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

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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.





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Colomer, Josep, Allison A Schmitt, Eric J Toone and Anthony R Means (2010). Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist. Biochemistry, 49(19). pp. 4244–4254. 10.1021/bi1001213 Retrieved from

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Eric John Toone

Professor Emeritus of Chemistry

Dr. Toone is a physical organic chemist who studies relationships between structure and activity in the context of biology. Currently active programs exist in biocatalysis/applied enzymology, ligand binding and the activity of water, and the synthesis of novel donors of nitric oxide. The study of these problems makes use of synthetic organic chemistry, traditional enzymology, isothermal titration microcalorimetry, and the techniques of directed evolution.

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