High throughout screening assay:glutamate receptor (RMI)

描述(申请人提供)：离子型谷氨酸受体是哺乳动物中枢神经系统兴奋性突触信号的主要媒介。谷氨酸与这一受体的结合触发了蛋白质中跨膜离子通道的形成，允许阳离子沿着其电化学梯度流过突触后膜，使其去极化，从而刺激接收细胞。另一方面，过量的谷氨酸过度激活了离子型谷氨酸受体，引发了一系列导致神经元死亡的事件。这种兴奋毒性途径被认为是与多种急慢性疾病如中风、肌萎缩侧索硬化症和阿尔茨海默病相关的神经退行性变的主要因素，因此离子型谷氨酸受体的拮抗剂已被确定为治疗这些疾病的良好候选药物。大量的初步工作集中在谷氨酸受体的NMDA亚型的拮抗剂上，因为这些亚型是钙离子渗透性的。然而，近来有证据表明，钙离子通透型AMPA受体亚型可能确实是神经元死亡的主要介质之一。例如，ALS中观察到的运动神经元选择性死亡被认为是通过激活运动神经元上高密度的钙离子通透性AMPA受体而介导的。然而，目前可用于AMPA受体的拮抗剂在治疗这些疾病方面效果不佳，主要是因为它们的不溶性。因此，人们正在寻找可溶性的AMPA受体拮抗剂，高通量的筛选试验将极大地促进这一受体的拮抗剂的鉴定。传统上，放射性配体结合分析一直被用来筛选与这种重要蛋白质结合的药物。然而，识别出的药物必须随后通过膜片钳方法进行表征，以确定它们是否具有拮抗特性，这是极其劳动密集型和低产量的。在这里，我们建议开发一种基于荧光的高通量分析方法，它不仅允许筛选具有高特异性的与受体结合的药物，而且还将直接读出药物结合的功能后果(激活或抑制)，从而允许选择性地筛选具有所需功能特性的药物。

DESCRIPTION (provided by applicant): Ionotropic glutamate receptors are the main mediators of excitatory synaptic signals in the mammalian central nervous system. Glutamate binding to this receptor triggers the formation of transmembrane ion channels in the protein, permitting cations to flow down their electrochemical gradients across the postsynaptic membrane, depolarizing it and thereby stimulating the receiving cell. Excess glutamate, on the other hand, over activates the ionotropic glutamate receptors triggering a cascade of events resulting in neuronal death. This excitotoxic pathway is thought to be a major factor in neurodegeneration associated with a variety of acute and chronic disorders such as stroke, ALS, and Alzheimer's disease, and therefore antagonists of the ionotropic glutamate receptors have been identified as good candidates for the treatment of these diseases. A large body of initial work focused on the antagonists of the NMDA subtype of the glutamate receptors since these are Ca 2+ permeable. However, more recently it has become apparent that Ca 2+ permeable subtype of AMPA receptors may indeed be one of the primary mediators of neuronal death. For instance, selective death of motor neurons observed in ALS is thought to be mediated by the activation of the high density of Ca 2+ permeable AMPA receptors in motor neurons. The antagonists currently available for the AMPA receptors, however, are not as effective in the treatment of these diseases mainly due to their insolubility. There is hence an ongoing search for soluble antagonists of the AMPA receptor, which would be greatly facilitated by a high throughput screening assay that would be able to identify antagonists of this receptor. Traditionally, radioactive ligand binding assays have been used to screen for drugs that bind to this important protein. However, the drugs identified have to then be characterized by patch clamp methods to determine if they exhibit antagonistic properties, which is extremely labor intensive and low throughput. Here we propose to develop a fluorescence based high throughput assay that will not only allow for the screening of drugs that bind to the receptor with high specificity, but will also provide a direct readout of the functional consequences (activation or inhibition) of the drug binding, thus allowing for a selective screening of drugs with the desired functional properties.