RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Fase G2 , Imidazóis/farmacologia , Indóis/farmacologia , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Domínio Catalítico , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Cisteína/química , Relação Dose-Resposta a Droga , Glutamina/química , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Indóis/antagonistas & inibidores , Concentração Inibidora 50 , Maleimidas/antagonistas & inibidores , Modelos Químicos , Modelos Moleculares , Nitrogênio/química , Fenótipo , Ligação Proteica , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
The antimitotic sponge tripeptide hemiasterlin (1) and a number of structural analogues have been synthesized and evaluated in cell-based assays for both cytotoxic and antimitotic activity in order to explore the SAR for this promising anticancer drug lead. One synthetic analogue, SPA110 (8), showed more potent in vitro cytotoxicty and antimitotic activity than the natural product hemiasterlin (1), and consequently it has been subjected to thorough preclinical evaluation and targeted for clinical evaluation. The details of the synthesis of hemiasterlin (1) and the analogues and a discussion of how their biological activities vary with their structures are presented in this paper.