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)，在大脑功能中起着关键作用。异常的NMDA受体信号转导与许多 中枢神经系统疾病和NMDA受体作为治疗靶点正受到广泛关注。尽管它已经 事实证明，很难将临床前的研究结果转化为临床疗效，来自 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 研究。大多数NMDA受体由两个甘氨酸结合的GluN1和两个谷氨酸结合的GluN2组成 亚单位。有四个不同的GluN2亚基(GluN2A-D)赋予NMDA受体不同的 中枢神经系统的功能特性和不同发育和区域表达。有选择地调制 因此，包含特定GluN2亚单位的NMDA受体可以靶向受体亚型的子集 在与疾病相关的神经元群体中表达。NMDA受体需要与甘氨酸同时结合 (或D-丝氨酸)到GluN1和谷氨酸到GluN2的激活，但主要依靠突触释放谷氨酸 对于中枢神经系统的激活，因为细胞外甘氨酸(或D-丝氨酸)持续存在。因此，谷氨酸 与GluN2的结合主要介导突触NMDA受体的时相激活，而激动剂在 GluN1具有调节反应幅度的作用。我们将研究NMDA受体的两种不同模式 调节，两者都表现出GluN2亚单位的选择性，并影响GluN1激动剂结合部位 调节NMDA受体的反应。在目标1中，我们将定义具有约束力的联系和作用机制 结合GluN1亚基界面的新型GluN2A型选择性负变构调节剂 和GluN2A激动剂结合域(ABD)，以负向调节激动剂与GluN1的结合。在目标2中，我们 将确定新型甘氨酸位点激动剂GluN2亚基特异性活性的结构基础 NMDA受体亚型之间的激动剂效力具有显著的高效性和前所未有的差异。通过 取代内源性甘氨酸或D-丝氨酸，这些依赖谷氨酸的激动剂可以调节NMDA受体 当被突触释放的谷氨酸激活时。在目标3中，我们将在急性脑缺血时调节神经元NMDA受体。 使用新型GluN2A选择性NAMS和GluN1激动剂的啮齿动物脑片。NMDA受体阳性神经元 在成年中枢神经系统中，至少表达两个不同的GluN2亚基，许多NMDA受体与 两个不同的GluN2亚基(即三异构体受体)。我们将对这部小说的活动进行评估 重组三异构体NMDA受体亚型(GluN1/2A/2B、GluN1/2B/2D和 GluN1/2A/2C)和天然NMDA受体在不同神经元群体中有明确的表达 Glun2亚基组合。这些调查将揭示NMDA以前未被识别的特征 受体调节，并为基础和翻译研究提供新的途径，以及促进 新型治疗剂的开发。

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.