Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking

Lucey, M.; Ashik, T.; Marzook, A.; Wang, Y.; Goulding, J.; Oishi, A.; Broichhagen, J.; Hodson, D.; Minnion, J.; Elani, Y.; Jockers, R.; Briddon, S. J.; Bloom, S. R.; Tomas, A.; Jones, B.

doi:10.1101/2021.04.01.438030

bioRxiv · 10.1101/2021.04.01.438030

Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking

Lucey, M.·Ashik, T.·Marzook, A.·Wang, Y.·Goulding, J.·Oishi, A.·Broichhagen, J.·Hodson, D.·Minnion, J.·Elani, Y.·Jockers, R.·Briddon, S. J.·Bloom, S. R.·Tomas, A.·Jones, B.

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Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to promote albumin binding in order to prolong their pharmacokinetics, but the potential for these ligand changes to influence GLP-1R signalling has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). In this work we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16, for their relative propensities to recruit and activate G proteins and {beta}-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes. Both ligands had similar cAMP potency but the exendin-4-C16 showed [~]2.5-fold bias towards G protein recruitment and a [~]60% reduction in {beta}-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with a reduced ability to promote the movement of the GLP-1R extracellular domain, as determined using a conformational biosensor approach, and a [~]70% increase in insulin secretion. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Overall, our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. Significance statementAcylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. Our work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking and interactions with the plasma membrane, which may be therapeutically important.

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Lucey, M., Ashik, T., Marzook, A., Wang, Y., Goulding, J., Oishi, A., Broichhagen, J., Hodson, D., Minnion, J., Elani, Y., Jockers, R., Briddon, S. J., Bloom, S. R., Tomas, A., Jones, B.. 2021-04-01. Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking. https://doi.org/10.1101/2021.04.01.438030

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