Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.
Abstract
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.
Type
Journal articleSubject
Binding SitesCalmodulin
Hydrogen-Ion Concentration
Substrate Specificity
Sulfonamides
Trifluoperazine
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https://hdl.handle.net/10161/4003Published Version (Please cite this version)
10.1021/bi1001213Publication Info
Colomer, Josep; Schmitt, Allison A; Toone, Eric J; & Means, Anthony R (2010). Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.
Biochemistry, 49(19). pp. 4244-4254. 10.1021/bi1001213. Retrieved from https://hdl.handle.net/10161/4003.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
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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