Design and synthesis of novel fluorescent allosteric modulators for the prostaglandin EP2 receptor

前列腺素E2受体(EP2R)是参与慢性炎症和癌症的重要靶点，属于G蛋白偶联受体(GPCRs)家族。GPCRs是一种复杂而灵活的膜结合蛋白，由约800个家族成员组成。EP2R是由内源性脂肪激素激活的，这种激素被称为前列腺素，通过与受体上的正构体结合部位结合而被激活。然而，最近，对于少数GPCR--最近的EP2R，发现了一个地形上不同的‘细胞内变构’结合位点(酱等人，2020)。到目前为止，只有一个小分子被报道与这个位点结合，并展示了有趣的药理学。虽然需要前列腺素E2等激动剂配体才能与变构配体结合，但结果是正构激动剂的疗效降低。因此，该配体是一种负变构调节剂(NAM)，其作用可能在控制炎症反应方面具有治疗作用。关于这类变构配体如何与GPCRs相互作用并影响对不同功能效应物(G蛋白或阻滞素)的信号传递的信息有限。例如，我们现在知道G蛋白和拦阻蛋白与GPCR胞内域的结合使用了不同的分子足迹，细胞内调节剂结合位点位于GPCR效应器界面。因此，更好的结构活性关系(SARS)有可能揭示新的配体类别--例如，选择性地调节G蛋白或arrestin相互作用以调节功能性前列腺素反应的“偏向”调节器。然而，良好的变构SAR的发展往往受到化合物类似物范围较小以及缺乏合适的探针配体来直接监测靶点结合的限制。为了探索EP2R上的变构SAR，学生因此将开发荧光高亲和力配体，并通过我们已建立的方法使用它们来监测配体结合和动力学(Sykes等人2017年)。结合受体模型研究，这将促进我们对该位点GPCR变构的性质，以及变构配体如何影响三伙伴激活的GPCR复合体中的前列腺素信使和效应蛋白结合的理解。在适当的时候，这些数据也将为未来专注于EP2R变构配体的药物发现项目提供信息。该项目的具体目标将是：1.设计、合成和表征EP2R 2的新型荧光变构配体。利用这些荧光调节剂直接探测EP2R调节剂SAR，建立基于tr-fret的竞争结合动力学分析(Sykes等人2017)。建立调节剂-EP2R相互作用的模型，并利用这些模型来理解前列腺素和效应器结合的变构调节。使用EP2R信号分析来表征新的化合物药理作用，以及通过建模研究了解受体突变的影响。这个以化学生物学为重点的项目将跨越合成化学、建模和药理学的学科，以增加我们对EP2R生物学的理解。随着越来越多的变构配体被发现用于更广泛的GPCR家族，结果将与许多其他重要的受体直接相关。

The prostaglandin E2 receptor (EP2R) is an important target involved in chronic inflammation and cancer and belongs to the family of G protein-coupled receptors (GPCRs). GPCRs are complex and flexible membrane-bound proteins comprising ~800 family members. The EP2R is activated by endogenous lipid hormones, called prostanoids through their binding to the orthosteric binding site on the receptor. However, recently, for a handful of GPCRs - most recently the EP2R, a topographically distinct 'intracellular allosteric' binding site has been discovered (Jiang et al, 2020).To date, only a single small molecule has been reported which is predicted to bind to this site and which exhibits intriguing pharmacology. Whilst an agonist ligand such as prostaglandin E2 is required to enable binding of the allosteric ligand, the consequence is a reduction in the efficacy of the orthosteric agonist. Thus the ligand is a negative allosteric modulator (NAM), whose effects may have therapeutic benefits in controlling inflammatory responses.There is limited information about how this class of allosteric ligands interacts with GPCRs and influences signalling to functionally different effectors (G proteins or arrestins). For example, we now know that binding of G proteins and arrestins to the GPCR intracellular domain exploits different molecular footprints, with the intracellular modulator binding sites positioned at this GPCR-effector interface. Better structure activity relationships (SARs) at this position thus have potential to reveal new ligand classes - for example "biased" modulators that selectively regulate G protein or arrestin interaction to tune the functional prostanoid response. However, the development of good allosteric SAR is often limited by the poor range of compound analogues and the lack of suitable probe ligands to monitor binding at the target site directly. To explore allosteric SAR at the EP2R, the student will therefore develop fluorescent high affinity ligands, and use them to monitor ligand binding and kinetics by our established methods (Sykes et al 2017). In conjunction with receptor modelling studies this will advance our understanding of the nature of GPCR allostery at this site, and how allosteric ligands influence both prostanoid messenger and effector protein binding in the three-partner activated GPCR complex. In due course, these data will also inform future drug discovery projects focused on EP2R allosteric ligands.Specific aims of the project will be: 1. To design, synthesis and characterise novel fluorescent allosteric ligands for the EP2R 2. To develop TR-FRET based competition binding kinetic assays using these fluorescent modulators to probe EP2R modulator SAR directly (Sykes et al 2017).3. To develop models of modulator- EP2R interaction and use these to understand the allosteric regulation of prostanoid and effector binding.4. To pharmacologically characterise novel compound pharmacology using EP2R signalling assays, and the effects of receptor mutants as informed by modelling studiesThis chemical biology-focused project will span the disciplines of synthetic chemistry, modelling and pharmacology to increase our understanding of EP2R biology. With increasing numbers of allosteric ligands being discovered for the wider GPCR family, the results will be of direct relevance to many other important receptors.