Abstract
Chk2 is a protein kinase involved in the ATM-dependent checkpoint pathway (http://discover.nci.nih.gov/mim). This pathway is activated by genomic instability and DNA damage and results in either cell cycle arrest, to allow DNA repair to occur, or cell death (apoptosis). Chk2 is activated by ATM-mediated phosphorylation and autophosphorylation and in turn phosphorylates its downstream targets (Cdc25A, Cdc25C, BRCA1, p53, Hdmx, E2F1, PP2A, and PML). Inhibition of Chk2 has been proposed to sensitize p53-deficient cells as well as protect normal tissue after exposure to DNA-damaging agents. We have developed a drug-screening program for specific Chk2 inhibitors using a fluorescence polarization assay, immobilized metal ion affinity-based fluorescence polarization (IMAP). This assay detects the degree of phosphorylation of a fluorescently linked substrate by Chk2. From a screen of over 100,000 compounds from the NCI Developmental Therapeutics Program, we identified a bis-guanylhydrazone [4,4′-diacetyldiphenylureabis(guanylhydrazone); NSC 109555] as a lead compound. In vitro data show the specific inhibition of Chk2 kinase activity by NSC 109555 using in vitro kinase assays and kinase-profiling experiments. NSC 109555 was shown to be a competitive inhibitor of Chk2 with respect to ATP, which was supported by docking of NSC 109555 into the ATP binding pocket of the Chk2 catalytic domain. The potency of NSC 109555 was comparable with that of other known Chk2 inhibitors, such as debromohymenialdisine and 2-arylbenzimidazole. These data define a novel chemotype for the development of potent and selective inhibitors of Chk2. This class of drugs may ultimately be useful in combination with current DNA-damaging agents used in the clinic.
Footnotes
-
This project was funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO12400. This research was supported in part by the Developmental Therapeutics Program in the Division of Cancer Treatment and Cancer Diagnosis and by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
-
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
-
doi:10.1124/mol.107.035832.
-
ABBREVIATIONS: DBH, debromohymenialdisine; NSC 109555, 4,4′-diacetyldiphenylurea-bis(guanylhydrazone); UCN-01, 7-hydroxystaurosporine; DMSO, dimethyl sulfoxide; PDB, Protein Data Bank; NSC 69432/MGBG, 2-[[(1E)-1-(diaminomethylidenehydrazinylidene)propan-2-ylidene] amino]guanidine; PAGE, polyacrylamide gel electrophoresis; VRX0466617, 3-hydroxy-N-isopropyl-5-(4-phenoxy-phenylamino)isothiazole-4-carboximidamine; Go6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole.
- Received March 7, 2007.
- Accepted July 6, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|