Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rationale drug development. To date, such understanding has been hampered by lack of direct, subtype selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R) or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin or DACRA peptides that was distinct at each receptor subtype. Of particular note, while both the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviours at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalisation of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimised therapeutics for treatment of metabolic diseases. Significance Statement Amylin receptors are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimised therapeutics for treatment of metabolic diseases.