Addressing GPCR Oligomers Using Peptide and Nanobody-ligand Conjugates

Abstract ID 92273 Poster Board 147 G-protein-coupled receptors (GPCRs), the largest family of transmembrane signalling proteins, are involved in a broad range of physiological processes, marking them as attractive drug targets. GPCRs were long hypothesized to exist as monomeric entities but in recent years, the evidence of GPCR assembly (dimerization and oligomerization) has sparked interest in addressing how receptor oligomerization modulates signalling. Targeting heteromeric GPCR assemblies also offers the prospect of engaging distinct signaling pathways engaged by each receptor simultaneously. Herein we aim to generate new approaches for the stimulation of insulin secretion, an outcome useful for the treatment of type-2 diabetes, by leveraging GPCR heterodimerization. Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted in response to glucose that potentiates insulin secretion from pancreatic β cells upon binding to its receptor (GLP-1R). Upon activation, GLP-1R couples with Gs protein to activate adenylate cyclase and stimulate cAMP production. The neurotransmitter acetylcholine can also induce insulin secretion by acting on the muscarinic receptor subtype M3 (M3R), which signals through Gq/G11 protein family. The compound iperoxo is a strong agonist for muscarinic acetylcholine receptors with higher potency. We aimed to assess whether we could activate both Gq and Gs signalling through the design of bivalent ligands that target both GLP1R and M3R. This was pursued through the synthesis of analogues of both a GLP1R agonist (GLP1 peptide with an azide click chemistry handle) and iperoxo (with an alkyne partner click handle) We systematically synthesized four ligands- Iperoxo, Iperoxo-alkyne, GLP-peptide-azide and GLP peptide-iperoxo conjugate and tested their activities on cells transfected with GLP1R, M3R, or both in signalling through Gq and Gs pathways. Among the compounds tested, only iperoxo induced the desired response through Gq signaling, suggesting a negative role of alkyne linkage to iperoxo. Both GLP-peptide-azide and GLP peptide-Iperoxo induced comparable responses through the Gs pathway relative to GLP-peptide in GLP1R expressing cells, with or without M3R expression. These initial results demonstrate better targeting of GLP1R than M3R for ligand conjugates and establish a need for a strategy for iperoxo linkage. Work is in progress to develop nanobody-based ligand conjugates to deliver iperoxo to cell populations that can modulate the insulin secretion pathway. This research was supported by the Intramural Research Program of the NIH, The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)