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

• Published in: Molecular cancer therapeutics 2004-10, Vol.3 (10), p.1221-1227
• Main Authors: Jiang, Xiuxian, Zhao, Baoguang, Britton, Robert, Lim, Lynette Y, Leong, Dan, Sanghera, Jasbinder S, Zhou, Bin-Bing S, Piers, Edward, Andersen, Raymond J, Roberge, Michel
• Format: Article
• Language: English
• ISSN: 1535-7163; 1538-8514

Inhibitors of the G sub(2) DNA damage checkpoint can selectively sensitize cancer cells with mutated p53 to killing by DNA-damaging agents. Isogranulatimide is a G sub(2) 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 (IC sub(50), 0.001 mu mol/L) and of the checkpoint kinase Chk1 (IC sub(50), 0.007 mu mol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC sub(50), 0.1 mu mol/L) but not protein kinase Cbeta. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3beta (IC sub(50), 0.5 mu mol/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 Glu super(85) and the amide nitrogen of Cys super(87). Unlike UCN-01, the basic N15 of isogranulatimide interacts with Glu super(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.