The Use Of Novel Polymer-Lipid Nanoparticles To Study G-Protein-Coupled Receptor Activation And Dynamics.

体内的大多数化学信使通过细胞表面的受体蛋白质发挥作用。最大的一类受体是G蛋白偶联受体(GPCRs)家族，它们对细胞信号和药物靶标都很重要。因此，GPCRs在基础生物学和制药业的商业开发方面都具有巨大的兴趣。了解GPCRs在分子水平上是如何工作的从根本上说是重要的，也是当今生物学中的“大问题”之一。对于少数几个受体，我们有一个晶体结构，当它与一种“阻断药物”结合时，它会显示出它们的三维形状。这加深了我们对受体结构的理解，这应该有助于未来的药物发现。然而，很明显，GPCRs是非常灵活的蛋白质，可以在结合激素和药物时改变其形状，而晶体结构提供了单一构象的快照，通常是非信号受体的构象。要完全理解GPCR，我们需要了解它们在信号传递过程中如何改变形状。这对与天然荷尔蒙受体的不同部分结合以改变受体活性的“变构”试剂也很重要。这为一种全新的毒品类别开辟了道路。在本申请中，我们描述了一种理解GPCRs的新方法。我们已经找到了一种新的方法，可以使大量的天然GPCRs在其膜环境中的非常小的颗粒中“增溶”，而不需要洗涤剂。此前，洗涤剂对受体的溶解一直是绝对要求的。这形成了这一领域进展的主要障碍，因为它的存在扰乱了受体的正常性质和形状，破坏了蛋白质的稳定。在任何地方，我们现在第一次可以绕过洗涤剂的障碍取得进步。这一重要进展为应用强大的生物物理技术提供关于GPCRs如何动态改变其形状以响应不同类别药物的详细信息开辟了前景。该项目汇集了来自伯明翰大学和阿斯顿大学的四名科学家，他们拥有互补的技能来开发我们的新技术。我们将以腺苷A2a受体(A2aR)为例研究GPCR家族。这是一种特性良好的受体，拥有大量文献、定义明确的药物和有用的工具，重要的是，它是为数不多的具有原子级晶体结构的受体之一。此外，我们的工业合作伙伴Heptares Treeutics将提供限制为功能定义形状(构象)的A2AR，这将对我们有很大帮助。最后，我们与埃塞克斯大学一位经验丰富的计算化学家进行了正式合作，他将根据我们的实验数据构建分子模型，并对受体的不同功能状态进行动态模拟。我们在该领域处于领先地位，我们的试点数据显示，我们目前有能力通过在酵母中培养A2aR来产生非常大量的A2aR，并将其自然状态下的活性受体快速‘溶解’到苯乙烯马来酸脂质颗粒(SMALP)中。我们将设计A2aR，使包括荧光基团在内的‘报告基团’能够在受体结构中的特定位置被引入。然后，我们可以用一系列技术来研究A2aR-SMALP。例如，当药物结合时，荧光可以告诉我们受体重要部分周围环境的变化。一种相关的技术也可以用来提供一种分子标尺，允许我们计算受体两个部分之间的距离，以确定它在不同信号转导能力的结合药物上或在Heptares Treeutics提供的构象受限的A2aRs中如何变化。总体而言，该项目将提供对GPCR激活背后的形状变化的洞察，并可能有助于未来的合理药物设计。

Most chemical messengers in the body, work through proteins on the cell surface known as receptors. The biggest class of receptors is the G-protein-coupled receptor (GPCRs) family, which are important for cell signalling and as drug targets. GPCRs are thus of enormous interest, both in terms of basic biology and also for commercial exploitation by the pharmaceutical industry. Understanding how GPCRs work at the molecular level is fundamentally important and is one of the 'big questions' in biology today. For a few receptors we have a crystal structure showing their 3-dimensional shape when bound to a 'blocking drug'. This has deepened our understanding of receptor architecture which should aid future drug discovery. However, it is clear that GPCRs are very flexible proteins that change their shape on binding hormones and drugs, whereas the crystal structures provide snap-shots of a single conformation, usually of the non-signalling receptor. To fully understand GPCRs, we need to understand how they change their shape during signalling. This is also important for 'allosteric' agents that bind to a different part of the receptor to the natural hormone to modify receptor activity. This opens the way to a whole new class of drugs. In this application we describe a new approach to understanding GPCRs. We have found a novel way to make large quantities of natural GPCRs 'solubilised' within very small particles of their membrane environment without the need for detergent. Previously, there has been an absolute requirement for detergent to solubilise receptors. This formed a major barrier to progress in this field as its presence disrupted the normal properties and shape of the receptor, de-stabilising the protein. For the first time anywhere, we can now circumvent the detergent barrier to progress. This important advance opens up the prospect of applying powerful biophysical techniques to provide detailed information on how GPCRs dynamically change their shape in response to drugs of different classes. The project brings together four scientists from the Universities of Birmingham and Aston, with complementary skills to exploit our new technologies. We will study the adenosine A2a receptor (A2aR) as an example of the GPCR family. This is a well-characterised receptor with a large literature, defined drugs, useful tools and importantly it is one of the few receptors for which there is an atomic level crystal structure. In addition, our industrial collaborators Heptares Therapeutics will provide A2aRs constrained into functionally-defined shapes (conformations) which will be a great help to us. Finally we have a formal collaboration with an experienced computational chemist at Essex University who will construct molecular models and dynamic simulations of different functional states of the receptor based on our experimental data. We are ahead of the field and our pilot data show that we have the capability at this very moment to produce very large amounts of A2aR by growing them in a yeast and to rapidly 'solubilise' active receptors in their natural state in a styrene maleic acid lipid particle (SMALP). We will engineer A2aR so that 'reporter groups', including fluorescent groups, can be introduced at defined locations in the receptor architecture. We can then study A2aR-SMALP by a range of techniques. For example, fluorescence can tell us about the changes in the environment around important parts of the receptor when drugs bind. A related technique can also be used to provide a molecular ruler that allows us to calculate the distance between two parts of the receptor to determine how it changes on binding drugs of different signalling capability or in conformationally-constrained A2aRs provided by Heptares Therapeutics. Overall, this project will provide insight into the changes of shape that underlie GPCR activation and may aid rational drug design in the future.