Inhibition of the G2 DNA Damage Checkpoint and of Protein Kinases Chk1 and Chk2 by the Marine Sponge Alkaloid Debromohymenialdisine

Abstract

Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G 2 phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G 2 checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G 2 checkpoint inhibitors. It inhibited the G 2 checkpoint with an IC 50 of 8 M and showed moderate cytotoxicity (IC 50 ‫؍‬ 25 M) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC 50 ‫؍‬ 3 M) and Chk2 (IC 50 ‫؍‬ 3.5 M) but not ataxiatelangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.

Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor

“…6 DBQ is an ATP-competitive inhibitor with an IC 50 below 183 nM for Chk2 but it is not specific for Chk2. 10,25,29 Overall, the global structures of the protein molecules in the two cocrystal structures are almost identical. The binding mode of DBQ (see Fig.…”

“…9,20,22 Additionally, because of the role of p53 in the initiation of apoptosis in response to DNA damage, inhibition of Chk2 in normal cells may also protect normal tissues, 1,23 which may reduce side effects due to chemotherapy or radiation therapy. 24 Because only a few specific inhibitors of Chk2 have been identified to date, [24][25][26][27][28][29][30] the discovery of additional inhibitors remains of considerable interest. A novel inhibitor of Chk2, NSC 109555 [ Fig.…”

The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe

“…This may explain why, at least in some cases, tumor cells are particularly sensitive to the induction of mitotic catastrophe. Chemical inhibitors of checkpoint kinases (such as DBH used in this study) might sensitize tumor cells to the induction of mitotic catastrophe (and subsequent apoptosis) (Graves et al, 2000;Curman et al, 2001;Hirose et al, 2001;Hu et al, 2001). Indeed, inhibition of Chk2 (and Chk1) by 7-hydroxystaurosporine (UCN-01) abrogates the g-radiation-induced G2-M checkpoint and thus enhances g-radiation-induced apoptosis (Yu et al, 2002).…”

“…Mitogenic effects were only observed for DBH as well as for its dibromo derivative. However, no such effect was observed when hymenial moiety of DBH was modified (Figure 4d) and the inhibition of check point kinase is lost (Barrios Sosa et al, 2000;Curman et al, 2001). In conclusion, genetic or chemical inhibition of Chk2 can abrogate the fusion-induced block in the prophase.…”

“…DN Chk2 had no inhibitory effect on the p53S15P phosphorylation (Figure 4a,b). A phenocopy of this genetic manipulation could be obtained by pharmacological inhibition of Chk2 using debromohymenialdisine (DBH) (Barrios Sosa et al, 2000;Curman et al, 2001), which acts in a dose-dependent manner to increase the frequency of syncytial mitosis (Figure 4c). To address the specificity of this effect, we took advantage of several chemical derivatives of DBH.…”