Aptamers for targeting Class B1 GPCRs and as modulators of receptor-induced cell signalling

BBSRC strategic theme: Bioscience for an integrated understanding of health

Aptamers 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.