Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.
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.
Colomer
Josep
J
Department of Pharmacology and Cancer Biology, Duke University Medical Center, P.O. Box 3813, Durham, North Carolina 27710, USA.
Schmitt
Allison A
AA
Toone
Eric J
EJ
Means
Anthony R
AR
D001665:Binding Sites; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity; D013449:Sulfonamides / Q000737:chemistry; D014268:Trifluoperazine / Q000737:chemistry
D002147:Calmodulin; D013449:Sulfonamides; D014268:Trifluoperazine; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide
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