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

dc.contributor.author

Colomer, Josep

dc.contributor.author

Schmitt, Allison A

dc.contributor.author

Toone, Eric J

dc.contributor.author

Means, Anthony R

dc.coverage.spatial

United States

dc.date.accessioned

2011-06-21T17:22:09Z

dc.date.issued

2010-05-18

dc.description.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.

dc.description.version

Version of Record

dc.identifier

http://www.ncbi.nlm.nih.gov/pubmed/20392081

dc.identifier.eissn

1520-4995

dc.identifier.uri

https://hdl.handle.net/10161/4003

dc.language

eng

dc.language.iso

en_US

dc.publisher

American Chemical Society (ACS)

dc.relation.ispartof

Biochemistry

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10.1021/bi1001213

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Biochemistry

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Binding Sites

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Calmodulin

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Hydrogen-Ion Concentration

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Substrate Specificity

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Sulfonamides

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Trifluoperazine

dc.title

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

dc.title.alternative
dc.type

Journal article

duke.date.pubdate

2010-5-18

duke.description.issue

19

duke.description.volume

49

pubs.author-url

http://www.ncbi.nlm.nih.gov/pubmed/20392081

pubs.begin-page

4244

pubs.end-page

4254

pubs.issue

19

pubs.organisational-group

Basic Science Departments

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Biochemistry

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Chemistry

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Duke

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Duke Cancer Institute

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Institutes and Centers

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School of Medicine

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Trinity College of Arts & Sciences

pubs.publication-status

Published

pubs.volume

49

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