Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore.
Br J Pharmacol. 2010 Jan 1;159(2):449-61. doi: 10.1111/j.1476-5381.2009.00549.x. Epub 2010 Jan 15.
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.
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.
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.
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.
N-甲基-D-天冬氨酸（NMDA）受体是治疗谷氨酸诱导的兴奋性毒性相关的神经和神经退行性疾病的有吸引力的药物靶点。本研究的目的是绘制高亲和力 5-取代苯并咪唑衍生物[N-｛2-[(4-苄基哌啶-1-基)甲基]苯并咪唑-5-基｝甲磺酰胺（XK1）和 N-[2-(4-苯氧基苄基)苯并咪唑-5-基]甲磺酰胺（XK2）]在 NMDA 受体 GluN2B 亚基上的结合域。
使用体外大鼠原代皮质神经元和表达异源 GluN1/GluN2B 受体的非洲爪蟾卵母细胞中的双电极电压钳，评估 XK1 和 XK2 的药理学拮抗特性。使用 GluN2B 的重组氨基末端结构域（ATD）直接测定配体结合。
XK1 和 XK2 有效防止了大鼠原代皮质神经元中 NMDA 诱导的兴奋性毒性。XK1（10 nM）和 XK2（1 nM）的低浓度显著逆转神经元死亡。两种化合物均未能抑制在异源表达 GluN1-1a 亚基与 GluN2B 亚基缺失的 ATD 共组装的卵母细胞中测量的电流。XK1 和 XK2 以低纳摩尔亲和力特异性结合重组 GluN2B ATD 蛋白。鉴定出 GluN2B ATD 中的几个残基对于赋予 XK1 和 XK2 敏感性是关键的。XK1 和 XK2 的抑制作用对 pH 敏感，在酸性 pH 值下增加。
这些结果表明 XK1 和 XK2 是体外有效的神经保护剂，并表明 5-取代苯并咪唑衍生物通过直接结合 GluN2B 亚基的 ATD 抑制 GluN1/GluN2B 受体。这些化合物作为研究含 GluN2B 的 NMDA 受体的药理学工具，是经典拮抗剂 ifenprodil 的有价值替代品。
