Wang JX , Irvine MW , Burnell ES , Sapkota K , Thatcher RJ , Li M , Simorowski N , Volianskis A , Collingridge GL , Monaghan DT , Jane DE , Furukawa H .

R01 NS111745 NINDS NIH HHS , BB/L001977/1 Biotechnology and Biological Sciences Research Council , R01 MH085926 NIMH NIH HHS, R01 MH060252 NIMH NIH HHS

	Fig. 1. NMDAR domain organization and ligands.a GluN1/GluN2 form tetrameric receptors with two GluN1 (dark gray) and two GluN2 (light gray) subunits. Each subunit has an ATD, LBD, TMD, and CTD domain (CTD not included). The LBD layer contains a dimer of GluN1–GluN2 heterodimers, one of which is highlighted by a surrounding red dashed line. The GluN1 upper lobe (D1) and lower lobe (D2) are colored in green and orange, and the GluN2 D1 and D2 in purple and magenta. The surface presentation is produced from the PDB structure 4PE5. b Schematic representation of GluN1 and GluN2 subunits. Glycine, l-glutamate, and competitive antagonists bind in the LBD clefts. The modular build of the subunits allows isolation of the GluN1 and GluN2A LBDs by replacing the M1–M3 transmembrane sequence with a Gly-Thr dipeptide linker. Color coding as in (a). c Chemical structures of GluN2 agonists L-glutamate and homoquinolinic acid and antagonists PPDA and UBP791 at pH ~7. PPDA and UBP791 are cis-racemic mixtures of (2S,3R)- and (2R,3S)-isomers.
	Fig. 2. UBP791 binds preferentially to GluN2C/2D-containing NMDARs over GluN2A/2B-containing NMDARs.a Representative TEVC dose-response traces of GluN1-4a/GluN2D (left panel) or GluN1-4a/GluN2A (right panel) NMDARs held at −60 mV. Currents were evoked by application of 100 μM glycine and 1 or 3 μM l-glutamate and inhibited by varying concentrations of UBP791 (threefold increments to max. 20 and 360 μM as shown). b Averaged dose-response curves (mean ± s.d.) for inhibition with UBP791 from ten GluN1-4a/GluN2D and 15 GluN1-4a/GluN2A recordings fit with the Hill equation to calculate IC50 and Hill coefficient (nH). c EC50 values for GluN1-4a/GluN2 (a–d) obtained from l-glutamate dose-response curves. d Inhibition potency (Ki) values calculated using the Cheng–Prusoff equation with the determined EC50 and IC50 values. Single data points are shown as open circles, the bar graph represents the mean with error bars for s.d., the number of recordings (n) and the fold difference to the Ki of GluN2D are as shown. Pairwise comparison shows subtypes have different potencies (p < 0.05 with two-tail t test and Bonferroni correction) except where stated (n.s.).
	Fig. 3. Site-directed mutagenesis of GluN2A binding pocket and its effect on l-glutamate and UBP791 sensitivity.a, b The GluN2A LBD of the GluN1–GluN2A LBD crystal structure complexed to PPDA (cyan sticks; PDB code: 4NF6) colored as in Fig. 1. Residues within the ligand-binding pocket (green dashed oval) which are not conserved among GluN2A-D in the sequence alignment (panel b arrows) are shown in yellow. Four mutations (Ala414Arg, Lys738Met, Gly740Arg, Arg741Lys) were introduced in GluN2A to generate GluN2A-4m. c EC50 values for l-glutamate and d Ki values for UBP791 for GluN1-4a/GluN2A-4m were obtained by TEVC. Dose-response for UBP791 was measured in the presence of 100 μM glycine and 1 μM l-glutamate with varying UBP791 concentrations (Supplementary Fig. 2). Single data points are shown as open circles, the bar graph represents the mean with error bars for s.d., and the number of recordings (n) and the fold-difference to EC50 and Ki of GluN2D are as shown. Pairwise comparison shows subtypes have different potencies (p < 0.0001 with two-tail t-test and Bonferroni correction).
