Inhibition of Chk1 by the G2 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 G(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 C beta (IC(50), 0.001 micromol/L) and of the checkpoint kinase Chk1 (IC(50), 0.007 micromol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC(50), 0.1 micromol/L) but not protein kinase C beta. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3beta (IC(50), 0.5 micromol/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(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.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Xiuxian
X
Jiang
Baoguang
B
Zhao
Robert
R
Britton
Lynette Y
LY
Lim
Dan
D
Leong
Jasbinder S
JS
Sanghera
Bin-Bing S
BB
Zhou
Edward
E
Piers
Raymond J
RJ
Andersen
Michel
M
Roberge
D000970:Antineoplastic Agents / Q000494:pharmacology*; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D016195:G2 Phase; D005973:Glutamine / Q000737:chemistry; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007211:Indoles / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D008301:Maleimides / Q000037:antagonists & inhibitors; D008956:Models, Chemical; D008958:Models, Molecular; D009584:Nitrogen / Q000737:chemistry; D010641:Phenotype; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D011494:Protein Kinases / Q000378:metabolism* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D013997:Time Factors
D000970:Antineoplastic Agents; D007093:Imidazoles; D007211:Indoles; D008301:Maleimides; C116644:isogranulatimide; D005973:Glutamine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D038362:Glycogen Synthase Kinase 3; D003545:Cysteine; C088060:bisindolylmaleimide; D009584:Nitrogen