Mechanism of action of novel subunit-selective NMDA receptor modulators

描述（由申请人提供）：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，介导脑和脊髓的兴奋性突触传递。NMDA受体是由2个甘氨酸结合NR1亚基和2个谷氨酸结合NR2亚基组成的异聚体，有4个家族成员（NR2A，B，C，D）。NMDA受体参与许多正常的脑功能，如神经元发育、学习、记忆，除了在中风、癫痫和神经精神疾病等神经病理条件中发挥作用外。自首次描述以来，nr2b选择性NMDA受体拮抗剂伊芬普罗地尔及其许多类似物已被用于大量研究，探索该亚基在脑功能的几乎每个方面的作用，包括行为、认知、突触可塑性、神经元发育和神经病理学。然而，对于NR2A-、NR2C-或nr2d受体，尚无高选择性拮抗剂被描述。因此，我们开发了一种检测方法来鉴定含有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种化合物作为新的亚单位选择性拮抗剂和增强剂。结果是鉴定出两类结构独特的化合物，与含有NR2A或nr2b的受体或AMPA/kainate受体相比，它们是NR1/NR2C和NR1/NR2D的100-500倍选择性抑制剂。我们还发现了两种结构上不同的增强剂，它们对NR2C/ d -含受体具有选择性。这些新的非竞争性变构调节剂为研究NR2C/D亚基在正常脑功能和神经系统疾病中的作用提供了一个突破性的机会。本建议使用这些亚单元选择性调制器解决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？我们将利用位点定向诱变，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？我们将在脑切片中评估皮质-丘脑下神经元突触中含有NR2C/ d的NMDA受体和海马间神经元传入兴奋突触中含有NR2C/ d的NMDA受体的抑制和增强作用。实验将测试NR2C/D调节剂存在时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神经元间和丘脑下神经元的兴奋性和峰值频率是否可以通过抑制或增强NR2C/ d -含受体而改变。公共卫生相关性：NMDA受体介导中枢神经系统神经元之间的通信，因此在几乎所有脑功能以及许多神经系统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亚基组成的四聚体组合，其中有四个亚型（NR2A-D）。由于不同的NR2亚基表达不同，具有不同的功能特性，它们在正常脑功能和疾病中发挥着不同的作用。虽然亚基选择性调节剂已被认为是许多神经系统疾病的有用治疗剂，但目前只发现了一种真正选择性的NR2亚基选择性拮抗剂。伊芬普罗地尔及其类似物对含nr2b受体的抑制作用比含NR2A、C、d的NMDA受体强500倍以上。自1993年首次描述伊芬普罗地尔的nr2b选择性以来，没有新的高选择性亚基特异性化合物被描述。由于缺乏药理学试剂，我们开始寻找作用于NR2C-和nr2d -受体的新配体。我们已经发现了至少三种NMDA受体拮抗剂，与NR2A-或nr2b受体相比，它们对NR2C-和nr2d受体的作用强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的影响。这一信息将有助于深入了解受体的功能，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.