	Fig. 4. Crystal structure of GluN1–GluN2A-4m LBD and comparison with GluN2D LBD.a Crystal structure of the GluN1–GluN2A-4m LBD heterodimer in complex with glycine (orange sphere) and l-glutamate (yellow sphere). Helices are labeled with underlined letters and the color coding is in accordance to Fig. 1. b Structures of the GluN2A-4m LBD and the GluN2D LBD (PDB: 3OEN) can be superposed with one another at an RMSD of 0.45 à over 224 Cα positions. c Comparison of the l-glutamate-binding sites of GluN2D and GluN2A-4m shows the similar mode of ligand-receptor interactions. Residue numbering shown for GluN2A-4m and GluN2D (in brackets). d Mutated residues in GluN2A-4m overlap well with the equivalent residues in GluN2D with minor differences in side chain orientations.
	Fig. 5. Structures of glycine/UBP791 complexed to GluN1–GluN2A (WT) and GluN1–GluN2A-4m LBD.a Overall structure of the GluN1–GluN2A LBD complexed to glycine (orange spheres) and UBP791 (cyan spheres) colored as in Fig. 1. Shown in mesh below is the Fo − Fc omit map of UBP791 contoured at 3σ. b, c The binding site of UBP791 (cyan sticks) showing polar (dashed lines) and hydrophobic interactions. GluN2A-Lys738 and -Glu714 form a hydrogen bond whereas GluN2A-Arg741 and –Thr797 form a water-mediated hydrogen bond. d Overall structure of the GluN1–GluN2A-4m LBD complexed to glycine (orange spheres) and UBP791 (cyan spheres). Note that the Fo − Fc omit map of UBP791 contoured at 3σ (green mesh) here is more ordered and continuous compared with that in the GluN1–GluN2A LBD in a. e The binding site of UBP791 (cyan sticks) showing similar polar (dashed lines) and hydrophobic interactions with the piperazine and phenanthrene moieties to those in the GluN1–GluN2A LBD in a. f In contrast to the GluN1–GluN2A LBD, GluN2A-4m-Met738 forms sulfur-aromatic interactions with the ligand and Tyr737, while GluN2A-4m-Lys741 forms a hydrogen bond with the carboxyethyl group of UBP791.
	Fig. 6. Effect of mutations in GluN2D on l-glutamate and UBP791 sensitivity.a Representative TEVC dose-response traces of single mutant GluN1-4a/GluN2D Met763Lys (left panel), Lys766Arg (middle panel) or double mutant Met763Lys/Lys766Arg (right panel) NMDARs held at −60 mV. Currents were evoked by application of 100 μM glycine and 1 μM l-glutamate and inhibited by varying concentrations of UBP791 (Met763Lys: concentration increments: 0.12/0.37/1.1/3.3/10/30/60 μM; for Lys766Arg and double mutant: three-fold increments from 0.08–60 μM). b Averaged dose-response curves (mean ± s.d.) for inhibition with UBP791 from eight, twelve, and six recordings for GluN1-4a/GluN2D Met763Lys, GluN1-4a/GluN2D Lys766Arg, and GluN1-4a/GluN2D Met763Lys/Lys766Arg, respectively, fit with the Hill equation to calculate IC50 and Hill coefficient (nH). c EC50 for l-glutamate and d Ki for UBP791 for the mutants were obtained by TEVC recordings as in Fig. 2. Single data points are shown as open circles, the bar graph represents the mean with error bars for s.d., the number of recordings (n) and the fold-difference to EC50 and Ki of GluN2D (WT) are as shown. Pairwise comparison shows WT and mutants have different potencies (p < 0.05 with two-tail t-test and Bonferroni correction) except where stated (n.s.).
