Inhibition of Chk1 by the G2 DNA damage checkpoint inhibitor isogranulatimide

Abstract

Abstract Inhibitors of the G2 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 Cβ (IC50, 0.001 μmol/L) and of the checkpoint kinase Chk1 (IC50, 0.007 μmol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC50, 0.1 μmol/L) but not protein kinase Cβ. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3β (IC50, 0.5 μmol/L). We determined the crystal structure of the Chk1 catalytic domain complexed with isogranulatimide. Like UCN-01, isogranulatimide binds in the ATP-binding pocket of Chk1 and hydrogen bonds with the backbone carbonyl oxygen of Glu85 and the amide nitrogen of Cys87. Unlike UCN-01, the basic N15 of isogranulatimide interacts with Glu17, 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.