Development of biotin-tagged affinity ligands and fluorophore-conjugated probes for the study of native kainate receptors

脑部疾病的社会负担和成本是巨大的，如果不能更好地了解中枢神经系统中神经活动的调节方式，就不可能在治疗或预防这些疾病方面取得重大进展。神经元通过被称为神经递质受体的蛋白质进行交流。大脑中几乎所有的兴奋性神经递质受体都被氨基酸谷氨酸激活。谷氨酸是以高度调节和活性依赖的方式从突触处的神经元释放出来的。有几种不同类别的受体可以对谷氨酸作出反应。其中最不典型的是红藻氨酸受体(KARs)。KARs调节神经元网络的活动，这对正常的大脑功能是必不可少的。它们还参与神经元发育和神经系统疾病，如癫痫、慢性疼痛、偏头痛、精神分裂症和神经退行性变，它们是治疗药物设计的潜在靶点。旨在确定新的和潜在的治疗性化合物的药物发现工作依赖于对新化合物所针对的神经递质受体的分子组成、结构和药理学特性的详细了解。在过去的几年里，很明显，受体蛋白在与其他蛋白质相互作用的复杂网络中运作。许多相互作用调节受体蛋白的运输、定位和移动，而其他相互作用则影响其急性功能和药理特性。已发现许多蛋白质与谷氨酸受体相互作用。这导致了复杂的蛋白质-蛋白质相互作用，可以对突触功能产生深远的影响。然而，在不同脑区的不同类型神经元中，天然KAR的相互作用伙伴对其功能和药理学特性的调节的一般原理尚不清楚。获得更多关于KAR相关蛋白的信息是很重要的，因为对KAR调节机制的深入了解将使它们参与正常的大脑功能和神经疾病得到更好的定义。在本研究项目中，我们旨在通过将新开发的化合物制成亲和探针来定义KAR在中枢神经系统中的分子组织、分布、蛋白质相互作用、药理特性和调节。基于KAR与各种药物相互作用的分子模拟，我们将设计、合成和表征新的药理试剂，这些试剂与KAR具有高亲和力，可用于选择性地鉴定、分离和分析脑样本中的天然受体和蛋白质相互作用伙伴。这些新的药理工具将用于分离KARs和相关蛋白质。对这些分离的蛋白质复合体的系统分析将提供关于大脑不同功能重要区域和神经元亚细胞室中KARs的分子组成、相互作用和调控的基本信息。一些相互作用的伙伴(称为辅助亚基)很可能显著影响KARs的药理性质，这可能对药物开发具有重要意义。因此，我们将建立辅助亚基对KAR药理性质的影响。建议的研究结果可能有助于未来开发治疗癫痫、慢性疼痛、偏头痛、精神分裂症和神经退行性疾病的药物。这些疾病中的许多目前还没有有效的治疗方法，对国家的健康和财富极其有害。

The social burden and cost of brain disorders is enormous, and no serious progress can be expected in the treatment or the prevention of these disorders without better understanding of the way neuronal activity is regulated in the central nervous system. Neurones communicate via proteins known as neurotransmitter receptors. Nearly all of the excitatory neurotransmitter receptors in the brain are activated by the amino acid glutamate. Glutamate is released from neurones at synapses in a highly regulated and activity dependent manner. There are several different classes of receptors which respond to glutamate. The least well characterised of these are the kainate receptors (KARs). KARs regulate the activity of neuronal networks, which is essential for normal brain function. They are also involved in neuronal development and neurological conditions such as epilepsy, chronic pain, migraine, schizophrenia and neurodegeneration and they represent a potential target for therapeutic drug design. Drug discovery efforts aimed at identifying new and potentially therapeutic compounds rely on detailed knowledge of the molecular composition, structure and pharmacological properties of the neurotransmitter receptors to which new compounds are targeted. Over the last few years, it has become clear that receptor proteins operate within a complex web of interactions with other proteins. Many interactions serve to modulate the transport, localisation and mobility of receptor proteins, while others affect their acute functioning and pharmacological properties. Many proteins have been found to interact with glutamate receptors. This gives rise to complex protein-protein interactions that can have a profound effect on synaptic function. However, the general principles that govern the modulation of the function and pharmacological properties of native KARs by their interaction partners in different types of neurones in various brain regions are not clear. It is important to gain more information about KAR associated proteins, because increased understanding of the mechanisms that regulate KARs will allow their involvement in normal brain function and neurological disorders to be better defined.In this research project we aim to define the molecular organisation, distribution, protein interactions, pharmacological properties and regulation of KARs in the central nervous system by using newly developed compounds into affinity probes. Based on molecular modelling of KAR interactions with various drugs we will design, synthesise and characterise new pharmacological reagents, which specifically bind to KARs with high affinity and can be used for the selective identification, isolation and analysis of the native receptors and protein interaction partners directly in brain samples. These new pharmacological tools will be used to isolate KARs and associated proteins. The systematic analysis of these isolated protein complexes will provide basic information about the molecular composition, interactions and regulation of KARs in different functionally important regions of the brain and subcellular compartments of neurones. It is very likely that some of the interaction partners (called auxiliary subunits) significantly influence the pharmacological properties of KARs and this may have important implications for drug development. Therefore, we will establish the effects of auxiliary subunits on the pharmacological properties of KARs.The results of the proposed studies may help the future development of therapeutic agents for the treatment of epilepsy, chronic pain, migraine, schizophrenia and neurodegenerative diseases. Many of these disorders currently have no effective treatments and are extremely deleterious to the health and wealth of the nation.