Mechanism of action of novel subunit-selective NMDA receptor modulators

描述(申请人提供)：N-甲基-D-天冬氨酸选择性谷氨酸受体(NMDA受体)是配体门控离子通道，在大脑和脊髓中介导兴奋性突触传递。NMDA受体是由两个甘氨酸结合的NR1亚基和两个谷氨酸结合的NR2亚基组成的异源多聚体，其中有四个家族成员(NR2A、B、C、D)。NMDA受体除了在中风、癫痫和神经精神障碍等神经病理疾病中发挥作用外，还参与许多正常的大脑功能，如神经元发育、学习和记忆。自从NR2B选择性NMDA受体拮抗剂ifenprodil及其许多类似物被首次描述以来，已有大量研究探索该亚单位在大脑功能的各个方面的作用，包括行为、认知、突触可塑性、神经元发育和神经病理学。然而，目前还没有针对含NR2A、NR2C或NR2D受体的高选择性拮抗剂的报道。因此，我们开发了一种方法来鉴定含有NR2C和NR2D的NMDA受体的非竞争性变构调节剂，并随后筛选了约60,000个化合物来寻找新的亚基选择性拮抗剂和增效剂。结果是鉴定了两类结构独特的化合物，它们是NR1/NR2C和NR1/NR2D的选择性抑制剂，是含有NR2A或NR2B的受体或AMPA/海人藻酸受体的100-500倍。我们还鉴定了两类结构不同的增强剂，它们对含有NR2C/D的受体具有选择性。这些新的非竞争性变构调节剂为研究NR2C/D亚单位在正常脑功能和神经疾病中的作用提供了一个突破性的机会。这项提案使用这些亚单位选择性调制器解决了3个问题。1.NR2C/D选择性抑制剂和增强剂的结构决定因素是什么？我们将利用定点突变，利用NR2A/B和NR2C/D之间的序列差异，确定介导三类NR2C/D选择性增强剂和抑制剂作用的关键残基。2.NR2C/D选择性抑制剂和增强剂的作用机制是什么？我们将分析在电压钳下记录的宏观和单通道电流，以确定非竞争性NR2C/NR2D抑制剂以及NR2C/NR2D增强剂的作用机制。3.NR2C/D调节剂如何改变突触信号和神经元兴奋性？我们将评估皮质-丘脑-底核神经元突触上含NR2D受体和传入兴奋性突触上含NR2C/D受体对脑片中海马神经元的抑制和增强作用。实验将测试在NR2C/D调制器存在的情况下，突触对刺激序列的反应。我们还将使用这些新的药理学工具来研究是否可以通过抑制或增强含有NR2C/D的受体来改变神经元和丘脑下层神经元的兴奋性和尖峰频率。与公共健康相关：NMDA受体介导中枢神经系统神经元之间的通讯，因此在几乎所有大脑功能和许多神经疾病中发挥重要作用。NMDA受体是由两个甘氨酸结合的NR1亚基和两个谷氨酸结合的NR2亚基组成的四聚体，其中有四种亚型(NR2A-D)。由于不同的NR2亚单位有不同的表达和不同的功能特性，它们在正常大脑功能和疾病中扮演着许多不同的角色。虽然亚单位选择性调节剂已被认为是一些神经系统疾病的有效治疗药物，但只有一种真正选择性的NR2亚单位选择性拮抗剂被发现。依芬地尔及其类似物对含NR2B受体的抑制作用是含NR2A、C、D受体的500倍以上。自从1993年首次描述依芬地尔的NR2B选择性以来，还没有新的高度选择性的亚基特异性化合物被描述过。由于缺乏药理试剂，我们不得不寻找作用于NR2C和NR2D受体的新配体。我们已经发现了至少三类NMDA受体功能的拮抗剂，它们在含有NR2C和NR2D的受体上的效力是含有NR2A或NR2B的受体的100-500倍。目前的拨款建议进行实验，以确定这些新化合物在NR2D亚基上的作用位置，它们如何控制NMDA受体功能，以及它们对突触传递和神经元兴奋性有什么影响。这些信息将提供对受体功能的洞察，并首次确定NR2C和NR2D亚基在突触传递中的作用。

DESCRIPTION (provided by applicant): N-methyl-D-aspartate-selective glutamate receptors (NMDA receptors) are ligand-gated ion channels that mediate excitatory synaptic transmission in the brain and spinal cord. NMDA receptors are heteromultimers comprised of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four family members (NR2A,B,C,D). NMDA receptors are involved in many normal brain functions such as neuronal development, learning, memory, in addition to their roles in neuropathological conditions such as stroke, epilepsy, and neuropsychiatric disorders. Since its first description, the NR2B-selective NMDA receptor antagonist ifenprodil and its many analogues have been used in a multitude of studies exploring the role of this subunit in virtually every aspect of brain function including behavior, cognition, synaptic plasticity, neuronal development, and neuropathology. However, no highly selective antagonists have yet been described for NR2A-, NR2C-, or NR2D-containing receptors. We therefore developed an assay to identify non-competitive allosteric modulators of NR2C- and NR2D-containing NMDA receptors, and subsequently screened ~60,000 compounds for new subunit-selective antagonists and potentiators. The result was the identification of two structurally unique classes of compounds that are 100-500 fold selective inhibitors of NR1/NR2C and NR1/NR2D compared to NR2A- or NR2B-containing receptors or AMPA/kainate receptors. We also identified two structurally distinct classes of potentiators that are selective for NR2C/D-containing receptors. These new non-competitive allosteric modulators represent a breakthrough opportunity to study the role of the NR2C/D subunits in normal brain function and in neurological diseases. This proposal addresses 3 questions using these subunit-selective modulators. 