title: Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue.
abstract: BACKGROUND AND PURPOSE Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I(f)  or I(h) ). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels. EXPERIMENTAL APPROACH HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings. KEY RESULTS In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I(f)  by 67% at -120 mV, while MEL57A (3 µM) reduced I(f)  by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I(h)  by 60% at -80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization. CONCLUSIONS Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.
authors.affiliation: CIMMBA, Department of Pharmacology, University of Florence, Firenze, Italy Department of Pharmaceutical Sciences, University of Florence, Florence, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Del Lungo
authors.forename: Michele
authors.initials: M
authors.lastname: Melchiorre
authors.forename: Luca
authors.initials: L
authors.lastname: Guandalini
authors.forename: Laura
authors.initials: L
authors.lastname: Sartiani
authors.forename: Alessandro
authors.initials: A
authors.lastname: Mugelli
authors.forename: Istvan
authors.initials: I
authors.lastname: Koncz
authors.forename: Tamas
authors.initials: T
authors.lastname: Szel
authors.forename: Andras
authors.initials: A
authors.lastname: Varro
authors.forename: Maria Novella
authors.initials: MN
authors.lastname: Romanelli
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Cerbai
mesh_terms: D000818:Animals; D001552:Benzazepines / Q000494:pharmacology*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000037:antagonists & inhibitors*; D003510:Cyclohexanes / Q000494:pharmacology*; D004285:Dogs; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology; D006168:Guinea Pigs; D057809:HEK293 Cells; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D032342:Muscle Cells / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000037:antagonists & inhibitors; D011690:Purkinje Fibers / Q000187:drug effects; D012849:Sinoatrial Node / Q000166:cytology
chemical_list: C574764:3-(3-((2-(3,4-dimethoxyphenyl)ethyl)-methylamino)cyclohexyl)-7,8-dimethoxy-1,3,4,5-tetrahydrobenzo(d)azepin-2-one; D001552:Benzazepines; D054815:Cyclic Nucleotide-Gated Cation Channels; D003510:Cyclohexanes; C574765:N,N-bis-((Z)-4-(7,8-dimethoxy-2-oxo-1,3-dihydrobenzo(d)azepin-3-yl)but-2-enyl)-2-(3,4-dimethoxyphenyl)-propanamine; D020033:Protein Isoforms
references.citation: EMBO Mol Med. 2011 May;3(5):266-78
references.pmid: 21438154
references.citation: Circulation. 1997 Feb 4;95(3):568-71
references.pmid: 9024140
references.citation: Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1948-55
references.pmid: 19060291
references.citation: Can J Physiol Pharmacol. 2009 Feb;87(2):84-90
references.pmid: 19234571
references.citation: Br J Pharmacol. 2012 Mar;165(5):1457-66
references.pmid: 21838751
references.citation: J Physiol. 2006 Apr 15;572(Pt 2):335-46
references.pmid: 16484306
references.citation: Pflugers Arch. 2002 Feb;443(4):532-40
references.pmid: 11907819
references.citation: J Cardiovasc Pharmacol. 1995 Jul;26(1):46-54
references.pmid: 7564364
references.citation: Circulation. 2003 Feb 18;107(6):817-23
references.pmid: 12591750
references.citation: Eur J Pharmacol. 2011 Oct 15;668(3):419-26
references.pmid: 21821019
references.citation: J Pain. 2005 Jul;6(7):417-24
references.pmid: 15993819
references.citation: Bioorg Med Chem. 2005 Feb 15;13(4):1211-20
references.pmid: 15670930
references.citation: J Mol Cell Cardiol. 2001 Mar;33(3):441-8
references.pmid: 11181013
references.citation: Curr Pharm Des. 2009;15(18):2149-68
references.pmid: 19519450
references.citation: Pflugers Arch. 2007 Jul;454(4):517-22
references.pmid: 17549513
references.citation: J Physiol. 2007 Jul 15;582(Pt 2):675-93
references.pmid: 17478540
references.citation: J Med Chem. 2010 Sep 23;53(18):6773-7
references.pmid: 20795648
references.citation: J Physiol. 1994 Dec 15;481 ( Pt 3):585-91
references.pmid: 7707227
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):12045-50
references.pmid: 18687886
references.citation: Br J Pharmacol. 2011 Nov;164 Suppl 1:S1-324
references.pmid: 22040146
references.citation: Am J Physiol Heart Circ Physiol. 2011 Jan;300(1):H366-73
references.pmid: 20952661
references.citation: J Physiol. 2008 Dec 15;586(24):5911-29
references.pmid: 18936078
references.citation: Circulation. 1996 Aug 1;94(3):562-70
references.pmid: 8759103
references.citation: Pflugers Arch. 1991 Jan;417(5):493-9
references.pmid: 1707158
references.citation: Biophys J. 2007 Mar 1;92(5):1559-72
references.pmid: 17142286
references.citation: Mol Pharmacol. 2006 Apr;69(4):1328-37
references.pmid: 16387796
references.citation: Lancet. 2010 Sep 11;376(9744):886-94
references.pmid: 20801495
references.citation: Circ Res. 2010 Feb 19;106(3):434-46
references.pmid: 20167941
references.citation: Curr Med Chem. 2011;18(24):3662-74
references.pmid: 21774761
references.citation: J Pharmacol Exp Ther. 2007 Sep;322(3):931-9
references.pmid: 17578902
references.citation: Annu Rev Physiol. 2003;65:453-80
references.pmid: 12471170
references.citation: J Am Coll Cardiol. 1995 Aug;26(2):305-12
references.pmid: 7608428
references.citation: Circ Res. 2002 Nov 1;91(9):790-7
references.pmid: 12411393
references.citation: Eur J Biochem. 2001 Mar;268(6):1646-52
references.pmid: 11248683
references.citation: Circ Res. 2002 Jun 28;90(12):1267-73
references.pmid: 12089064
references.citation: Physiol Rev. 2009 Jul;89(3):847-85
references.pmid: 19584315
references.citation: Pharmacol Res. 2006 May;53(5):435-9
references.pmid: 16644235
references.citation: Vision Res. 1999 May;39(10):1767-74
references.pmid: 10343868
references.citation: Pharmacol Ther. 2005 Jul;107(1):59-79
references.pmid: 15963351
references.citation: Br J Pharmacol. 2007 Feb;150(4):383-90
references.pmid: 17211458
references.citation: Am J Cardiol. 1997 May 15;79(10):1401-5
references.pmid: 9165168