	Fig. 7. Designed compounds with increased GluN2C/2D selectivity and potency.a UBP1700, 1701, and 1702 have carboxylate moieties of different length, bulkiness, and rigidity. The chiral center in the R-group of UBP1701 is marked with an asterisk. At physiological pH the amine group of the piperazine ring is protonated and therefore positively charged and the three carboxylic acid groups are negatively charged. b Ki values for UBP1700 inhibition were measured on GluN1-4a/GluN2 (a–d) NMDARs and GluN1-4a/GluN2A-4m NMDAR as described in Fig. 2. Inhibition dose-response was observed in the presence of 100 μM glycine, 1 or 3 μM l-glutamate, and varying concentrations of UBP1700 (Supplementary Fig. 4). Pairwise comparison shows subtypes have different potencies (p < 0.05 with two-tail t-test and Bonferroni correction) except where stated (n.s.). c Potencies of UBP1701 and UBP1702 on GluN1-4a/GluN2A and GluN1-4a/GluN2D were obtained as in Fig. 2.

Acker, Mechanism for noncompetitive inhibition by novel GluN2C/D N-methyl-D-aspartate receptor subunit-selective modulators. 2011, Pubmed

Acker, Mechanism for noncompetitive inhibition by novel GluN2C/D N-methyl-D-aspartate receptor subunit-selective modulators. 2011, Pubmed
Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed
Akazawa, Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats. 1994, Pubmed
Armstrong, Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. 2000, Pubmed
Banerjee, Double dissociation of spike timing-dependent potentiation and depression by subunit-preferring NMDA receptor antagonists in mouse barrel cortex. 2009, Pubmed
Bettini, Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors. 2010, Pubmed
Bouvier, Presynaptic NMDA receptors: Roles and rules. 2015, Pubmed
Cheng, Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. 1973, Pubmed
Cohen, The impact of NMDA receptor hypofunction on GABAergic neurons in the pathophysiology of schizophrenia. 2015, Pubmed
Costa, N-methyl-D-aspartate (NMDA) receptor NR2 subunit selectivity of a series of novel piperazine-2,3-dicarboxylate derivatives: preferential blockade of extrasynaptic NMDA receptors in the rat hippocampal CA3-CA1 synapse. 2009, Pubmed , Xenbase
Edman, TCN 201 selectively blocks GluN2A-containing NMDARs in a GluN1 co-agonist dependent but non-competitive manner. 2012, Pubmed , Xenbase
Emsley, Coot: model-building tools for molecular graphics. 2004, Pubmed
Evans, Scaling and assessment of data quality. 2006, Pubmed
Evans, How good are my data and what is the resolution? 2013, Pubmed
Feng, Structure-activity analysis of a novel NR2C/NR2D-preferring NMDA receptor antagonist: 1-(phenanthrene-2-carbonyl) piperazine-2,3-dicarboxylic acid. 2004, Pubmed , Xenbase
Freire, Do enthalpy and entropy distinguish first in class from best in class? 2008, Pubmed
Furukawa, Subunit arrangement and function in NMDA receptors. 2005, Pubmed
Furukawa, Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core. 2003, Pubmed
Gielen, Mechanism of differential control of NMDA receptor activity by NR2 subunits. 2009, Pubmed
Hackos, Positive Allosteric Modulators of GluN2A-Containing NMDARs with Distinct Modes of Action and Impacts on Circuit Function. 2016, Pubmed
Hansen, Structural and mechanistic determinants of a novel site for noncompetitive inhibition of GluN2D-containing NMDA receptors. 2011, Pubmed , Xenbase
Hansen, Structural determinants of agonist efficacy at the glutamate binding site of N-methyl-D-aspartate receptors. 2013, Pubmed , Xenbase
Hansen, Subunit-selective allosteric inhibition of glycine binding to NMDA receptors. 2012, Pubmed
Hardingham, The Yin and Yang of NMDA receptor signalling. 2003, Pubmed
Hardingham, Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. 2010, Pubmed
