Abrogation of IR-induced apoptosis by PV1019 in mouse thymocytes. Wild-type (A) or Chk2(−/−) (B) mouse thymocytes were isolated and exposed to 5 Gy IR (or control) and incubated for 16 h in the presence or absence of 1 μM PV1019 (preincubated for 1 h). Cells were stained with propidium iodide and analyzed by flow cytometry. Representative graphs show the number of cells in G₁ and sub-G₁ cell cycle phases. The percentage of cells in either the G₁ or sub-G₁cell cycle phase is shown.

PV1019 is a selective and potent inhibitor of Chk2 in vitro. A, structures of NSC 109555 (Jobson et al., 2007) and PV1019. The differences are shown by dotted boxes. B, Chk2 was incubated with histone H1 and various concentrations of either NSC 109555 or PV1019 for the indicated times in an in vitro kinase assay. A representative gel of a Chk2 inhibitor experiment for PV1019 is shown. C, effect of increasing concentrations of PV1019 on Chk2 and GST-Cdc25C (top) and on Chk1 and histone H1 phosphorylation (bottom). Representative gels are shown. D, competitive inhibition of Chk2 by PV1019 with respect to ATP. The data are plotted using double reciprocal (Lineweaver-Burk) plots. E, effect of 1 mM ATP on PV1019-mediated inhibition of Chk2. Chk2 was incubated with histone H1 and various concentrations of PV1019 in an in vitro kinase assay containing 1 mM ATP. The percentage inhibition of Chk2-mediated histone H1 phosphorylation is represented graphically.

PV1019 causes growth inhibition in a panel of colon and ovarian human tumor cell lines that mimics the effect of Chk2 RNAi in ovarian human tumor cells. A, the human colon carcinoma cell lines from the NCI-60 were incubated with varying concentrations of PV1019 for 48 h. The cells were subjected to the sulforhodamine assay and GI₅₀ values were determined. B, the human tumor ovarian cell lines from the NCI-60 were incubated with varying concentrations of PV1019 for 48 h. The cells were subjected to the sulforhodamine B assay and GI₅₀ values were determined. C and D, Chk2 protein levels measured by Western blot in the colon (C) and ovarian (D) cell lines from the National Cancer Institute Developmental Therapeutics Program screen. E, OVCAR-4 cells were transfected with either control siRNA or CHEK2 siRNA and incubated for 96 h in 96-well plates. MTS was added to the plates and the absorbance at 495 nm was measured. Representative experiments are shown. F, OVCAR-8 cells were transfected with either control siRNA or CHEK2 siRNA and incubated for 96 h in 96-well plates. MTS was added to the plates, and the absorbance at 495 nm was measured. Representative experiments are shown.

Cocrystal structure of PV1019 bound in the ATP-binding pocket of the catalytic domain of Chk2. A, coordinates of PV1019 superimposed on the final 2F_o − F_c electron density maps at 2.07 Å resolution contoured at the 1σ level. Atoms are colored as carbon (gray), nitrogen (blue), and oxygen (red). B, crystal structure of the catalytic domain of Chk2 in complex with PV1019. The protein is illustrated in ribbons, and the inhibitor is shown in space-filling spheres with carbon (gray), nitrogen (blue), and oxygen (red). C, LIGPLOT schematic diagram of the molecular interactions of PV1019 with the ATP-binding pocket of Chk2. Residues involved in hydrogen bonding to the ligand are illustrated in stick format, water molecules are shown as cyan spheres, and hydrogen bonds are shown as green dashes. The “half-suns” represent hydrophobic interactions between the protein and ligand. D, superposition of the Chk2-PV1019 [red sticks, Protein Data Bank (PDB) code: 2w7x] complex with Chk2-NSC 109555 (green sticks, PDB code: 2w0j), and Chk2-ADP (cyan sticks, PDB code: 2cn5) complexes illustrating the binding conformations of the two inhibitors and ADP. The protein side chain coordinates are from the Chk2-PV1019 structure with carbon (gray), nitrogen (blue), oxygen (red), and sulfur (yellow).

Evidence for cellular inhibition of Chk2 by PV1019. A, abbreviated molecular interaction map depicting the phosphorylations of Chk2, Cdc25C, and HDMX after Chk2 activation. The conventions used for the interactions (Kohn, 1999) are as follows: the line with an open circle indicates enzymatic stimulation and phosphorylation, the line with an open arrowhead indicates general stimulation, and the open circle with a line crossing through indicates degradation. For details of the molecular interaction map for Chk2 see Pommier et al. (2006). B, OVCAR-4 cells were exposed to 10 Gy of IR followed by a 1-h incubation in the presence or absence of PV1019 or ABI (preincubated for 1 h). The levels of Chk2 phosphorylated on Ser⁵¹⁶ and total Chk2 were detected by Western blotting. Actin was used as a loading control. C, OVCAR-5 cells were exposed to PV1019 and TPT (or control) for 1 h. The levels of Chk2 phosphorylated on Ser⁵¹⁶ were detected by Western blotting. Actin was used as a loading control. D, OVCAR-5 cells were pretreated with TPT for 1 h, the drug was washed off, and the cells were exposed to PV1019 (or control) for an additional hour. The levels of Chk2 phosphorylated on Ser⁵¹⁶ were detected by Western blotting. Actin was used as loading control. E, MCF7 cells were exposed to either 1 μM TPT for 4 h or 10 Gy of IR followed by a 4-h incubation in the presence or absence of PV1019 (preincubated for 2.5 h). Western blotting using anti-HDMX antibodies was performed to measure the levels of HDMX. Representative gels are depicted, and the quantified levels of HDMX are shown in the graphs. F, MCF7 cells were exposed to 10 Gy of IR followed by a 1-h incubation in the presence or absence of PV1019 (preincubated for 2.5 h). The levels of Chk2 phosphorylated on Ser⁵¹⁶ were detected by Western blotting (top panel). The levels of Cdc25C phosphorylated on Ser⁵¹⁶ were detected by Western blotting (bottom panel). Actin was used as a loading control for each experiment.

PV1019 (PV) synergistically inhibits cellular proliferation with topoisomerase I inhibitors in ovarian human tumor cell lines and synergizes the effect of radiation in the brain tumor cell line, U251. Cells from the indicated cell lines OVCAR-5 (A) and OVCAR-4 (C and E) were incubated with various concentrations of TPT (A and C) and CPT (E) alone or coincubated with various concentrations of TPT or CPT and fixed concentrations of PV1019 for 48 h in 96-well plates. MTS was added to the plates and the absorbance at 495 nm was measured. Combination indices for A and C were calculated using CalcuSyn software, and the values were tabulated (B and D, respectively). F, U251 cells were seeded in six-well tissue culture plates, and radiation was delivered 16 h later. U251 cells were treated with 5 μM PV1019 1 h before different dose (0, 2, 4, 6, and 8 Gy) of IR. All cells including DMSO vehicle control were rinsed with PBS, and cultures were replaced with fresh growth medium 2 h after IR. The number of colonies was determined 10 days later, and survival curves were generated after normalizing for cell killing by PV1019 alone. Dose enhancement factor was calculated at a surviving fraction of 0.1. The average plating efficiency with 5 μM PV1019 alone was approximately 67%. Data represent the mean ± S.E. from four independent experiments in triplicate.