Partial agonism improves the anti-hyperglycaemic efficacy of an oxyntomodulin-derived GLP-1R/GCGR co-agonist.

OBJECTIVE: Glucagon-like peptide-1 and glucagon receptor (GLP-1R/GCGR) co-agonism can maximise weight loss and improve glycaemic control in type 2 diabetes and obesity. In this study, we investigated the cellular and metabolic effects of modulating the balance between G protein and ß-arrestin-2 recruitment at GLP-1R and GCGR using oxyntomodulin (OXM)-derived co-agonists. This strategy has been previously shown to improve the duration of action of GLP-1R mono-agonists by reducing target desensitisation and downregulation. METHODS: Dipeptidyl dipeptidase-4 (DPP-4)-resistant OXM analogues were generated and assessed for a variety of cellular readouts. Molecular dynamic simulations were used to gain insights into the molecular interactions involved. In vivo studies were performed in mice to identify the effects on glucose homeostasis and weight loss. RESULTS: Ligand-specific reductions in ß-arrestin-2 recruitment were associated with slower GLP-1R internalisation and prolonged glucose-lowering action in vivo. The putative benefits of GCGR agonism were retained, with equivalent weight loss compared to the GLP-1R mono-agonist liraglutide despite a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and ß-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. CONCLUSIONS: Diminishing ß-arrestin-2 recruitment may be an effective way to increase the therapeutic efficacy of GLP-1R/GCGR co-agonists. These benefits can be achieved by partial rather than biased agonism.

Evaluation of biased agonism mediated by dual agonists of the GLP-1 and glucagon receptors.

Metabolic diseases such as obesity, diabetes, and their comorbidities have converged as one of the most serious health concerns on a global scale. Selective glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists are one of the major therapeutics for type 2 diabetes and obesity. Polypharmacological approaches that enable modulation of multiple metabolic targets in a single drug have emerged as a potential avenue to improve therapeutic outcomes. Among numerous peptides under development are those targeting the GLP-1R and either the glucagon receptor (GCGR), glucose-dependent insulinotropic peptide receptor (GIPR) or all 3 receptors, as dual- or tri- peptide agonists. Despite many of them entering into clinical trials, current development has been based on only a limited understanding of the spectrum of potential pharmacological properties of these ligands beyond binding selectivity. In the present study, we examined the potential for agonists that target both GLP-1R and GCGR to exhibit biased agonism, comparing activity across proximal activation of Gs protein, cAMP accumulation, pERK1/2 and ß-arrestin recruitment. Three distinct dual agonists that have different relative cAMP production potency for GLP-1R versus GCGR, "peptide 15", MEDI0382 and SAR425899, and one triagonist of the GLP-1R, GCGR and GIPR were examined. We demonstrated that all novel peptides have distinct biased agonism profiles relative to either of the cognate agonists of the receptors, and to each other. This is an important feature of the pharmacology of this drug class that needs to be considered alongside selectivity, bioavailability and pharmacokinetics for rational optimization of new therapeutics.

Degradation and Stabilization of Peptide Hormones in Human Blood Specimens.

Plasma hormone peptides, including GLP-1, GIP, Glucagon, and OXM, possess multiple physiological roles and potential therapeutic and diagnostic utility as biomarkers in the research of metabolic disorders. These peptides are subject to proteolytic degradation causing preanalytical variations. Stabilization for accurate quantitation of these active peptides in ex vivo blood specimens is essential for drug and biomarker development. We investigated the protease-driven instability of these peptides in conventional serum, plasma, anticoagulated whole blood, as well as whole blood and plasma stabilized with protease inhibitors. The peptide was monitored by both time-course Matrix-Assisted Laser Desorption Ionization Time-to-Flight Mass Spectrometry (MALDI -TOF MS) and Ab-based assay (ELISA or RIA). MS enabled the identification of proteolytic fragments. In non-stabilized blood samples, the results clearly indicated that dipeptidyl peptidase-IV (DPP-IV) removed the N-terminal two amino acid residues from GLP-1, GIP and OXM(1-37) and not-yet identified peptidase(s) cleave(s) the full-length OXM(1-37) and its fragments. DPP-IV also continued to remove two additional N-terminal residues of processed OXM(3-37) to yield OXM(5-37). Importantly, both DPP-IV and other peptidase(s) activities were inhibited efficiently by the protease inhibitors included in the BD P800* tube. There was preservation of GLP-1, GIP, OXM and glucagon in the P800 plasma samples with half-lives > 96, 96, 72, and 45 hours at room temperature (RT), respectively. In the BD P700* plasma samples, the stabilization of GLP-1 was also achieved with half-life > 96 hours at RT. The stabilization of these variable peptides increased their utility in drug and/or biomarker development. While stability results of GLP-1 obtained with Ab-based assay were consistent with those obtained by MS analysis, the Ab-based results of GIP, Glucagon, and OXM did not reflect the time-dependent degradations revealed by MS analysis. Therefore, we recommended characterizing the degradation of the peptide using the MS-based method when investigating the stability of a specific peptide.

The role of gut hormones in the regulation of body weight and energy homeostasis.

Obesity is one of the greatest public health challenges of the 21st century with 1.6 billion adults currently classified as being overweight and 400 million as obese. Obesity is causally associated with type 2 diabetes, hypertension, cardiovascular disease, obstructive sleep apnoea and certain forms of cancer and is now one of the leading causes of mortality and morbidity worldwide. The gastrointestinal tract is the largest endocrine organ in the body producing hormones that have important sensing and signaling roles in regulating body weight and energy expenditure. The last decade has witnessed a marked increase in our understanding of the role of gut hormones in energy homeostasis. Consequently, strategies aimed at modulating circulating gut hormone concentrations or targeting their receptors are being developed as potential pharmacotherapies for obesity. This review summarizes the current knowledge regarding the mechanisms, sites of action and effects of the anorectic gut hormones peptide tyrosine-tyrosine (PYY), pancreatic polypeptide (PP), oxyntomodulin, and amylin and of the unique orexigenic hormone, ghrelin.