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.

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.

Fig. 1

Concentration-response data for glycine at NMDA receptors with wild type or mutated GluN1 subunits. A–C, concentration-response data for glycine at GluN1/N2A (A), GluN1/N3A (B), and GluN1/N3B (C) receptors. Receptors were expressed in Xenopus oocytes and current responses recorded from two-electrode voltage-clamp electrophysiology. Data are mean ± S.E.M. from 6–28 oocytes. EC₅₀-values and parameters for bell-shaped fitting are listed in Table 1 and Table 2. D, representative two-electrode voltage-clamp recording of responses from GluN1/N3A (top), GluN1(F484A)/N3A (upper middle), GluN1(T518L)/N3A (lower middle), and GluN1(F484A/T518L)/N3A (bottom) receptors to increasing concentration of glycine.

Fig. 2

Co-expression of GluN1(F484A/T518L) and GluN3A or GluN3B subunits results in surface expression of functional receptors in Xenopus oocytes. A–F, representative two-electrode voltage-clamp recordings of current responses to application of 10 μM, 100 μM, and 1000 μM glycine to Xenopus oocytes co-injected with GluN1(F484A/T518L) and GluN3A (A) or GluN3B (B), and to Xenopus oocytes injected with the individual subunits alone: GluN1 (C), GluN1(F484A/T518L) (D), GluN3A (E), or GluN3B (F). Data are from 10–13 oocytes from 3 different batches of oocytes.

Fig. 3

The LBD of GluN1 and GluN3A subunits – crystal structures and homology model. A and B, crystal structures of the agonist binding pockets of the GluN1 and GluN3A LBDs in complex with D-serine (PDB: 1PB8 and PBD: 2RCB, respectively) (Furukawa and Gouaux, 2003; Yao et al., 2008). Carbon atoms of D-serine are shown in yellow and residues that interact with D-serine in the binding pocket are shown as sticks. Black dashes indicate polar interactions between D-serine and residues in the binding pockets. C, crystal structure of the GluN1 LBD in a closed conformation with the agonist D-serine bound (PDB: 1PB8) (Furukawa and Gouaux, 2003) (top-left), crystal structure of the GluN1 LBD in an open conformation with the antagonist DCKA bound (PDB: 1PBQ) (Furukawa and Gouaux, 2003) (top-right), crystal structure of the GluN3A LBD in a closed conformation with the agonist D-serine bound (PDB: 2RCB) (Yao et al., 2008) (bottom-left), model of the GluN3A LBD in an open conformation (bottom-right).

Fig. 4

Hit identification in the antagonist screen of library compounds. A, 99 compounds from the virtual screen were examined for antagonistic activity at GluN1(F484A/T518L)/N3A and GluN1(F484A/T518L)/N3B receptors expressed in Xenopus oocytes. Responses to co-application of 100 μM test compounds and 100 μM glycine were recorded using two-electrode voltage-clamp recordings. Data are given as percentage of control response (100 μM glycine) in the same recording and are averaged from two oocytes. Compounds displaying > 25% inhibition of the maximal response were classified as hits and are highlighted in red. B, structure of the 8 identified hits.

Fig. 5

Inhibition data for TK13, TK30, and TK80. A, representative two-electrode voltage-clamp recording of responses from GluN1(F484A/T518L)/N3A (left) and GluN1(F484A/T518L)/N3B (right) receptors expressed in Xenopus oocytes showing inhibition by increasing concentration of TK80 in the continuous presence of 100 μM glycine at the GluN1(F484A/T518L)/N3B receptor. No inhibition was observed at the GluN1(F484A/T518L)/N3A receptor. B–D, concentration-response data for TK13 (B), TK30 (C), and TK80 (D) at receptors determined by two-electrode voltage-clamp recordings. Data at GluA1 and GluK2 receptors are presented as percent of 30 μM glutamate response in 300 μM of the compound. Data are mean ± S.E.M. from 3–9 oocytes. IC₅₀-values are listed in Table 3 and Table 4. The compounds were co-applied with 100 μM glycine at GluN1(F484A/T518L)/N3 receptors, with 100 μM glutamate and 0.5 μM glycine at GluN1/N2 receptors, and with 30 μM glutamate at GluA1 and GluK2 receptors. Oocytes expressing GluK2 were incubated for 5 min. in 10 μM concanavalin A.

Fig. 6

Mechanism of inhibition for TK13, TK30, and TK80 at GluN3-containing NMDA receptors. A, representative two-electrode voltage-clamp recording of response from GluN1(F484A/T518L)/N3A receptors expressed in Xenopus oocytes showing that increasing the concentration of glycine from 100 μM to 3000 μM did not reduce the extent of inhibition by 100 μM TK13. B, representative two-electrode voltage-clamp recording of response from GluN1(F484A/T518L)/N3B receptors expressed in Xenopus oocytes showing that increasing the concentration of glycine from 100 μM to 3000 μM surmounted inhibition by 100 μM TK80. C, bar graph showing inhibition of responses to 100 μM glycine or 3000 μM glycine at GluN1(F484A/T518L)/N3A receptors by 100 μM TK13 and 30 μM TK30, and at GluN1(F484A/T518L)/N3B receptors by 100 μM TK80. Data are shown as percentage of the response to 100 μM or 3000 μM glycine, respectively, and are mean ± S.E.M. from 4–7 oocytes. *, P < 0.05 (Student’s t test). D–F, glycine concentration-response curves in the absence (0 μM) and presence of increasing concentrations of TK13 (D), TK30 (E), and TK80 (F) at GluN1(F484A/T518L)/N3A or GluN1(F484A/T518L)/N3B receptors. EC₅₀-values and maximal responses are listed in Table 5. Data are from 4–6 oocytes.

Fig. 7

Mechanism of inhibition for TK13, TK30, and TK80 at GluN3-containing NMDA receptors. A–C, bar graph and current-voltage relationship curves showing inhibition of GluN1(F484A/T518L)/N3A responses at membrane potentials of −60 mV to +30 mV by 100 μM TK13 (A) and 30 μM TK30 (B), and inhibition of GluN1(F484A/T518L)/N3B responses at membrane potentials of −60 mV and +30 mV by 100 μM TK80 (C). Data are shown as percentage of the response to 100 μM glycine (control) and are mean ± S.E.M. from 4–5 oocytes. The current-voltage relationship curves are normalized to the response to 100 μM glycine at −60 mV. P > 0.05 (Student’s t test).

Fig. 8

Effects of TK13, TK30, and TK80 at wild-type GluN1/N3B receptors. A–C, representative two-electrode voltage-clamp recordings of responses from GluN1/N3B receptors expressed in Xenopus oocytes showing inhibition of 10 μM glycine-activated current response by 100 μM TK13 (A), 30 μM TK30 (B), and 100 μM TK80 (C). D, bar graph showing inhibition of responses to 10 μM glycine at GluN1/N3B receptors by 100 μM TK13, 30 μM TK30, and by 100 μM TK80. Data are mean ± S.E.M. from 6–8 oocytes.