACS.

Figure 4. Effect of CCT241533 on PARP inhibitor cytotoxocity in different tumor cell lines.

(A) HeLa or (B) HT-29 cells were treated with AG14447 alone or in combination with a fixed dose (GI₅₀) of CCT241533. (C) HeLa cells treated with either olaparib alone or in combination with 3 μM CCT241533 in a growth delay assay. Values are mean±SD (n=4). Similar results were obtained in repeat experiments and are summarized in Table 1. (D) HeLa cells were treated with olaparib alone or in combination with a fixed dose of CCT241533 in a colony forming assay. Values are mean±SD (n=3). Similar results were obtained in repeat experiments.

Figure 5. (A) Effect of CCT241533 on olaparib cytotoxicity and CHK2 activation in HeLa cells.

HeLa cells were treated with olaparib alone (3 or 10μM) or in combination with 3 μM CCT241533 as indicated. Similar results were obtained in repeat experiments. Et = 50 μM etoposide. (B) Effects of olaparib and CCT241533 on apoptosis in HeLa cells. Cells were treated with olaparib or CCT241533 alone or in combination and apoptosis determined using Annexin V staining. FACS data shown in Figure S4B. Similar results were obtained in repeat experiments. (C) A schematic representation of the possible effects of CHK2 inhibition and PARP inhibition on the DNA damage, repair and survival pathways in cells.