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

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. British Journal of Pharmacology (2010) 159, 449-461; doi:10.1111/j.1476-5381.2009.00549.x; published online 15 January 2010