Zeng Yue, Zheng Yueming, Zhang Tongtong, Ye Fei, Zhan Li, Kou Zengwei, Zhu Shujia, Gao Zhaobing
Center for Neurological and Psychiatric Research and Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
College of Pharmacy, University of Chinese Academy of Sciences, Beijing, China.
Front Pharmacol. 2022 Jun 9;13:888308. doi: 10.3389/fphar.2022.888308. eCollection 2022.
N-methyl-D-aspartate receptors (NMDARs) are Ca-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca-permeability and lower Mg-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 μM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.
N-甲基-D-天冬氨酸受体（NMDARs）是中枢神经系统中可通透钙离子的离子型谷氨酸受体（iGluRs），在神经元发育和突触可塑性中发挥重要作用。传统的NMDARs通常由GluN1和GluN2亚基组成，其生物物理特性与含GluN3的NMDARs不同：含GluN3的NMDARs比传统NMDARs具有更小的单位电导、更低的钙离子通透性和更低的镁离子敏感性。然而，针对含GluN3的NMDARs的特异性调节剂非常少。在此，我们开发了一种基于细胞的高通量钙检测方法，并鉴定出3-氟-1,2-亚苯基双（3-羟基苯甲酸）（WZB117）作为GluN1/GluN3受体的相对选择性抑制剂。WZB117对HEK-293细胞中表达的GluN1/GluN3A受体的IC值为1.15±0.34μM。一致地，在存在CGP-78608的情况下，WZB117对甘氨酸诱导的电流表现出强烈的抑制活性，但对急性分离的大鼠海马神经元中NMDA、KA和AMPA诱导的电流仅有轻微影响。在这四种内源性电流中，只有第一种主要由GluN1/GluN3受体介导。机制研究表明，WZB117以不依赖甘氨酸、电压和pH的方式抑制GluN1/GluN3A受体，表明它是一种变构调节剂。定点突变和嵌合体构建进一步揭示，WZB117可能作用于GluN3A的M1前区，其关键决定因素与先前鉴定的调节剂不同。总之，我们的研究开发了一种发现含GluN3的NMDARs调节剂的有效方法，并将WZB117表征为GluN1/GluN3受体的新型变构抑制剂。
