Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.


Autoria(s): Colomer, J; Schmitt, AA; Toone, EJ; Means, AR
Data(s)

18/05/2010

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