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 | ||
dc.identifier.eissn | 1520-4995 | |
dc.identifier.uri | ||
dc.language | eng | |
dc.language.iso | en_US | |
dc.publisher | American Chemical Society (ACS) | |
dc.relation.ispartof | Biochemistry | |
dc.relation.isversionof | 10.1021/bi1001213 | |
dc.relation.journal | Biochemistry | |
dc.subject | Binding Sites | |
dc.subject | Calmodulin | |
dc.subject | Hydrogen-Ion Concentration | |
dc.subject | Substrate Specificity | |
dc.subject | Sulfonamides | |
dc.subject | 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 | ||
pubs.begin-page | 4244 | |
pubs.end-page | 4254 | |
pubs.issue | 19 | |
pubs.organisational-group | Basic Science Departments | |
pubs.organisational-group | Biochemistry | |
pubs.organisational-group | Chemistry | |
pubs.organisational-group | Duke | |
pubs.organisational-group | Duke Cancer Institute | |
pubs.organisational-group | Institutes and Centers | |
pubs.organisational-group | School of Medicine | |
pubs.organisational-group | Trinity College of Arts & Sciences | |
pubs.publication-status | Published | |
pubs.volume | 49 |