Probing the dynamics of agonist drug interaction with Cys-loop channels by single-molecule recording

通过单分子记录探讨激动剂药物与 Cys 环通道相互作用的动力学

• 批准号: MR/J007110/1
• 负责人: Lucia Sivilotti
• 金额: $ 68.56万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FJ007110%2F1
• 关键词: Probing; dynamics; agonist; drug; interaction

Ion channels are proteins that act as "nanoswitches" to translate voltage or chemical stimuli into electrical currents. They are essential to many bodily functions, including information processing in neurones and cell-to cell communication at synapses outside the brain which allow nerve impulses to move muscles and regulate blood pressure and heart rate. Unsurprisingly, inherited channel mutations can produce human disease, from cystic fibrosis to neurological conditions. Channels are targeted by many drugs. The nicotinic-type channels we work on mediate the effects of sleeping pills, drugs for epilepsy, nicotine, alcohol, insecticides and antiparasitic drugs. If we were better at designing drugs to activate or modulate nicotinic channel function, they could be useful in other hard-to-treat conditions, from chronic pain to spasticity after stroke, especially if we could exploit the great diversity of channel subtypes to design agents selective for single subtypes. To this day, drugs are discovered by large scale screening of chemicals to find if they are effective on a human drug receptor. The process is expensive and wasteful, and few new drugs become available every year. Instead of that, it would be ideal to be able to design chemicals to have a specific action on a particular human channel. For this we need to understand two steps: how a chemical binds to the channel and how it then changes the shape and function of the channel protein. Part of the problem is that, working as a switch, the channel itself changes shape and we don't know how this affects the binding of the drug.This is what we want to find out. At UCL we perfected a technique to see and interpret the tiny current (more than a billion times smaller than the current in a kettle) produced by one channel protein. This analysis is the only one that can tell us how tightly the drug binds to different states of the protein and how quickly the protein moves between these different states, with and without the drug. We will use in our work the glycine channel as a model for the nicotinic family. This channel has ideal properties for single molecule recording and has recently allowed us to see why some drugs are less effective than others in turning the receptor on, a result we found to be applicable to other nicotinic channels. We will extend our work and obtain these measurements for chemicals that activate the receptor, and differ from each other in their chemical structure in a systematic way. We will also change the protein itself, by mutating appropriate positions. Combining this information will allow us to see where the drugs "touch" the protein most closely. Glycine channels are also the mammalian channel that is closest to a well-resolved X-ray structure (that of an invertebrate channel, GluCl, published in June 2011). This makes it possible to use the structural data in relation to channel function. At Oxford we will model the structure of the glycine channel by homology to GluCl by computer calculations and use this work to plan and interpret the experiments on channel function in terms of channel 3-D shape. Ultimately our work should lead us to understand what features in a chemical determine its affinity and efficacy for a nicotinic channel and how the different parts of the channel move with activation. It should give us indications on how the structure of drugs should be modified, in order to make them more effective. Hence this fundamental research will be useful to lay the basis for future drug development and hopefully enable rational drug design, in the glycine receptor itself (a therapeutic orphan) and in the nicotinic superfamily as a whole. Our results will also help our drug industry colleagues interpret their data that come from the quick assay techniques used by in high-throughput screening of libraries of compounds, such as binding and macroscopic functional measurements.

