mitoguazone and diacetyldiphenylurea-bisguanylhydrazone

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.

Topics: Adenosine Triphosphate; Carbanilides; Catalytic Domain; Checkpoint Kinase 2; Fluorescence Polarization Immunoassay; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Recombinant Proteins

The interactions of naturally occurring polyamines: putrescine, spermidine and spermine, with anticancer bis-guanylhydrazones: methylglyoxal-bis(guanylhydrazone) (MGBG) and 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (DDUG) were investigated at the level of mitochondrial membrane. The effects of bis-guanylhydrazones on intact rat liver mitochondria were readily prevented or reversed by polyamines and these interactions were also affected by the mitochondrial transmembrane potential. Magnesium cations enhanced the protective action of polyamines. The data indicate that competition exists between the essential anticancer bis(guanylhydrazone) and polyamines for low affinity negatively charged binding sites at the outer surface of inner mitochondrial membrane. The study of drug interactions was extended to the level of isolated tumor mitochondria from rat HTC hepatoma and murine L1210 leukemia cells. A complicated pattern of interactions between the anticancer bis-guanylhydrazones and phenethylbiguanide was obtained.

Topics: Animals; Antineoplastic Agents; Carbanilides; Dinitrophenols; Drug Interactions; Guanidines; In Vitro Techniques; Intracellular Membranes; Leukemia L1210; Liver Neoplasms, Experimental; Mitochondria; Mitochondria, Liver; Mitoguazone; Oxygen Consumption; Polyamines; Putrescine; Rats; Spermidine; Spermine

The ability of methylglyoxal-bis(guanylhydrazone) (MGBG) and 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) to interact with the hypoglycemic agent, phenethylbiguanide (DBI), in affecting the bioenergetic functions of isolated rat liver mitochondria was studied. DBI was found to increase markedly the inhibitory effect of either 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) or MGBG on respiration of isolated rat liver mitochondria. Conversely, these bis(guanylhydrazones) enhanced the inhibitory potency of DBI and increased the apparent affinity of mitochondria for the drug. As with MGBG and 4,4'-diacetyldiphenylurea-bis(guanylhydrazone), the potassium cationophore, valinomycin, increased the sensitivity of mitochondrial respiration to DBI. It is suggested that the enhancement of bis(guanylhydrazone) inhibition of mitochondrial respiration by DBI involves inhibition of proton fluxes across the inner mitochondrial membrane and the subsequent alkalinization of the mitochondrial matrix. This drug interaction was extended to the level of antiproliferative activity in which DBI was found to potentiate the growth-inhibitory effects of MGBG on murine L1210 leukemia in vivo.

Topics: Animals; Carbanilides; Dose-Response Relationship, Drug; Drug Synergism; Guanidines; Leukemia L1210; Mitochondria, Liver; Mitoguazone; Oxygen Consumption; Phenformin; Polyamines; Rats; Valinomycin

Topics: Adenosine Triphosphate; Animals; Carbanilides; Dose-Response Relationship, Drug; Guanidines; Microscopy, Electron; Mitochondria, Liver; Mitochondrial Swelling; Mitoguazone; Monoamine Oxidase; Oxygen Consumption; Rats

Topics: Animals; Autophagy; Carbanilides; Cell Division; Cells, Cultured; Leukemia L1210; Mice; Microscopy, Electron; Mitochondria; Mitoguazone; Phagocytosis