Mechanism of Action of Novel Subunit-Selective NMDA Receptor Modulators

描述（由申请人提供）：与突触后去极化同时发生的谷氨酸的突触释放允许NMDA受体介导兴奋性突触电流的缓慢的Ca 2+可渗透成分。由此产生的Ca 2+流入可以触发突触强度的变化，这被认为是学习和记忆的细胞相关性。NMDA受体功能的降低阻断了许多形式的突触可塑性，而GluN 2B亚基表达的增强增强了突触可塑性。由于这些原因，GluN 2B亚基的变构增效剂长期以来被预测为认知增强剂，在广泛的病症中具有潜在的临床用途，包括阿尔茨海默病、损伤后运动学习和精神分裂症。上一个资助周期支持的工作使我们能够定义2种新型负变构NMDA受体调节剂和3种新型变构增效剂的结构决定因素和作用机制，这些变构增效剂对含有GluN 2C或GluN 2C/D亚基的NMDA受体具有高度选择性。在上一个融资周期中，我们还首次确定了几种药物样GluN 2B变构增效剂。GluN 2B增效作用的位点和机制与GluN 2C/D增效剂不同。当GluN 1的可变剪接外显子5包含在氨基末端结构域中时，不存在增强作用，并且这些新化合物显示对次最大激动剂反应的反应的强增强作用（>5倍），这部分是由于谷氨酸和/或甘氨酸效力的变构增强。这些GluN 2B增效剂不作用于任何已知的调节剂结合位点，因此在NMDA受体调节方面开辟了新天地。我们提出了3个实验，将推进我们的理解NMDA受体门控，这些调制器的网站和机制的作用，GluN 2B在突触可塑性的作用，和GluN 2B增强学习/记忆的影响。 目的1：GluN 2B增强的作用机制是什么？我们将使用全细胞电压钳和单通道记录从重组和神经元GluN 2B-含有NMDA受体，以阐明两类化合物的代表性成员增强受体功能的机制。 目的2：GluN 2B增效剂的结构决定因素是什么？我们将利用嵌合策略和诱变通过前M1/M1区的GluN 1和GluN 2B，以确定结构决定因素的行动。我们已经开发了叠氮化物和二苯甲酮的光亲和标签，是积极的，应该共价标记残基附近的His标记的GluN 1和GluN 2亚基的结合口袋。 目的3：GluN 2B功能增强是否改变突触可塑性？我们将评估GluN 2B增强改变海马切片中诱导可塑性改变（LTP，LTD）所需阈值的能力。 目的4：增强GluN 2B功能是否改变体内学习和记忆？我们将评估在体内学习的一类GluN 2B变构增效剂，是脑渗透的影响。

DESCRIPTION (provided by applicant): Synaptic release of glutamate coincident with postsynaptic depolarization allows NMDA receptors to mediate a slow, Ca2+-permeable component of the excitatory synaptic current. The resulting influx of Ca2+ can trigger changes in synaptic strength that have been proposed as a cellular correlate of learning and memory. Reduction of NMDA receptor function blocks many forms of synaptic plasticity, and enhancement of the GluN2B subunit expression enhances synaptic plasticity. For these reasons, allosteric potentiators of the GluN2B subunit have long been predicted to act as cognitive enhancers, with potential clinical utility in a wide range of conditions including Alzheimer's disease, post-injury motor learning, and schizophrenia. Work supported by the previous funding cycle allowed us to define the structural determinants and mechanism of action of 2 new classes of negative allosteric NMDA receptor modulators and 3 new classes of allosteric potentiators that are highly selective for NMDA receptors containing the GluN2C or GluN2C/D subunits. During the previous funding cycle, we also identified for the first time several drug-like GluN2B allosteric potentiators. The site and mechanism of action of GluN2B potentiation is distinct from that observed for GluN2C/D potentiators. Potentiation is absent when the alternatively spliced exon5 of GluN1 is included in the amino terminal domain, and these new compounds show strong potentiation (>5-fold) of responses to submaximal agonist responses in part due to allosteric enhancement of glutamate and/or glycine potency. These GluN2B potentiators do not act at any known modulator binding site, and thus break new ground in terms of NMDA receptor regulation. We propose 3 experiments that will advance our understanding of NMDA receptor gating, the site and mechanism of action of these modulators, the role of GluN2B in synaptic plasticity, and the effects of GluN2B potentiation in learning/memory. Aim 1: What is the mechanism of action of GluN2B potentiation? We will use whole cell voltage clamp and single channel recordings from recombinant and neuronal GluN2B-containing NMDA receptors to elucidate the mechanism by which representative members of two classes of compounds potentiate receptor function. Aim 2: What are the structural determinants of GluN2B potentiators? We will utilize a chimeric strategy and mutagenesis through of preM1/M1 regions in GluN1 and GluN2B to identify the structural determinants of action. We have developed azide and benzophenone photoaffinity labels that are active and should covalently label residues near the binding pocket of His-tagged GluN1 and GluN2 subunits. Aim 3: Does potentiation of GluN2B function alter synaptic plasticity? We will assess the ability of GluN2B potentiation to shift the threshold necessary to induce plasticit (LTP, LTD) in hippocampal slices. Aim 4: Does potentiation of GluN2B function alter learning and memory in vivo? We will assess in vivo the effects on learning for the class of GluN2B allosteric potentiator that is brain permeable.