Rotation 1: Aptamers as allosteric modulators of G Protein-Coupled Receptors

适体是具有明确二级结构的单链寡核苷酸。它们通过指数富集配体的系统进化（SELEX）针对选定的靶标提出，从而对所选靶标具有高亲和力和选择性，并使适体在治疗学领域作为抗体的替代品获得了很大的吸引力。G蛋白偶联受体（gpcr）是一种结合细胞外激动剂的跨膜蛋白，然后将这些信号传递到质膜上，通过G蛋白和-阻滞蛋白激活细胞内信号级联反应。B1类gpcr对肽激素有反应，并具有大量的细胞外结构域，以促进其激动剂配体的结合和随后的构象转移，从而实现跨膜信号转导。这个大的细胞外区域可以作为一个大的表面，适体可以形成广泛的和选择性的相互作用，而不需要穿过质膜。针对GPCR的适体很少，只有一类B1 GPCR被靶向：胰高血糖素受体（GCGR）。这种GPCR介导胰高血糖素在胰腺中的高血糖作用。甚至更少的这些GPCR靶向适配体在其影响GPCR信号传导的能力方面被表征；然而，针对2肾上腺素受体的适配体对该受体的不同构象状态具有选择性。这表明适体可以驱动gpcr进入具有独特信号谱的受体状态，从而允许严格控制靶受体信号。对于与疾病相关的受体，这可以阻止致病性信号传导并促进促进健康的信号传导途径。本项目一开始将研究已知gcgr靶向适配体的药理作用。这将利用基于生物发光共振能量转移（BRET）的分析，例如使用标记的G蛋白亚基来研究GCGR激活的信号级联如何受到添加该适体的影响。为了测试该适体对某些GCGR构象的选择性，这些检测可以在受体活性修饰蛋白（RAMPs）存在的情况下进行，RAMPs可以改变GCGR的信号谱，从而改变GCGR的确认状态。这也将扩展到其他B1类gpcr，如胰高血糖素样肽-1受体（GLP1R），以测量该适体对其靶受体的选择性。SELEX的过程将在我们手中得到验证；在HEK293细胞中表达GCGR，将其暴露于含有GCGR靶向适配体的DNA文库中。经过少量严格的SELEX轮后，将评估已知适体的流行程度，以开发和验证我们的协议。一旦验证，SELEX过程可以应用于瞬时转染到HEK293细胞中的新受体，如GLP1R。该受体的激动剂已被确定为关键的肥胖治疗；然而，由于价格高昂，这些治疗方法在很大程度上是无法获得的。与肽生产相比，DNA适体的生产相对容易和低成本意味着glp1r激活适体可能是一种更容易获得的治疗方法。一旦SELEX和适体的药理学特征验证了这个平台，它可以更广泛地用于靶向各种疾病相关的gpcr。

BBSRC strategic theme: Bioscience for an integrated understanding of healthAptamers are single stranded oligonucleotides with defined secondary structures. They are raised against select targets by Systematic Evolution of Ligands by Exponential enrichment (SELEX), resulting in high affinity and selectivity for their chosen target and allowing aptamers to gain much traction as alternatives to antibodies in the field of theranostics. G protein-coupled receptors (GPCRs) are transmembrane proteins that bind extracellular agonists, then transduce these signals across the plasma membrane to activate intracellular signalling cascades via G proteins and -arrestins. Class B1 GPCRs respond to peptide hormones and have substantial extracellular domains to facilitate the binding of their agonist ligands and subsequent conformational shifts that allow transmembrane signal transduction. This large extracellular region could act as a large surface across which aptamers may form extensive and selective interactions without need to cross the plasma membrane. Few aptamers have been raised against GPCRs, with only one Class B1 GPCR having been targeted: the glucagon receptor (GCGR). This GPCR mediates the hyperglycaemic effects of glucagon in the pancreas. Even fewer of these GPCR-targeting aptamers have been characterised in their ability to affect GPCR signalling; however, aptamers raised against the 2 adrenoceptor are selective for distinct conformational states of this receptor. This suggests that aptamers could drive GPCRs into receptor states with unique signalling profiles, allowing for tight control of target receptor signalling. For disease-implicated receptors, this could prevent pathogenic signalling and encourage health-promoting signalling pathways. The beginning of this project will investigate the pharmacological effects of the known GCGR-targeting aptamer. This will utilise bioluminescence resonance energy transfer (BRET)-based assays, for example using labelled G protein subunits to investigate how signalling cascades activated by the GCGR are affected by addition of this aptamer. To test the selectivity of this aptamer for certain GCGR conformations, these assays can be performed in the presence of receptor activity-modifying proteins (RAMPs) that alter the signalling profile, and therefore the confirmational states, of the GCGR. This will also be extended to other Class B1 GPCRs, such as the glucagon-like peptide-1 receptor (GLP1R), to measure selectivity of this aptamer for its target receptor. The process of SELEX will then be validated in our hands; expressing the GCGR in HEK293 cells exposing it to a DNA library spiked with GCGR-targeting aptamer. After a small number of SELEX rounds with increasing stringency, the prevalence of the known aptamer will be assessed to develop and validate our protocol. Once validated, the SELEX process can be applied to new receptors transiently transfected into HEK293 cells, such as the GLP1R. Agonists of this receptor have been identified as key obesity treatments; however, these treatments are vastly inaccessible due to their high price. The relative ease and low cost of DNA aptamer production when compared to peptide production means a GLP1R-activating aptamer may be a more accessible treatment. Once SELEX and pharmacological characterisation of aptamers have validated this platform, it can be used more widely to target a variety of disease-associated GPCRs.