Inhibition of Chk1 by the G(2) DNA damage checkpoint inhibitor isogranulatimide

Inhibitors of the G(2) DNA damage checkpoint can selectively sensitize cancer cells with mutated p53 to killing by DNA-damaging agents. Isogranulatimide is a G2 checkpoint inhibitor containing a unique indole/maleimide/ imidazole skeleton identified in a phenotypic cell-based screen; however, the mechanism of action of isogranulatimide is unknown. Using natural and synthetic isogranulatimide analogues, we show that the imide nitrogen and a basic nitrogen at position 14 or 15 in the imidazole ring are important for checkpoint inhibition. Isogranulatimide shows structural resemblance to the aglycon of UCN-01, a potent bisindolemaleimide inhibitor of protein kinase Cbeta (IC50, 0.001 mumol/L) and of the checkpoint kinase Chk1 (IC50, 0.007 mumol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC50, 0.1 mumol/L) but not protein kinase Cbeta. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3beta (IC50, 0.5 mumol/L). We determined the crystal structure of the Chk1 catalytic domain complexed with isogranulatimicle. Like UCN-01, isogranulatimide binds in the ATP-binding pocket of Chk1 and hydrogen bonds with the backbone carbonyl oxygen of Glu(85) and the amide nitrogen Of Cys(87). Unlike UCN-01, the basic N15 of isogranulatimide interacts with Glu(17), causing a conformation change in the kinase glycine-rich loop that may contribute importantly to inhibition. The mechanism by which isogranulatimide inhibits Chk1 and its favorable kinase selectivity profile make it a promising candidate for modulating checkpoint responses in tumors for therapeutic benefit.