Irvine, Piperazine-2,3-dicarboxylic acid derivatives as dual antagonists of NMDA and GluK1-containing kainate receptors. 2012, Pubmed , Xenbase
Jespersen, Structural insights into competitive antagonism in NMDA receptors. 2014, Pubmed , Xenbase
Jullienne, Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo. 2011, Pubmed
Kabsch, XDS. 2010, Pubmed
Karakas, Crystal structure of a heterotetrameric NMDA receptor ion channel. 2014, Pubmed
Karakas, Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors. 2011, Pubmed , Xenbase
Khatri, Structural determinants and mechanism of action of a GluN2C-selective NMDA receptor positive allosteric modulator. 2014, Pubmed , Xenbase
Koutsilieri, Excitotoxicity and new antiglutamatergic strategies in Parkinson's disease and Alzheimer's disease. 2007, Pubmed
Krogsgaard-Larsen, Structure-Activity Relationship Study of Ionotropic Glutamate Receptor Antagonist (2S,3R)-3-(3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid. 2015, Pubmed
Lancelot, Glutamate toxicity in chronic neurodegenerative disease. 1998, Pubmed
Laurie, Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA. 1994, Pubmed
Lee, NMDA receptor structures reveal subunit arrangement and pore architecture. 2014, Pubmed , Xenbase
Lind, Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits. 2017, Pubmed , Xenbase
McCoy, Phaser crystallographic software. 2007, Pubmed
Monyer, Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. 1994, Pubmed
Mosley, Quinazolin-4-one derivatives: A novel class of noncompetitive NR2C/D subunit-selective N-methyl-D-aspartate receptor antagonists. 2010, Pubmed , Xenbase
Nakazawa, Spatial and temporal boundaries of NMDA receptor hypofunction leading to schizophrenia. 2017, Pubmed
Otwinowski, Processing of X-ray diffraction data collected in oscillation mode. 1997, Pubmed
Paoletti, NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. 2013, Pubmed
Romero-Hernandez, Novel Mode of Antagonist Binding in NMDA Receptors Revealed by the Crystal Structure of the GluN1-GluN2A Ligand-Binding Domain Complexed to NVP-AAM077. 2017, Pubmed , Xenbase
Ruben, Overcoming roadblocks in lead optimization: a thermodynamic perspective. 2006, Pubmed
Sheng, Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. 1994, Pubmed
Standaert, Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurons in the rat neostriatum, neocortex and hippocampus. 1996, Pubmed
Swanger, A Novel Negative Allosteric Modulator Selective for GluN2C/2D-Containing NMDA Receptors Inhibits Synaptic Transmission in Hippocampal Interneurons. 2018, Pubmed , Xenbase
Swanger, NMDA Receptors Containing the GluN2D Subunit Control Neuronal Function in the Subthalamic Nucleus. 2015, Pubmed
Vance, Ligand-specific deactivation time course of GluN1/GluN2D NMDA receptors. 2011, Pubmed
Volianskis, Different NMDA receptor subtypes mediate induction of long-term potentiation and two forms of short-term potentiation at CA1 synapses in rat hippocampus in vitro. 2013, Pubmed
von Engelhardt, GluN2D-containing NMDA receptors-mediate synaptic currents in hippocampal interneurons and pyramidal cells in juvenile mice. 2015, Pubmed
Vonrhein, Data processing and analysis with the autoPROC toolbox. 2011, Pubmed
Wienen-Schmidt, Paradoxically, Most Flexible Ligand Binds Most Entropy-Favored: Intriguing Impact of Ligand Flexibility and Solvation on Drug-Kinase Binding. 2018, Pubmed
Williams, Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors. 1993, Pubmed , Xenbase
Winn, Overview of the CCP4 suite and current developments. 2011, Pubmed
Winter, Automated data collection for macromolecular crystallography. 2011, Pubmed
Yuan, Control of NMDA receptor function by the NR2 subunit amino-terminal domain. 2009, Pubmed , Xenbase
Zhou, NMDA receptors in nervous system diseases. 2013, Pubmed