Allosteric Modulators of Glutamate Receptors

描述(申请人提供)：离子型谷氨酸受体控制多种正常的神经元过程，包括学习和记忆。这些重要的神经递质受体的激活与许多神经退行性疾病有关，特别是中风和癫痫。此外，增强AMPA亚型谷氨酸受体(变构调节剂)谷氨酸活性的药物已被证明可以改善认知，并可能对阿尔茨海默病等神经退行性疾病有好处。与谷氨酸结合的AMPA受体结构域(S1S2结构域)可以在细菌中以可溶性蛋白质的形式产生，并可以用各种高分辨率的生物物理方法进行研究。S1S2结构域已被证明是研究药物与AMPA受体相互作用的一个很好的模型系统，因为至少在激动剂结合部位，结合亲和力与膜结合的全长四聚体蛋白相同，结合时的构象转变似乎也相似。该蛋白在6 mM以下为单体，但变构调节剂可以促进S1S2结构域的二聚化，同时阻止膜结合蛋白的脱敏和减慢失活速度。我们测定了一个AMPA受体结合域(GluR2 S1S2)与六种不同的变构激活剂的晶体结构，并用核磁共振波谱研究了其中一些配合物。考虑到在所有结构中产生的结合相互作用，可以在二聚体界面处定义相对较大的结合表面。尽管在变构调节剂的功能效应方面做了大量的工作，但对结合的机制，导致功能结果的结构相互作用，甚至在某些情况下，结合的化学计量比知之甚少。我们建议使用X射线结晶学、核磁共振光谱、等温滴定量热法和分析性超速离心法，结合全细胞记录来详细定义结合作用。目标将是了解药物如何结合到这个表面(单体和二聚体，是什么控制着亚位点的特异性)，如何放大亲和力，结合相互作用如何与功能结果相关，以及如何在这个位点提供尽可能多的特异性。此外，我们已经设计并正在合成二聚体形式的变构激活剂，我们预计它们将以比目前可用的药物更高的亲和力和特异性结合。我们相信，对本申请中描述的这些药物与AMPA受体相互作用的详细研究将为设计有效的增强认知药物提供新的线索。 公共卫生相关性：AMPA受体介导了中枢神经系统中大部分快速兴奋性突触传递。AMPA受体活性的增强已被证明对提高认知能力是有益的，例如，放大谷氨酸(变构激活剂)作用的药物可能在治疗阿尔茨海默病和其他神经疾病方面有效。这项应用的目标是了解变构激活剂的结合部位，目的是确定这些药物的作用机制和功能效应的结构相关性。

DESCRIPTION (provided by applicant): Ionotropic glutamate receptors control a wide variety of normal neuronal processes including learning and memory. Activation of these important neurotransmitter receptors is involved in a number of neurodegenerative diseases, notably stroke and epilepsy. In addition, drugs that enhance the activity of glutamate at the AMPA subtype of glutamate receptors (allosteric modulators) have been shown to improve cognition and may have benefits in neurodegenerative diseases such as Alzheimer's disease. The domain of AMPA receptors that binds glutamate (S1S2 domain) can be produced as a soluble protein in bacteria and studied with a variety of high-resolution biophysical methods. The S1S2 domain has been shown to be an excellent model system for the study of drugs that interact with AMPA receptors in that, at least at the agonist binding site, the binding affinity is the same as the membrane-bound, full-length tetrameric protein and the conformational transitions upon binding seem to be similar as well. The protein is monomeric below 6 mM, but allosteric modulators can promote dimerization of the S1S2 domain, while blocking desensitization and slowing the rate of deactivation in the membrane-bound protein. We have determined the crystal structure of one AMPA receptor-binding domain (GluR2 S1S2) with six different allosteric activators and studied some of the complexes using NMR spectroscopy. Considering the binding interactions made in all of the structures, a relatively large binding surface at the dimer interface can be defined. Despite considerable work on the functional effects of allosteric modulators, very little is known about the mechanism of binding, the structural interactions that lead to a functional outcome, and, in some cases, even the stoichiometry of binding. We propose to define the binding interaction in detail using X-ray crystallography, NMR spectroscopy, isothermal titration calorimetry, and analytical ultracentrifugation, in conjunction with whole cell recording. The goal will be to understand how drugs bind to this surface (monomer vs. dimer, what controls the subsite specificity), how to amplify affinity, how binding interactions are related to functional outcomes, and how to provide as much specificity as possible at this site. In addition, we have designed and are synthesizing dimeric forms of allosteric activators that we anticipate should bind with higher affinity and specificity than currently available drugs. We believe that detailed studies of the interaction of these drugs with AMPA receptors described in this application will lead to new clues as to the design of effective drugs for enhancing cognition. PUBLIC HEALTH RELEVANCE: AMPA receptors mediate the majority of fast excitatory synaptic transmission in the central nervous system. Enhancement of the activity of AMPA receptors has been shown to be beneficial in increasing cognition, such that drugs that amplify the effect of glutamate (allosteric activators) may be effective in treatment of Alzheimer's disease and other neurological disorders. This application is targeted toward understanding the binding site for allosteric activators with the goal of defining the mechanism of action and the structural correlates of the functional effects of these drugs.