A Novel Negative Allosteric Modulator Selective for GluN2C/2D-Containing NMDA Receptors Inhibits Synaptic Transmission in Hippocampal Interneurons

Abstract

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in numerous neurological disorders. NMDARs typically comprise two GluN1 and two GluN2 subunits. The four GluN2 subtypes (GluN2A-GluN2D) have distinct functional properties and gene expression patterns, which contribute to diverse functional roles for NMDARs in the brain. Here, we present a series of GluN2C/2D-selective negative allosteric modulators built around a N-aryl benzamide (NAB) core. The prototypical compound, NAB-14, is >800-fold selective for recombinant GluN2C/GluN2D over GluN2A/GluN2B in Xenopus oocytes and has an IC₅₀ value of 580 nM at recombinant GluN2D-containing receptors expressed in mammalian cells. NAB-14 inhibits triheteromeric (GluN1/GluN2A/GluN2C) NMDARs with modestly reduced potency and efficacy compared to diheteromeric (GluN1/GluN2C/GluN2C) receptors. Site-directed mutagenesis suggests that structural determinants for NAB-14 inhibition reside in the GluN2D M1 transmembrane helix. NAB-14 inhibits GluN2D-mediated synaptic currents in rat subthalamic neurons and mouse hippocampal interneurons, but has no effect on synaptic transmission in hippocampal pyramidal neurons, which do not express GluN2C or GluN2D. This series possesses some druglike physical properties and modest brain permeability in rat and mouse. Altogether, this work identifies a new series of negative allosteric modulators that are valuable tools for studying GluN2C- and GluN2D-containing NMDAR function in brain circuits, and suggests that the series has the potential to be developed into therapies for selectively modulating brain circuits involving the GluN2C and GluN2D subunits.

Keywords: GluN2C; GluN2D; NAB-14; NMDA receptor; NMDAR; glutamate receptor; negative allosteric modulator.

Figure 1

NAB-14 is a GluN2C/2D-selective NMDAR antagonist. The chemical structures are shown for (a) compound 1, (b) the regions evaluated in the NAB series structure-activity relationship, and (c) NAB-14. (d) Current responses to maximal concentrations of glutamate (100 μM) and glycine (30 μM) co-applied with increasing concentrations of NAB-14 were recorded by two-electrode voltage-clamp (TEVC) in Xenopus oocytes expressing GluN1 and GluN2A, GluN2B, GluN2C, or GluN2D. (e) Concentration-response data for NAB-14 were plotted as the percent of the maximal response to glutamate and glycine (mean ± s.e.m.) and fit by the Hill equation. (f) Representative current responses to 100 μM glutamate and 30 μM glycine co-applied with increasing concentrations of NAB-14 are shown for Xenopus oocytes expressing GluN1 and wild type (WT) GluN2C, GluN2C_C1/2C_C2, GluN2A_C1/2C_C2, or GluN2A_C1/2A_C2. (g) Concentration-response data for NAB-14 were plotted as the percent of the maximal glutamate and glycine response (mean ± s.e.m.) and fit by the Hill equation. The pIC50 values for 2A_C1/2C_C2 and 2C_C1/2C_C2 groups were compared by an F test [F (1,118) = 28.65, p < 0.001].

Figure 2

Structural determinants of NAB-14 reside in the M1 transmembrane helix. (a) A sequence alignment of the M1 transmembrane helix across rat GluN2 subunits shows four residues that differ between GluN2A/2B and GluN2C/2D (gray shading). Site-directed mutagenesis was used to switch these GluN2A and GluN2D residues and to mutate all M1 residues to alanine or cysteine. (b) A representative trace depicts the current response to 100 μM glutamate and 30 μM glycine recorded by TEVC in Xenopus oocytes in the absence and presence of 10 μM NAB-14. (c,d) The peak amplitudes of current responses were measured and expressed as the percent of the maximal response to glutamate and glycine. The data were analyzed by one-way ANOVA and post hoc (c) Bonferroni tests or (d) Dunnett’s tests. GluN2A mutants were compared to 2A WT, and GluN2D mutants were compared to 2D WT [c: F(9,42) = 38.155, p < 0.001; d: F(23,243) = 95.155, p < 0.001; *p < 0.05; see Supplementary Table 12 for mean comparison p-values]. (e) A homology model of the GluN1/GluN2D receptor based on crystal structures of the GluN1/GluN2B receptor^, shows the GluN2 M1 helix in yellow and illustrates the residues that affect NAB-14 activity when mutated; blue residues resulted in a nearly complete loss of inhibition and green residues modestly affected inhibition at 10 μM NAB-14, and Cys590 in magenta was involved in GluN2 selectivity as shown by the effects of switching the analogous GluN2A and GluN2D residues at this position in panel c.

Figure 3

Association and dissociation kinetics of NAB-14. (a) Glutamate (100 μM) and glycine (30 μM) plus increasing concentrations of NAB-14 were co-applied to HEK cells transiently expressing GluN1/GluN2D using a rapid solution exchange system, as shown in a representative current response recorded by whole-cell voltage-clamp. (b) The current responses during NAB-14 association and dissociation were fit with single exponentials. The inverse of these time constants were plotted vs. NAB-14 concentration and fit by linear regression to determine the rate constants K_ON, the slope of the 1/τ_ON line, and KOFF, the y-intercept of the 1/τ_OFF line. (c) Glycine plus increasing concentrations of NAB-14 were applied to HEK cells, then glutamate was applied for either 15 s or 5 ms in the continued presence of glycine and NAB-14. (d) Concentration-response data from 15 s (peak and steady-state) and 5 ms (peak) applications were plotted. pIC₅₀ values from the fitted curves were compared by an F-test [F(2,72) = 31.50, p <0.001]. (e) NAB-14 concentration-response data were acquired for responses to five NMDA/glycine pressure pulses applied to HEK cells at 1 Hz. The respresentative trace shows responses for control and 3 μM NAB-14. (f) The peak amplitude of the 1^st and 5^th responses were measured, and pIC₅₀ values from the fitted curves were compared by an F-test [F(1,20) = 8.86, p = 0.008].

Figure 4

NAB-14 inhibits GluN2C/2D-containing NMDARs in cultured neurons and brain slices. (a) Cultured rat cortical neurons were dissociated and transfected with GFP, GFP and GluN2C, or GFP and GluN2D, and then cultured for 2-5 days. NMDAR current responses were evoked by pressure pulses of NMDA and glycine and recorded by whole-cell voltage-clamp in the absence and presence of NAB-14 (20 μM). (b) The peak amplitude of the current responses were measured and compared between the three transfection groups by one-way ANOVA and post hoc Dunnett’s test [F(2,14) = 16.257, p < 0.001, *GluN2C: p < 0.001, *GluN2D: p = 0.005]. (c,d) NMDAR responses were evoked with pressure pulses of NMDA and glycine at 30 s intervals in acute slices of the rat STN. 20 μM NAB-14 was bath-applied after stable control responses were obtained, then washed out, and 400 μM D,L-APV was applied to ensure the response was NMDAR-mediated. (e) The peak amplitudes of responses in NAB-14 and D,L-APV were plotted as the percent of the control response, and the amplitudes in control and NAB-14-treated conditions were compared by paired t-test (*p < 0.001).

Figure 5

NAB-14 inhibits EPSCs in subthalamic neurons. (a) EPSCs were evoked in rat brain slices of the STN, and the NMDAR component was isolated by application of CNQX (10 μM) and bicuculline (10 μM). EPSCs recorded by whole-cell voltage-clamp (hold = −40 mV) are shown for representative neurons in aCSF (control), vehicle (0.1% DMSO), and D,L-APV (100 μM) conditions as well as control, NAB-14 (10 μM), and D,L-APV (100 μM) conditions. (b) Paired values for EPSC peak amplitude, charge transfer, and τ_W were plotted for each neuron under control and treated (vehicle or NAB-14) conditions. (c) Values were plotted as the percent of the control response for each cell (gray circles) with the group mean ± s.e.m. Data were compared between vehicle and NAB-14 groups by unpaired t-tests. The significance level was corrected for family-wise error: α = 0.05/3 = 0.016 (*p < 0.001).

Figure 6

GluN2D-containing NMDARs mediate synaptic transmission at CA3-CA1 synapses in hippocampal interneurons. (a) Electrically evoked NMDAR EPSCs were recorded from CA1 interneurons or pyramidal neurons by whole-cell voltage-clamp (hold = +40 mV) in aCSF with NBQX (10 μM) and bicuculline (10 μM). EPSCs were recorded in aCSF (control), aCSF containing 0.1% DMSO (vehicle) or 10 μM NAB-14, and 100 μM D,L-APV. The peak amplitude, charge transfer, and τ_W were measured for (b) interneuron and (c) pyramidal neuron EPSCs, and the paired values for control and vehicle or NAB-14 conditions were plotted for each neuron. (d) The data were normalized to baseline and plotted as the percent of the control responses for each cell (gray circles) with the mean ± s.e.m. The effect of NAB-14 on each parameter was compared between interneuron (IN) and pyramidal neuron (PN) groups by unpaired t-tests. The significance level was corrected for family-wise error: α = 0.05/3 = 0.016 (^†p = 0.009, ^‡p = 0.011, and *p = 0.005).