Mechanism of Action of Novel Subunit-Selective NMDA Receptor Modulators

描述(由申请人提供)：突触释放谷氨酸与突触后去极化一致，允许NMDA受体介导兴奋性突触电流中缓慢的、钙离子渗透的成分。由此产生的钙离子内流可以触发突触强度的变化，这被认为是学习和记忆的细胞关联。NMDA受体功能的降低阻断了多种形式的突触可塑性，而GluN2B亚单位表达的增强则增强了突触的可塑性。由于这些原因，GluN2B亚单位的变构增强剂一直被预测为认知增强剂，在包括阿尔茨海默病、损伤后运动学习和精神分裂症在内的广泛情况下具有潜在的临床应用价值。在前一个资助周期的支持下，我们确定了2类新的负变构NMDA受体调节剂和3类新的变构增强剂的结构决定因素和作用机制，它们对含有GluN2C或GluN2C/D亚单位的NMDA受体具有高度的选择性。在之前的资助周期中，我们还首次确定了几种类似药物的GluN2B变构增强剂。GluN2B增强剂的作用部位和作用机制与GluN2C/D增强剂不同。当GluN1的选择性剪接外显子5包含在氨基末端结构域中时，没有增强作用，这些新化合物显示出对次极大激动剂反应的强烈增强(&gt；5倍)，部分原因是谷氨酸和/或甘氨酸的变构增强。这些GluN2B增强子不作用于任何已知的调节剂结合部位，因此在NMDA受体调控方面具有新的突破。我们提出了三个实验，这些实验将促进我们对NMDA受体门控、这些调节剂的作用部位和机制、GluN2B在突触可塑性中的作用以及GluN2B增强在学习/记忆中的作用的理解。目的1：GluN2B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的重组和神经元型含GluN2B的NMDA受体来阐明两类化合物的代表性成员增强受体功能的机制。目标2：GluN2B增强剂的结构决定因素是什么？我们将利用嵌合策略和通过GluN1和GluN2B中的前M1/M1区域的突变来确定作用的结构决定因素。我们已经开发了叠氮和二苯甲酮光亲和标记，它们是活性的，应该共价标记His标记的GluN1和GluN2亚基结合口袋附近的残基。目的3：GluN2B功能增强是否会改变突触的可塑性？我们将评估GluN2B增强作用改变诱导海马片塑性蛋白(LTP，LTD)所需阈值的能力。目的4：GluN2B功能的增强是否会改变体内的学习和记忆？我们将在体内评估GluN2B变构增强剂对学习的影响，这种增强剂是脑部通透性的。

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.