Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.
Data(s) |
18/05/2010
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Formato |
4244 - 4254 |
Identificador |
http://www.ncbi.nlm.nih.gov/pubmed/20392081 Biochemistry, 2010, 49 (19), pp. 4244 - 4254 http://hdl.handle.net/10161/4003 1520-4995 |
Idioma(s) |
ENG en_US |
Relação |
Biochemistry 10.1021/bi1001213 Biochemistry |
Tipo |
Journal Article |
Cobertura |
United States |
Resumo |
We developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM. |
Palavras-Chave | #Binding Sites #Calmodulin #Hydrogen-Ion Concentration #Substrate Specificity #Sulfonamides #Trifluoperazine |