Wang JX et al. (2020), Structural basis of subtype-selective competiti...

XB-ART-56895

Nat Commun 2020 Jan 22;111:423. doi: 10.1038/s41467-020-14321-0.

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Structural basis of subtype-selective competitive antagonism for GluN2C/2D-containing NMDA receptors.

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

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N-Methyl-D-aspartate receptors (NMDARs) play critical roles in the central nervous system. Their heterotetrameric composition generates subtypes with distinct functional properties and spatio-temporal distribution in the brain, raising the possibility for subtype-specific targeting by pharmacological means for treatment of neurological diseases. While specific compounds for GluN2A and GluN2B-containing NMDARs are well established, those that target GluN2C and GluN2D are currently underdeveloped with low potency and uncharacterized binding modes. Here, using electrophysiology and X-ray crystallography, we show that UBP791 ((2S*,3R*)-1-(7-(2-carboxyethyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) inhibits GluN2C/2D with 40-fold selectivity over GluN2A-containing receptors, and that a methionine and a lysine residue in the ligand binding pocket (GluN2D-Met763/Lys766, GluN2C-Met736/Lys739) are the critical molecular elements for the subtype-specific binding. These findings led to development of UBP1700 ((2S*,3R*)-1-(7-(2-carboxyvinyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) which shows over 50-fold GluN2C/2D-selectivity over GluN2A with potencies in the low nanomolar range. Our study shows that the L-glutamate binding site can be targeted for GluN2C/2D-specific inhibition.

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	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 Å 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.

References [+] :

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