title: Structure-based discovery of antagonists for GluN3-containing N-methyl-D-aspartate receptors.
abstract: NMDA receptors are ligand-gated ion channels that assemble into tetrameric receptor complexes composed of glycine-binding GluN1 and GluN3 subunits (GluN3A-B) and glutamate-binding GluN2 subunits (GluN2A-D). NMDA receptors can assemble as GluN1/N2 receptors and as GluN3-containing NMDA receptors, which are either glutamate/glycine-activated triheteromeric GluN1/N2/N3 receptors or glycine-activated diheteromeric GluN1/N3 receptors. The glycine-binding GluN1 and GluN3 subunits display strikingly different pharmacological selectivity profiles. However, the pharmacological characterization of GluN3-containing receptors has been hampered by the lack of methods and pharmacological tools to study GluN3 subunit pharmacology in isolation. Here, we have developed a method to study the pharmacology of GluN3 subunits in recombinant diheteromeric GluN1/N3 receptors by mutating the orthosteric ligand-binding pocket in GluN1. This method is suitable for performing compound screening and characterization of structure-activity relationship studies on GluN3 ligands. We have performed a virtual screen of the orthosteric binding site of GluN3A in the search for antagonists with selectivity for GluN3 subunits. In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands.
authors: Dept. of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, DK-2100 Copenhagen, Denmark.
authors: Trine
authors: T
authors: Kvist
authors: Schrödinger, Inc., 120 West 45th St., New York, NY 10036, USA.
authors: Jeremy R
authors: JR
authors: Greenwood
authors: Dept. of Pharmacology, Emory University School of Medicine, 5062 Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322, USA.
authors: Kasper B
authors: KB
authors: Hansen
authors: Dept. of Pharmacology, Emory University School of Medicine, 5062 Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322, USA.
authors: Stephen F
authors: SF
authors: Traynelis
authors: Dept. of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, DK-2100 Copenhagen, Denmark. Electronic address: hbo@sund.ku.dk.
authors: Hans
authors: H
authors: Bräuner-Osborne
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry* / Q000494:pharmacology*; D005998:Glycine / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008564:Membrane Potentials / Q000187:drug effects* / Q000235:genetics; D008958:Models, Molecular; D009865:Oocytes; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D018691:Excitatory Amino Acid Antagonists; C584082:GluN3A protein, mouse; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D054852:Small Molecule Libraries; D005998:Glycine
keywords: 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid; 6-cyano-7-nitroquinoxaline-2,3-dione; 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one; AMPA; Antagonist; CNQX; DCKA; GluN3 subunit; LBD; N-methyl-d-aspartate; NMDA; NMDA receptor; Selectivity; TEVC; TK13; TK30; TK80; Virtual screening; Xenopus oocyte electrophysiology; [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid; dichlorokynurenic acid; iGluR; ionotropic glutamate receptor; ligand-binding domain; two-electrode voltage-clamp; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
references: BMC Neurosci. 2010 Oct 19;11:133
references: 20958962
references: J Pharmacol Exp Ther. 2009 Dec;331(3):975-84
references: 19726695
references: J Pharmacol Exp Ther. 2007 Aug;322(2):739-48
references: 17502428
references: Nature. 2002 Feb 14;415(6873):793-8
references: 11823786
references: Adv Drug Deliv Rev. 2001 Mar 1;46(1-3):3-26
references: 11259830
references: Pharmacol Rev. 2010 Sep;62(3):405-96
references: 20716669
references: EMBO J. 2003 Jun 16;22(12):2873-85
references: 12805203
references: Neuron. 1994 Jun;12(6):1291-300
references: 8011339
references: Mol Pharmacol. 2007 Jan;71(1):112-22
references: 17047094
references: Brain Res Mol Brain Res. 2002 Apr 30;100(1-2):43-52
references: 12008020
references: Nat Neurosci. 2011 Mar;14(3):338-44
references: 21297630
references: PLoS One. 2012;7(8):e42327
references: 22870318
references: Trends Neurosci. 2012 Apr;35(4):240-9
references: 22240240
references: Proteins. 2008 Jul;72(1):434-46
references: 18214958
references: J Neurosci. 1995 Oct;15(10):6509-20
references: 7472413
references: J Neurosci. 2006 Apr 26;26(17):4559-66
references: 16641235
references: J Neurosci. 1998 Aug 15;18(16):6163-75
references: 9698310
references: Nature. 2005 Dec 22;438(7071):1167-71
references: 16372012
references: J Neurosci. 2001 Feb 15;21(4):1228-37
references: 11160393
references: Nat Methods. 2007 Apr;4(4):319-21
references: 17369835
references: J Mol Biol. 2006 Apr 7;357(4):1184-201
references: 16483599
references: J Neurosci. 2012 May 2;32(18):6197-208
references: 22553026
references: Science. 2007 Nov 2;318(5851):815-7
references: 17975069
references: Mol Pharmacol. 2010 Jul;78(1):1-11
references: 20363861
references: Nat Neurosci. 2006 May;9(5):611-21
references: 16617342
references: Neurobiol Dis. 2012 Dec;48(3):290-8
references: 22801082
references: Neuron. 2009 Aug 13;63(3):342-56
references: 19679074
references: J Neurosci. 2009 Apr 22;29(16):5260-5
references: 19386922
references: Nature. 1998 May 28;393(6683):377-81
references: 9620802
references: J Neurosci. 2001 Dec 1;21(23):RC185
references: 11717388
references: Trends Pharmacol Sci. 2007 Nov;28(11):561-6
references: 17961731
references: Biochem Biophys Res Commun. 2007 Mar 2;354(1):102-8
references: 17214961
references: Nature. 2006 Feb 23;439(7079):988-92
references: 16372019
references: EMBO J. 2008 Aug 6;27(15):2158-70
references: 18636091
references: Nature. 2005 Dec 22;438(7071):1162-6
references: 16372011
references: Mol Neurobiol. 2008 Aug;38(1):16-26
references: 18654865
references: J Neurosci. 1995 Oct;15(10):6498-508
references: 7472412
references: Mol Pharmacol. 2007 Oct;72(4):907-20
references: 17622578
references: Biochem Pharmacol. 1973 Dec 1;22(23):3099-108
references: 4202581
references: J Neurophysiol. 2002 Apr;87(4):2052-63
references: 11929923
references: Adv Drug Deliv Rev. 2002 Mar 31;54(3):255-71
references: 11922947
references: J Neurosci. 1998 Apr 15;18(8):2954-61
references: 9526012
references: Genomics. 2001 Dec;78(3):178-84
references: 11735224
references: Methods Mol Biol. 2012;796:399-421
references: 22052503
references: Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):14163-8
references: 18779583
references: Prog Neurobiol. 2010 May;91(1):23-37
references: 20097255