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Chemical modification of ion channels: development of a novel and fast binding assay for ion channel inhibitors

离子通道的化学修饰：开发离子通道抑制剂的新型快速结合测定法

基本信息

批准号：
EP/E042139/1
负责人：
Mike Shipman
金额：
$ 24.94万
依托单位：
University of Warwick
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2008
资助国家：
英国
起止时间：
2008 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=EP%2FE042139%2F1
关键词：
Chemical modification ion channels development

项目摘要

Ligand-gated ion channels are proteins located at nerve terminals, also called synapses. They are responsible for the rapid transmission of nerve impulses from one nerve cell to the next one. Ligand-gated ion channels are transmembrane proteins and mostly comprise several subunits. The crucial physiological importance of ligand-gated ion channel becomes apparent when the channel function is impaired. In fact, numerous mutations in ligand-gated ion channel genes are known to cause neurological diseases. In addition, ligand-gated ion channels are the site of action of many therapeutic drugs as well as toxins from snakes, spiders and wasps.Small organic molecules (neurotransmitters) are released into the synaptic cleft from the pre-synaptic cell and they bind to the ligand-gated ion channel resulting in a conformational transition from a non-conducting closed state to a conducting open state. High ion flux across the biological membrane in the open channel state triggers further events in the post-synaptic cell and finally leads to the generation of a new action potential and transmission of the nerve impulse.There is evidence that small molecules which block channel function specifically may be useful for treatment of certain psychiatric disorders such as anxiety, drug-dependence, schizophrenia and cognitive dysfunction. Although there have been considerable achievements in the past ten years to solve the exact three-dimensional structure of these large proteins these structures are not accurate enough to allow rational structure-based drug design. As a result many different compounds or libraries of compounds are being synthesised and have to be tested against the ion channel receptors. Current screening methods are time consuming and some even rely on using radioactive compounds.The serotonin 5-HT3 ion channel receptor is the focus of this project. It functions as a pentamer of five identical subunits. Each subunit has an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain is predominantly located in the synaptic cleft and is responsible for forming the neurotransmitter binding site. This research proposal describes the development of a fast and efficient binding assay which is based on detection of fluorescence and which would be amenable for high-throughput screening. In the proposed project the serotonin 5-HT3 receptor, which can be expressed in mammalian cells using standard molecular biological techniques, will be chemically modified with a fluorescent dye near the binding site using a photochemical reaction and designed synthetic modifier compounds. Specially designed small organic molecules will be synthesised which will bind to the modified receptor and quench its fluorescence. Any new compound which has a higher binding affinity than the quencher compound will displace the quencher and as a result alter the fluorescence of the modified receptor. The restoration of fluorescence could be detected using an appropriate read-out. Hence increase in fluorescence upon addition of a new compound would mean it is binding to the ion channel receptor and accordingly represents a potential receptor inhibitor. Once set up the binding assay would be tested using known 5-HT3 receptor inhibitors in order to compare the results with data obtained by traditional analysis methods.Apart from being used in the proposed binding assay, undoubtedly, a fluorescent 5-HT3 receptor could prove a very useful tool to address fundamental questions of how these ligand-gated ion channels work and how the binding event is structurally linked to the channel opening event. Such improved knowledge about the function and structure would be very valuable for drug design and ultimately will lead to better and more selective compounds. Furthermore, if successfully developed, the same concept could be used to tackle more complex but pharmacologically more important ion channel receptors.

配体门控离子通道是位于神经末梢的蛋白质，也称为突触。它们负责神经冲动从一个神经细胞到下一个神经细胞的快速传递。配基门控离子通道是一种跨膜蛋白，主要由几个亚基组成。当配体门控离子通道的功能受损时，其重要的生理意义就变得明显起来。事实上，已知配体门控离子通道基因的大量突变会导致神经系统疾病。此外，配体门控离子通道是许多治疗药物以及蛇、蜘蛛和黄蜂毒素的作用部位，小分子(神经递质)从突触前细胞释放到突触间隙，与配体门控离子通道结合，导致构象从非传导闭合状态转变为传导开放状态。在开放通道状态下，穿过生物膜的高离子流量会在突触后细胞中触发进一步的事件，最终导致新的动作电位的产生和神经脉冲的传递。有证据表明，特异性阻断通道功能的小分子可能有助于治疗某些精神疾病，如焦虑、药物依赖、精神分裂症和认知功能障碍。虽然在过去的十年里，在解决这些大蛋白的精确三维结构方面已经取得了相当大的成就，但这些结构还不够精确，不足以进行合理的基于结构的药物设计。因此，许多不同的化合物或化合物库正在被合成，并必须与离子通道受体进行测试。目前的筛选方法费时费力，有些甚至依赖于放射性化合物。5-羟色胺5-HT3离子通道受体是本项目的重点。它的功能相当于五个相同亚基的五聚体。每个亚基都有一个胞外区、一个跨膜区和一个胞内区。胞外区主要位于突触间隙，负责形成神经递质结合部位。这项研究计划描述了一种基于荧光检测的快速有效的结合分析的发展，它将适用于高通量筛选。在拟议的项目中，可以使用标准分子生物学技术在哺乳动物细胞中表达的5-HT3受体，将通过光化学反应在结合部位附近用荧光染料进行化学修饰，并设计合成修饰剂化合物。将合成专门设计的小有机分子，这些小分子将结合到修改后的受体上，并猝灭其荧光。任何比猝灭剂化合物具有更高结合亲和力的新化合物都会取代猝灭剂，从而改变修饰受体的荧光。可以使用适当的读出来检测荧光的恢复。因此，加入新化合物后荧光增强意味着它与离子通道受体结合，从而代表了潜在的受体抑制剂。一旦建立，将使用已知的5-HT3受体抑制剂来测试结合试验，以便将结果与传统分析方法获得的数据进行比较。毫无疑问，除了用于拟议的结合试验之外，荧光5-HT3受体可能被证明是一个非常有用的工具来解决这些配体门控离子通道如何工作以及结合事件如何在结构上与通道开放事件相联系的基本问题。这种对功能和结构的改进将对药物设计非常有价值，最终将导致更好和更具选择性的化合物。此外，如果成功开发，同样的概念可以用来处理更复杂但在药理上更重要的离子通道受体。