离子通道是一种蛋白质，它充当“纳米开关”，将电压或化学刺激转化为电流。它们对许多身体功能都是必不可少的，包括神经元的信息处理和大脑外突触的细胞对细胞通信，这些突触允许神经冲动移动肌肉，调节血压和心率。毫不奇怪，遗传的通道突变会导致人类疾病，从囊性纤维化到神经疾病。渠道是许多药物的靶子。我们研究的尼古丁类通道调节安眠药、治疗癫痫的药物、尼古丁、酒精、杀虫剂和抗寄生虫药物的效果。如果我们能更好地设计药物来激活或调节尼古丁通道功能，它们可能会对其他难以治疗的疾病有用，从慢性疼痛到中风后的痉挛，特别是如果我们能够利用通道亚型的巨大多样性来设计针对单一亚型的选择性药物。直到今天，药物是通过对化学物质进行大规模筛选来发现的，以确定它们是否对人类药物受体有效。这一过程既昂贵又浪费，而且每年几乎没有新药可用。取而代之的是，能够设计出对特定人类通道具有特定作用的化学物质将是理想的。为此，我们需要了解两个步骤：化学物质如何与通道结合，以及它如何改变通道蛋白的形状和功能。部分问题是，作为一个开关，通道本身会改变形状，我们不知道这会如何影响药物的结合。这是我们想要找出的。在伦敦大学学院，我们完善了一项技术，可以观察和解释由单通道蛋白质产生的微小电流(比水壶中的电流小10亿倍以上)。这个分析是唯一可以告诉我们药物与蛋白质不同状态的结合有多紧密，以及蛋白质在这些不同状态之间移动的速度，无论有没有药物。我们将在我们的工作中使用甘氨酸通道作为尼古丁家族的模型。这种通道具有理想的单分子记录特性，最近使我们能够看到为什么一些药物在打开受体方面比其他药物效果更差，我们发现这一结果适用于其他尼古丁通道。我们将扩大我们的工作，并获得这些测量激活受体的化学物质，并以系统的方式在化学结构上相互不同。我们还将通过突变适当的位置来改变蛋白质本身。结合这些信息，我们可以看到药物与蛋白质的接触最紧密的地方。甘氨酸通道也是最接近分辨率良好的X射线结构的哺乳动物通道(2011年6月发表的无脊椎动物通道GluCl)。这使得能够使用与通道功能相关的结构数据。在牛津大学，我们将通过计算机计算来模拟甘氨酸通道的结构，并利用这项工作来计划和解释通道三维形状方面的通道功能实验。最终，我们的工作应该引导我们理解一种化学物质中的哪些特征决定了它对尼古丁通道的亲和力和有效性，以及该通道的不同部分是如何随着激活而移动的。它应该给我们提供一些迹象，说明应该如何修改药物的结构，以便使它们更有效。因此，这项基础研究将有助于为未来的药物开发奠定基础，并有望在甘氨酸受体本身(治疗性孤儿)和整个尼古丁超家族中进行合理的药物设计。我们的结果还将帮助我们的制药业同行解释他们的数据，这些数据来自于在化合物文库的高通量筛选中使用的快速分析技术，例如结合和宏观功能测量。

项目成果

Alternative Binding Mode of Full and Partial Agonists in a Pentameric Ligand-Gated Ion Channel Stabilises Loop C in an Open Conformation

五聚体配体门控离子通道中完全和部分激动剂的替代结合模式使环 C 稳定在开放构象中

• DOI: 10.1016/j.bpj.2017.11.1694
• 发表时间: 2018
• 期刊: Biophysical Journal
• 影响因子: 3.4
• 作者: Dämgen M

The Kinetic Properties of the Human Glycine Receptor in Response to Different Agonists

人甘氨酸受体响应不同激动剂的动力学特性

• 发表时间: 2015
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Hurdiss Elliot J.

Interaction of the Glycine Receptor Alpha 1 Binding Site with Partial Agonists

甘氨酸受体 Alpha 1 结合位点与部分激动剂的相互作用

• DOI: 10.1016/j.bpj.2013.11.3047
• 发表时间: 2014
• 期刊: Biophysical Journal
• 影响因子: 3.4
• 作者: Greiner T

Interactions of the human glycine receptor binding site with different agonists: a single channel approach

人甘氨酸受体结合位点与不同激动剂的相互作用：单通道方法

• 发表时间: 2014
• 期刊: Proceedings of the Physiological Society
• 作者: Hurdiss E

Mechanism of loop C closure in the glycine receptor and its relevance for partial agonism

甘氨酸受体中C环闭合机制及其与部分激动的相关性

• 发表时间: 2017
• 期刊: EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS
• 影响因子: 2
• 作者: Damgen M. A.