title: 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.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, P.O. Box 3813, Durham, North Carolina 27710, USA.
authors.forename: Josep
authors.initials: J
authors.lastname: Colomer
authors.forename: Allison A
authors.initials: AA
authors.lastname: Schmitt
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Toone
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Means
mesh_terms: 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
chemical_list: D002147:Calmodulin; D013449:Sulfonamides; D014268:Trifluoperazine; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide
references.citation: J Biol Chem. 1999 Jul 16;274(29):20215-22
references.pmid: 10400638
references.citation: Biochem J. 2006 Feb 1;393(Pt 3):797-805
references.pmid: 16201971
references.citation: Science. 1998 Jul 24;281(5376):533-8
references.pmid: 9677190
references.citation: J Mol Recognit. 1999 Jan-Feb;12(1):3-18
references.pmid: 10398392
references.citation: Biophys Chem. 2005 Apr 1;115(2-3):89-97
references.pmid: 15752588
references.citation: J Biol Chem. 2004 Apr 9;279(15):15411-9
references.pmid: 14754892
references.citation: J Biol Chem. 2002 Dec 27;277(52):50403-8
references.pmid: 12393914
references.citation: J Biol Chem. 1995 Mar 10;270(10):5418-26
references.pmid: 7890656
references.citation: J Biol Chem. 2005 Jun 10;280(23):21804-9
references.pmid: 15831494
references.citation: Mini Rev Med Chem. 2006 Dec;6(12):1383-9
references.pmid: 17168814
references.citation: Cell Metab. 2008 May;7(5):377-88
references.pmid: 18460329
references.citation: Methods Enzymol. 1999;306:145-53
references.pmid: 10432452
references.citation: Endocr Rev. 2003 Dec;24(6):719-36
references.pmid: 14671000
references.citation: Cancer Res. 1989 May 1;49(9):2398-404
references.pmid: 2706627
references.citation: Mol Endocrinol. 2000 Aug;14(8):1125-36
references.pmid: 10935538
references.citation: J Mol Biol. 1997 Mar 14;266(5):1050-62
references.pmid: 9086281
references.citation: J Biol Chem. 1987 Feb 25;262(6):2746-9
references.pmid: 3818620
references.citation: J Biol Chem. 1992 Feb 15;267(5):2864-7
references.pmid: 1346611
references.citation: Cell. 1986 Nov 7;47(3):423-31
references.pmid: 3533275
references.citation: Cell. 1986 Nov 7;47(3):381-9
references.pmid: 2876781
references.citation: Biochemistry. 1994 Dec 27;33(51):15259-65
references.pmid: 7803388
references.citation: Cell. 1991 Jun 14;65(6):949-59
references.pmid: 2044154
references.citation: Methods Mol Biol. 2009;535:201-8
references.pmid: 19377994
references.citation: J Biol Chem. 2006 Oct 27;281(43):32207-16
references.pmid: 16943194
references.citation: Biochemistry. 2007 Dec 25;46(51):14899-906
references.pmid: 18052208
references.citation: Protein Expr Purif. 2003 Aug;30(2):230-7
references.pmid: 12880772
references.citation: Cancer Res. 1986 Jan;46(1):229-32
references.pmid: 3940193
references.citation: J Mol Biol. 1998 Feb 13;276(1):165-76
references.pmid: 9514729
references.citation: Methods Enzymol. 1983;102:185-94
references.pmid: 6139736
references.citation: J Biol Chem. 2006 Sep 1;281(35):25336-43
references.pmid: 16835226
references.citation: J Biol Chem. 2001 Apr 27;276(17):14083-91
references.pmid: 11278815
references.citation: Annu Rev Genet. 2001;35:647-72
references.pmid: 11700296
references.citation: Nat Rev Mol Cell Biol. 2000 Oct;1(1):11-21
references.pmid: 11413485
references.citation: Nucleic Acids Res. 1996 Jul 1;24(13):2519-24
references.pmid: 8692690
references.citation: Annu Rev Biochem. 1998;67:821-55
references.pmid: 9759505
references.citation: Trends Cell Biol. 2000 Aug;10(8):322-8
references.pmid: 10884684
references.citation: Cell. 1982 Jan;28(1):41-50
references.pmid: 7066986
references.citation: Biochemistry. 2001 Nov 20;40(46):13925-32
references.pmid: 11705382
references.citation: Biochim Biophys Acta. 2009 Jun;1793(6):1068-77
references.pmid: 19133300
references.citation: Proc Natl Acad Sci U S A. 1986 Dec;83(24):9497-501
references.pmid: 3467320
references.citation: Biochemistry. 2007 Apr 3;46(13):4017-27
references.pmid: 17352496
references.citation: Neuromolecular Med. 2006;8(3):279-306
references.pmid: 16775381
references.citation: Eur J Cancer Clin Oncol. 1989 Jan;25(1):19-26
references.pmid: 2920766
references.citation: J Struct Funct Genomics. 2000;1(1):8-14
references.pmid: 12836676
references.citation: Biochem Pharmacol. 1982 Jul 1;31(13):2217-26
references.pmid: 6127079
references.citation: FEBS Lett. 2003 Jul 3;546(1):113-20
references.pmid: 12829246
references.citation: J Biol Chem. 1998 Nov 27;273(48):31880-9
references.pmid: 9822657
references.citation: Curr Biol. 2003 Aug 5;13(15):1299-305
references.pmid: 12906789
references.citation: J Biol Chem. 2005 Aug 12;280(32):29060-6
references.pmid: 15980064
references.citation: Nat Struct Biol. 1994 Nov;1(11):795-801
references.pmid: 7634090
references.citation: Cell Metab. 2005 Jul;2(1):21-33
references.pmid: 16054096
references.citation: Science. 2005 Dec 9;310(5754):1642-6
references.pmid: 16308421