title: A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex.
abstract: The Ca(2+) dependent interaction between troponin I (cTnI) and troponin C (cTnC) triggers contraction in heart muscle. Heart failure is characterized by a decrease in cardiac output, and compounds that increase the sensitivity of cardiac muscle to Ca(2+) have therapeutic potential. The Ca(2+)-sensitizer, levosimendan, targets cTnC; however, detailed understanding of its mechanism has been obscured by its instability. In order to understand how this class of positive inotropes function, we investigated the mode of action of two fluorine containing novel analogs of levosimendan; 2',4'-difluoro(1,1'-biphenyl)-4-yloxy acetic acid (dfbp-o) and 2',4'-difluoro(1,1'-biphenyl)-4-yl acetic acid (dfbp). The affinities of dfbp and dfbp-o for the regulatory domain of cTnC were measured in the absence and presence of cTnI by NMR spectroscopy, and dfbp-o was found to bind more strongly than dfbp. Dfbp-o also increased the affinity of cTnI for cTnC. Dfbp-o increased the Ca(2+)-sensitivity of demembranated cardiac trabeculae in a manner similar to levosimendan. The high resolution NMR solution structure of the cTnC-cTnI-dfbp-o ternary complex showed that dfbp-o bound at the hydrophobic interface formed by cTnC and cTnI making critical interactions with residues such as Arg147 of cTnI. In the absence of cTnI, docking localized dfbp-o to the same position in the hydrophobic groove of cTnC. The structural and functional data reveal that the levosimendan class of Ca(2+)-sensitizers work by binding to the regulatory domain of cTnC and stabilizing the pivotal cTnC-cTnI regulatory unit via a network of hydrophobic and electrostatic interactions, in contrast to the destabilizing effects of antagonists such as W7 at the same interface.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Robertson
authors.forename: Yin-Biao
authors.initials: YB
authors.lastname: Sun
authors.forename: Monica X
authors.initials: MX
authors.lastname: Li
authors.forename: Brian D
authors.initials: BD
authors.lastname: Sykes
mesh_terms: D000818:Animals; D002118:Calcium; D006801:Humans; D006835:Hydrazones / Q000737:chemistry* / Q000494:pharmacology*; D066298:In Vitro Techniques; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011724:Pyridazines / Q000737:chemistry* / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D000077464:Simendan; D055672:Static Electricity; D013329:Structure-Activity Relationship; D017186:Titrimetry; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D006835:Hydrazones; D008024:Ligands; D011724:Pyridazines; D019209:Troponin C; D019210:Troponin I; D000077464:Simendan; D002118:Calcium
references.citation: Curr Top Med Chem. 2006;6(14):1473-82
references.pmid: 16918463
references.citation: J Biomol NMR. 1994 Sep;4(5):603-14
references.pmid: 22911360
references.citation: Proc Natl Acad Sci U S A. 2000 May 9;97(10):5140-5
references.pmid: 10792039
references.citation: Eur Biophys J. 2002 Jul;31(4):245-56
references.pmid: 12122471
references.citation: J Comput Chem. 2009 Dec;30(16):2785-91
references.pmid: 19399780
references.citation: J Mol Cell Cardiol. 2000 Mar;32(3):479-91
references.pmid: 10731446
references.citation: J Physiol. 2009 Jan 15;587(1):155-63
references.pmid: 19015190
references.citation: J Biomol NMR. 1995 Nov;6(3):277-93
references.pmid: 8520220
references.citation: Biochem Biophys Res Commun. 2008 Apr 25;369(1):88-99
references.pmid: 18162171
references.citation: Br J Pharmacol. 2007 Apr;150(7):826-8
references.pmid: 17325657
references.citation: Biochemistry. 2000 Oct 17;39(41):12731-8
references.pmid: 11027154
references.citation: J Biol Chem. 1994 Nov 18;269(46):28584-90
references.pmid: 7961805
references.citation: Biochemistry. 1997 Oct 7;36(40):12138-46
references.pmid: 9315850
references.citation: Biochemistry. 2008 Jul 15;47(28):7485-95
references.pmid: 18570382
references.citation: Nature. 2003 Jul 3;424(6944):35-41
references.pmid: 12840750
references.citation: Eur J Biochem. 2000 Nov;267(22):6665-72
references.pmid: 11054120
references.citation: J Biomol NMR. 1999 Mar;13(3):289-302
references.pmid: 10212987
references.citation: J Biol Chem. 2002 Aug 23;277(34):31124-33
references.pmid: 12060657
references.citation: J Am Chem Soc. 2006 Oct 4;128(39):12910-6
references.pmid: 17002387
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Aug;60(Pt 8):1355-63
references.pmid: 15272157
references.citation: Biochemistry. 2009 Jun 23;48(24):5541-52
references.pmid: 19419198
references.citation: Circ Res. 1995 Jul;77(1):107-13
references.pmid: 7788868
references.citation: Eur J Pharmacol. 2004 Feb 13;486(1):1-8
references.pmid: 14751401
references.citation: J Biol Chem. 1994 Sep 23;269(38):23731-5
references.pmid: 8089144
references.citation: J Biomol NMR. 2003 Dec;27(4):313-21
references.pmid: 14512729
references.citation: Arch Pharm Res. 2010 Jan;33(1):25-46
references.pmid: 20191341
references.citation: Prog Cardiovasc Dis. 2004 Nov-Dec;47(3):159-76
references.pmid: 15736582
references.citation: Drug Metab Dispos. 1990 Nov-Dec;18(6):1060-4
references.pmid: 1981513
references.citation: Proc Natl Acad Sci U S A. 2005 Apr 5;102(14):5038-43
references.pmid: 15784741
references.citation: Chembiochem. 2004 May 3;5(5):637-43
references.pmid: 15122635
references.citation: Proteins. 2003 Feb 15;50(3):496-506
references.pmid: 12557191
references.citation: Methods Mol Biol. 2004;278:353-78
references.pmid: 15318003
references.citation: J Biol Chem. 2001 Mar 23;276(12):9337-43
references.pmid: 11113122
references.citation: J Biol Chem. 1998 Apr 3;273(14):8153-60
references.pmid: 9525919
references.citation: Biochemistry. 1985 Jan 15;24(2):544-50
references.pmid: 2983759
references.citation: J Biomol NMR. 1996 Dec;8(4):477-86
references.pmid: 9008363
references.citation: J Mol Cell Cardiol. 1995 Sep;27(9):1859-66
references.pmid: 8523447
references.citation: J Mol Cell Cardiol. 2010 May;48(5):925-33
references.pmid: 20116385
references.citation: Eur J Pharmacol. 2004 Feb 13;486(1):67-74
references.pmid: 14751410
references.citation: FEBS Lett. 1994 Aug 15;350(1):87-90
references.pmid: 8062930
references.citation: J Biomol NMR. 1996 Dec;8(4):492-8
references.pmid: 20859780
references.citation: Mol Cell Biochem. 2004 Nov;266(1-2):87-107
references.pmid: 15646030
references.citation: Biochemistry. 1994 Dec 6;33(48):14434-42
references.pmid: 7981203
references.citation: IUBMB Life. 2002 Dec;54(6):323-33
references.pmid: 12665242
references.citation: J Muscle Res Cell Motil. 2004;25(7):559-79
references.pmid: 15711886
references.citation: Biochemistry. 1999 Jun 29;38(26):8289-98
references.pmid: 10387074
references.citation: Circulation. 2006 Jan 17;113(2):305-15
references.pmid: 16418450
references.citation: J Cardiovasc Pharmacol. 1995 May;25(5):794-801
references.pmid: 7630157