High throughout screening assay:glutamate receptor (RMI)

描述（由申请人提供）：离子型谷氨酸受体是哺乳动物中枢神经系统中兴奋性突触信号的主要介质。 谷氨酸与该受体结合，触发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离子型谷氨酸受体，引发导致神经元死亡的级联事件。 这种兴奋性毒性途径被认为是与各种急性和慢性疾病如中风、ALS和阿尔茨海默病相关的神经变性的主要因素，因此离子型谷氨酸受体的拮抗剂已被鉴定为治疗这些疾病的良好候选物。 大量的初始工作集中在谷氨酸受体的NMDA亚型的拮抗剂上，因为它们是Ca 2+可渗透的。 然而，最近的研究表明，AMPA受体的Ca2+渗透亚型可能确实是神经元死亡的主要介质之一。 例如，在ALS中观察到的运动神经元的选择性死亡被认为是由运动神经元中高密度的Ca 2+渗透性AMPA受体的激活介导的。 然而，目前可用于AMPA受体的拮抗剂在治疗这些疾病中不那么有效，主要是由于它们的不溶性。 因此，对AMPA受体的可溶性拮抗剂的研究正在进行中，这将通过能够鉴定该受体的拮抗剂的高通量筛选测定而大大促进。 传统上，放射性配体结合试验已被用于筛选与这种重要蛋白质结合的药物。 然而，所鉴定的药物必须随后通过膜片钳方法表征以确定它们是否表现出拮抗性质，这是极其劳动密集型和低通量的。 在这里，我们建议开发一种基于荧光的高通量测定，其不仅允许以高特异性筛选与受体结合的药物，而且还将提供药物结合的功能后果（激活或抑制）的直接读出，从而允许选择性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.