Kvist, Trine7; Greenwood, Jeremy R4; Hansen, Kasper B8; Traynelis, Stephen F5; Bräuner-Osborne, Hans9
1 Medicinal Chemistry Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet2 Department of Drug Design and Pharmacology, Faculty of Pharmaceutical Sciences, Københavns Universitet3 Drug Research Academy M, Drug Research Academy, Faculty of Pharmaceutical Sciences, Københavns Universitet4 Schrödinger, Inc.5 Emory University School of Medicine6 Experimental Pharmacology, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet7 Drug Research Academy M, Drug Research Academy, Faculty of Pharmaceutical Sciences, Københavns Universitet8 Department of Drug Design and Pharmacology, Faculty of Pharmaceutical Sciences, Københavns Universitet9 Experimental Pharmacology, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
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.