title: STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase.
abstract: STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) was synthesized, and its inhibitory properties were investigated both in vitro and in vivo. STO-609 inhibits the activities of recombinant CaM-KK alpha and CaM-KK beta isoforms, with K(i) values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activities. Comparison of the inhibitory potency of the compound against various protein kinases revealed that STO-609 is highly selective for CaM-KK without any significant effect on the downstream CaM kinases (CaM-KI and -IV), and the IC(50) value of the compound against CaM-KII is approximately 10 microg/ml. STO-609 inhibits constitutively active CaM-KK alpha (glutathione S-transferase (GST)-CaM-KK-(84-434)) as well as the wild-type enzyme. Kinetic analysis indicates that the compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 suppresses the Ca(2+)-induced activation of CaM-KIV in a dose-dependent manner. In agreement with this observation, the inhibitor significantly reduces the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 microg/ml (approximately 80% inhibitory rate). Taken together, these results indicate that STO-609 is a selective and cell-permeable inhibitor of CaM-KK and that it may be a useful tool for evaluating the physiological significance of the CaM-KK-mediated pathway in vivo as well as in vitro.
authors.affiliation: Department of Chemistry, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. tokumit@kms.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokumitsu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Inuzuka
authors.forename: Yumi
authors.initials: Y
authors.lastname: Ishikawa
authors.forename: Masahiko
authors.initials: M
authors.lastname: Ikeda
authors.forename: Ikutaro
authors.initials: I
authors.lastname: Saji
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kobayashi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015723:Gene Library; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007546:Isoquinolines; D007700:Kinetics; D053644:Naphthalimides; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013379:Substrate Specificity
chemical_list: D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D007546:Isoquinolines; D053644:Naphthalimides; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C458525:STO 609; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase