Mapping the high-affinity binding domain of 5-substituted benzimidazoles to the proximal N-terminus of the GluN2B subunit of the NMDA receptor

Abstract

Background and purpose: N-methyl-D-aspartate (NMDA) receptors represent an attractive drug target for the treatment of neurological and neurodegenerative disorders associated with glutamate-induced excitotoxicity. The aim of this study was to map the binding domain of high affinity 5-substituted benzimidazole derivatives [N-{2-[(4-benzylpiperidin-1-yl)methyl]benzimidazol-5-yl}methanesulphonamide (XK1) and N-[2-(4-phenoxybenzyl)benzimidazol-5-yl]methanesulphonamide (XK2)] on the GluN2B subunit of the NMDA receptor.

Experimental approach: The pharmacological antagonistic profiles of XK1 and XK2 were assessed using in vitro rat primary cerebrocortical neurones and two-electrode voltage clamp on Xenopus oocytes expressing heterologous GluN1/GluN2B receptors. Direct ligand binding was determined using the recombinant amino-terminal domain (ATD) of GluN2B.

Key results: XK1 and XK2 effectively protected against NMDA-induced excitotoxicity in rat primary cortical neurones. Low concentrations of XK1 (10 nM) and XK2 (1 nM) significantly reversed neuronal death. Both compounds failed to inhibit currents measured from oocytes heterologously expressing GluN1-1a subunit co-assembled with the ATD-deleted GluN2B subunit. XK1 and XK2 showed specific binding to recombinant protein of GluN2B ATD with low nanomolar affinities. Several residues in the recombinant ATD of GluN2B were identified to be critical for conferring XK1 and XK2 sensitivity. The inhibitory effects of XK1 and XK2 were pH-sensitive, being increased at acidic pH.

Conclusions and implications: These results demonstrate that XK1 and XK2 are effective neuroprotective agents in vitro and indicate that 5-substituted benzimidazole derivatives inhibit GluN1/GluN2B receptors via direct binding to the ATD of the GluN2B subunit. These compounds represent valuable alternatives to the classical antagonist ifenprodil as pharmacological tools for studying GluN2B-containing NMDA receptors.

Figure 1

Functional organization of the GluN2B subunit and its selective antagonists. (A) GluN2B subunit consists of the antagonist-binding domain – amino-terminal domain (ATD) and the glutamate-binding domain (S1-S2) for various antagonists. These ligand binding domains are linked to three transmembrane domains (I, III and IV) with a re-entrant loop (II). The C-terminal tail (-COOH) resides in the cytoplasmic face of the plasma lipid bilayer. (B) Chemical structures of the classical reference GluN2B-selective antagonist – ifenprodil and two 5-substituted benzimidazole derivatives (XK1 and XK2). (C) Chemical synthesis of XK1. Reagents and conditions: (a) methanesulphonyl chloride, N,N-diisopropylethylamine, r.t., 4 h; (b) H₂, 10% Pd/C, r.t., 2 h; (c) ethyl chloroacetate, triethylamine, anhydrous toluene, microwave-assisted condition, 170°C, 10 min; (d) 6M HCl, microwave-assisted condition, 150°C, 4 min; (e) Mukaiyama reagent (2-chloro-1-methyl-pyridinium iodide), 4-dimethylaminopyridine, triethylamine, anhydrous dimethylformamide, 50°C, 1h; (f) glacial acetic acid, 140°C, 15 min; (g) ether/HCl. Pd/C, palladium on carbon catalyst; r.t., room temperature condition.

Figure 2

Neuroprotection of rat cerebrocortical neurones by NMDA receptor antagonists. (A) NMDA induced significant degree of neuronal death (^#P < 0.001, compared with -NMDA, -MK801, -ifenprodil) which was reversed completely in the presence of the open channel blocker MK-801 (10 µM) and GluN2B-selective non-competitive antagonist ifenprodil (P < 0.001). Blockers alone did not affect neuronal viability (P > 0.05). Concentration-dependent antagonism of NMDA-induced neuronal cell death by XK1 (B) and XK2 (C). Data are the mean of at least three independent experiments on cerebrocortical neuronal cultures harvested from three separate pregnant rats (±SEM). Triplicates per concentration tested were performed on each independent experiment. XK2 showed relatively higher potency than XK1 in reversing neuronal death (compare at XK2 1 nM, *P < 0.05 and XK1 3 nM, ***P < 0.001). NMDA, N-methyl-D-aspartate.

Figure 3

Inhibitory activities of XK1 and XK2 mediated via ATD of GluN2B. (A) Representative current traces recorded from Xenopus oocytes co-expressing rat GluN1-1a/GluN2Bwt (left traces) and GluN1-1a/GluN2BΔM394 (right traces). The horizontal lines show applications of solutions containing 0.3, 1, 3, 10, 30 or 100 nM XK1 or XK2 in the presence of maximal glutamate (Glu, 100 µM) and glycine (Gly, 100 µM). Schematic representation of the wt and ATD-truncated GluN2B subunits are depicted above the current traces; grey box denotes signal peptide, black rectangle denotes ATD and black sticks denote three transmembrane domains and a re-entrant loop. The holding potential was –50 mV. Calibration: current in nA and time in min. (B) Composite XK1 and XK2 inhibition curves are shown for oocytes expressing GluN1-1a/GluN2Bwt and GluN1-1a/GluN2BΔM394. Response (%) represents the percentage of current measured at each antagonist concentration to the maximum current elicited by glutamate (100 µM) and glycine (100 µM). Each point is the mean ± SEM value of four to seven oocytes. The mean IC₅₀ values for XK1 and XK2 on GluN1-1a/GluN2Bwt are 37.6 nM and 15.7 nM respectively. The IC₅₀ values for the high affinity binding of XK1 and XK2 on GluN1-1a/GluN2BΔM394 could not be determined within the antagonists' concentration range studied. ATD, amino-terminal domain.

Figure 4

XK1 and XK2 bind to recombinant ATD protein of GluN2B. (A) Ifenprodil, (B) XK1 and (C) XK2 bound to 6xHis-ATD2B in a concentration-dependent manner yielding K_D values 91 nM, 1.2 nM and 1.0 nM respectively. The x axes were broken between the last two highest antagonist concentrations to highlight the curve fitting of data at the lower concentrations as well as saturated bindings. ATD, amino-terminal domain.

Figure 5

The XK1 and XK2 inhibition of recombinant GluN1/GluN2Bwt receptors is pH sensitive. (A) (Left panel) The % inhibition produced by 1 nM, 30 nM and 300 nM XK1 and (B) XK2 were measured at pH 6.8 (solid squares), pH 7.3 (open circles) and pH 8.0 (solid triangles) from oocytes injected with GluN1-1a/GluN2Bwt. (Right panel) Histograms comparing the potency of 30 nM XK1 or XK2 at three pHs tested. **Indicates a significant increase in inhibition by XK1 and XK2 at acidic pH (P < 0.01). Values are mean ± SEM from n= 4–16 for each condition tested and are expressed as a percentage of the control response to glutamate (100 µM) and glycine (100 µM) at each pH.