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

BBSRC战略主题：生物科学用于对健康的综合理解适体是具有确定的二级结构的单链寡核苷酸。它们通过指数富集配体系统进化（SELEX）针对选择的靶点产生，从而对其选择的靶点产生高亲和力和选择性，并允许适体作为治疗诊断学领域中抗体的替代品获得更大的吸引力。G蛋白偶联受体（GPCR）是跨膜蛋白，其结合细胞外激动剂，然后将这些信号传递穿过质膜以通过G蛋白和β-arrestins激活细胞内信号级联。B1类GPCR响应肽激素，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允许跨膜信号转导的构象转变。这个大的细胞外区域可以作为一个大的表面，在其上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。很少有适体针对GPCR产生，只有一种B1类GPCR被靶向：胰高血糖素受体（GCGR）。该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靶向适体的特征在于其影响GPCR信号传导的能力;然而，针对2肾上腺素受体产生的适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进入具有独特信号传导谱的受体状态，从而允许对靶受体信号传导进行严格控制。对于疾病相关的受体，这可以防止致病信号传导并促进促进健康的信号传导途径。本项目的开始将研究已知的GCGR靶向适体的药理作用。这将利用基于生物发光共振能量转移（BRET）的测定，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这种适体的影响。为了测试这种适体对某些GCGR构象的选择性，这些测定可以在受体活性修饰蛋白（RAMP）存在下进行，所述受体活性修饰蛋白改变GCGR的信号传导谱，并因此改变其构象状态。这也将扩展到其他B1类GPCR，例如胰高血糖素样肽-1受体（GLP 1 R），以测量该适体对其靶受体的选择性。然后将在我们手中验证SELEX的过程;在HEK 293细胞中表达GCGR，将其暴露于加标GCGR靶向适体的DNA文库。在增加严格性的少量SELEX轮之后，将评估已知适体的流行率以开发和验证我们的方案。一旦验证，SELEX过程可以应用于瞬时转染到HEK 293细胞中的新受体，例如GLP 1 R。这种受体的激动剂已被确定为关键的肥胖治疗;然而，这些治疗由于其高昂的价格而难以获得。与肽生产相比，DNA适体生产的相对容易和低成本意味着GLP 1 R活化适体可能是更容易获得的治疗方法。一旦适体的SELEX和药理学表征验证了这个平台，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.