1. What are the structural determinants of NR2C/D-selective inhibitors and potentiators? We will utilize site-directed mutagenesis, exploiting sequence differences between NR2A/B and NR2C/D, to identify key residues that mediate the actions of three classes of NR2C/D-selective potentiators and inhibitors. 2. What is the mechanism of action of NR2C/D selective inhibitors and potentiators? We will analyze macroscopic and single channel currents recorded under voltage clamp to define the mechanism of action of the non-competitive NR2C/NR2D inhibitors as well as NR2C/NR2D potentiators. 3. How do NR2C/D modulators alter synaptic signaling and neuronal excitability? We will evaluate the effect of inhibition and potentiation of NR2D-containing NMDA receptors at the cortical-subthalamic neuron synapse and NR2C/D-containing NMDA receptors at afferent excitatory synapses onto hippocampal interneurons in brain slices. Experiments will test the synaptic response to stimulus trains in the presence of NR2C/D modulators. We will also use these new pharmacological tools to investigate whether interneuron and subthalamic neuron excitability and spiking frequency can be altered through inhibition or potentiation of NR2C/D-containing receptors. PUBLIC HEALTH RELEVANCE: NMDA receptors mediate communication between neurons in the central nervous system, and thus play an important role in virtually all brain functions as well as numerous neurological diseases. The NMDA receptors are tetrameric assemblies comprised of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four subtypes (NR2A-D). Because different NR2 subunits are differentially expressed and have different functional properties, they serve a host of divergent roles in both normal brain function and disease. Although subunit-selective modulators have been proposed to be useful therapeutic agents in a number of neurological diseases, only one truly selective NR2 subunit-selective antagonist has been discovered. Ifenprodil and its analogues inhibit NR2B-containing receptors over 500-fold more potently than NR2A,C,D-containing NMDA receptors. Since the first description of the NR2B-selectivity of ifenprodil in 1993, no new highly selective subunit- specific compounds have been described. This lack of pharmacological reagents has led us to search for new ligands that act at NR2C- and NR2D-containing receptors. We have found at least three classes of antagonists of NMDA receptor function that are 100-500 fold more potent at NR2C- and NR2D-containing receptors when compared to NR2A- or NR2B-containing receptors. The current grant proposes experiments that define where on the NR2D subunit these novel compounds act, how they control NMDA receptor function, and what effect they have on synaptic transmission and neuronal excitability. This information will provide insight into receptor function, and define for the first time the role of the NR2C and NR2D subunits in synaptic transmission.