Structural and functional investigation of allosteric NMDA receptor modulation

NMDA型离子型谷氨酸受体介导中枢兴奋性神经传递 中枢神经系统（CNS），并在大脑功能中发挥关键作用。异常的NMDA受体信号转导与许多 CNS疾病和NMDA受体作为治疗靶点受到广泛关注。虽然 由于临床前研究结果难以转化为临床疗效，因此许多NMDA受体配体 这些研究创造了一个有价值的药理学工具箱，继续支持神经科学 research.大多数NMDA受体由两个结合甘氨酸的GluN 1和两个结合谷氨酸的GluN 2组成 亚单位。有四种不同的GluN 2亚基（GluN 2 A-D）赋予NMDA受体不同的功能， 功能特性以及CNS中不同的发育和区域表达。选择性调节 因此，含有特定GluN 2亚基的NMDA受体可以靶向受体亚型的子集 在疾病相关的神经元群体中表达。NMDA受体需要同时结合甘氨酸 (or D-丝氨酸）转化为GluN 1和谷氨酸转化为GluN 2进行激活，但主要依赖于谷氨酸的突触释放 因为细胞外甘氨酸（或D-丝氨酸）持续存在，所以在CNS中激活。因此，谷氨酸 与GluN 2的结合主要介导突触NMDA受体的阶段性激活，而与GluN 2的结合主要介导突触NMDA受体的激动剂占据， GluN 1可以调节反应幅度。我们将研究NMDA受体的两种不同模式， 调节，这两者都显示GluN 2亚基选择性，并影响GluN 1激动剂结合位点， 调节NMDA受体反应。在目标1中，我们将定义绑定触点和作用机制， 一类新的GluN 2A选择性负变构调节剂，其结合GluN 1之间的亚基界面 和GluN 2A激动剂结合结构域（ABD）以负调节激动剂与GluN 1的结合。在目标2中， 将确定新型甘氨酸位点激动剂GluN 2亚基特异性活性的结构基础， 在NMDA受体亚型中具有显著的高效力和前所未有的激动剂功效变化。通过 取代内源性甘氨酸或D-丝氨酸，这些GluN 2依赖性激动剂可以调节NMDA受体 当被突触谷氨酸释放激活时。在目的3中，我们将调节急性脑缺血时神经元NMDA受体， 使用新型GluN 2A选择性NAMs和GluN 1激动剂的啮齿动物脑切片。NMDA受体阳性神经元 在成人中枢神经系统中表达至少两种不同的GluN 2亚基，许多NMDA受体与 两种不同的GluN 2亚基（即三异聚体受体）。我们将评估小说的活跃程度 重组三异聚体NMDA受体亚型（GluN 1/2A/2B、GluN 1/2B/2D和GluN 2/2A/2B）的调节剂， GluN 1/2A/2C）和天然NMDA受体在不同的神经元群体中的表达，这些 GluN 2亚基组合。这些研究将揭示NMDA以前未被认识的特征 受体调节，并为基础和转化研究提供新的途径，以及促进 开发新的治疗药物。

NMDA-type ionotropic glutamate receptors mediate excitatory neurotransmission in the central nervous system (CNS) and play critical roles in brain functions. Aberrant NMDA receptor signaling is implicated in many CNS diseases, and NMDA receptors are receiving widespread interest as therapeutic targets. Although it has proven difficult to translate preclinical findings into clinical efficacy, the many NMDA receptor ligands from these studies have created a valuable pharmacological toolbox that continues to support neuroscience research. Most NMDA receptors are composed of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. There are four different GluN2 subunits (GluN2A-D) that endow NMDA receptors with distinct functional properties and different developmental and regional expression in the CNS. Selective modulation of NMDA receptors that contain a specific GluN2 subunit can therefore target a subset of receptor subtypes expressed in disease-relevant neuronal populations. NMDA receptors require simultaneous binding of glycine (or D-serine) to GluN1 and glutamate to GluN2 for activation, but mainly rely on synaptic release of glutamate for activation in the CNS, since extracellular glycine (or D-serine) is continuously present. Thus, glutamate binding to GluN2 primarily mediates phasic activation of synaptic NMDA receptors, while agonist occupancy at GluN1 can modulate response amplitude. We will investigate two distinct modes of NMDA receptor modulation, both of which display GluN2 subunit-selectivity and affect the GluN1 agonist binding site to modulate NMDA receptor responses. In Aim 1, we will define binding contacts and mechanism of action for a new class of GluN2A-selective negative allosteric modulators that bind the subunit interface between GluN1 and GluN2A agonist binding domains (ABDs) to negatively modulate agonist binding to GluN1. In Aim 2, we will determine the structural basis for GluN2 subunit-specific activity of novel glycine site agonists with remarkably high potency and unprecedented variation in agonist efficacy among NMDA receptor subtypes. By replacing endogenous glycine or D-serine, these GluN2-dependent agonists can modulate NMDA receptors when activated by synaptic glutamate release. In Aim 3, we will modulate neuronal NMDA receptors in acute rodent brain slices using novel GluN2A-selective NAMs and GluN1 agonists. NMDA receptor-positive neurons in the adult CNS express at least two different GluN2 subunits and many NMDA receptors are assembled with two different GluN2 subunits (i.e. triheteromeric receptors). We will evaluate the activity of the novel modulators at recombinant triheteromeric NMDA receptor subtypes (GluN1/2A/2B, GluN1/2B/2D, and GluN1/2A/2C) and at native NMDA receptors in distinct neuronal populations with defined expression of these GluN2 subunit combinations. These investigations will uncover previously unrecognized features of NMDA receptor modulation and provide new avenues for basic and translational research, as well as facilitate the development of novel therapeutic agents.