Distinct roles of the extracellular surface residues of glucagon-like peptide-1 receptor in ß-arrestin 1/2 signaling.

Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with ß-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in ß-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated ß-arrestin 1/2 recruitment for diverse ligands, and ß-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased ß-arrestin 1 recruitment but increased ß-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected ß-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in ß-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive ß-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in ß-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in ß-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on ß-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without ß-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and ß-arrestins. Our study offers valuable information about ligand induced ß-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.

OXM-104, a potential candidate for the treatment of obesity, NASH and type 2 diabetes.

OBJECTIVE: Dual glucagon-like peptide-1 (GLP-1) and glucagon receptor agonists are therapeutic agents with an interesting liver-specific mode of action suitable for metabolic complications. In this study, dual GLP-1 and glucagon receptor agonist OXM-104 is compared head-to-head with the once-daily dual GLP-1 and glucagon receptor agonist cotadutide and GLP-1 receptor agonist semaglutide to explore the metabolic efficacy of OXM-104. METHODS: The in vitro potencies of OXM-104, cotadutide and semaglutide were assessed using reporter assays. In addition, in vivo efficacy was investigated using mouse models of diet-induced obesity (DIO mice), diabetes (db/db mice) and diet-induced NASH mice (MS-NASH). RESULTS: OXM-104 was found to only activate the GLP-1 and glucagon with no cross-reactivity at the (GIP) receptor. Cotadutide was also found to activate the GLP-1 and glucagon receptors, whereas semaglutide only showed activity at the GLP-1 receptor. OXM-104, cotadutide, and semaglutide elicited marked reductions in body weight and improved glucose control. In contrast, hepatoprotective effects, i.e., reductions in steatosis and fibrosis, as well as liver fibrotic biomarkers, were more prominent with OXM-104 and cotadutide than those seen with semaglutide, demonstrated by an improved NAFLD activity score (NAS) by OXM-104 and cotadutide, underlining the importance of the glucagon receptor. CONCLUSION: These results show that dual GLP-1 and glucagon receptor agonism is superior to GLP-1 alone. OXM-104 was found to be a promising therapeutic candidate for the treatment of metabolic complications such as obesity, type 2 diabetes and NASH.

Self-assembled GLP-1/glucagon peptide nanofibrils prolong inhibition of food intake.

Introduction: Oxyntomodulin (Oxm) hormone peptide has a number of beneficial effects on nutrition and metabolism including increased energy expenditure and reduced body weight gain. Despite its many advantages as a potential therapeutic agent, Oxm is subjected to rapid renal clearance and protease degradation limiting its clinical application. Previously, we have shown that subcutaneous administration of a fibrillar Oxm formulation can significantly prolong its bioactivity in vivo from a few hours to a few days. Methods: We used a protease resistant analogue of Oxm, Aib2-Oxm, to form nanfibrils depot and improve serum stability of released peptide. The nanofibrils and monomeric peptide in solution were characterized by spectroscopic, microscopic techniques, potency assay, QCM-D and in vivo studies. Results: We show that in comparison to Oxm, Aib2-Oxm fibrils display a slower elongation rate requiring higher ionic strength solutions, and a higher propensity to dissociate. Upon subcutaneous administration of fibrillar Aib2-Oxm in rodents, a 5-fold increase in bioactivity relative to fibrillar Oxm and a significantly longer bioactivity than free Aib2-Oxm were characterized. Importantly, a decrease in food intake was observed up to 72-hour post-administration, which was not seen for free Aib2-Oxm. Conclusion: Our findings provides compelling evidence for the development of long-lasting peptide fibrillar formulations that yield extended plasma exposure and enhanced in vivo pharmacological response.

Emerging roles of oxyntomodulin-based glucagon-like peptide-1/glucagon co-agonist analogs in diabetes and obesity.

Oxyntomodulin (OXM) is an endogenous peptide hormone secreted from the intestines following nutrient ingestion that activates both glucagon-like peptide-1 (GLP-1) and glucagon receptors. OXM is known to exert various effects, including improvement in glucose tolerance, promotion of energy expenditure, acceleration of liver lipolysis, inhibition of food intake, delay of gastric emptying, neuroprotection, and pain relief. The antidiabetic and antiobesity properties have led to the development of biologically active and enzymatically stable OXM-based analogs with proposed therapeutic promise for metabolic diseases. Structural modification of OXM was ongoing to enhance its potency and prolong half-life, and several GLP-1/glucagon dual receptor agonist-based therapies are being explored in clinical trials for the treatment of type 2 diabetes mellitus and its complications. In the present article, we provide a brief overview of the physiology of OXM, focusing on its structural-activity relationship and ongoing clinical development.

Discovery of novel OXM-based glucagon-like peptide 1 (GLP-1)/glucagon receptor dual agonists.

Novel glucagon receptor (GCGR) and glucagon-like peptide 1 receptor (GLP-1R) dual agonists are reported to have improved efficacy over GLP-1R mono-agonists in treating type 2 diabetes (T2DM) and obesity. Here, we describe the discovery of a novel oxyntomodulin (OXM) based GLP-1R/GCGR dual agonist with potent and balanced potency toward GLP-1R and GCGR. The lead peptide OXM-7 was obtained via stepwise rational design and long-acting modification. In ICR and db/db mice, OXM-7 exhibited prominent acute and long-acting hypoglycemic effects. In diet-induced obesity (DIO) mice, twice-daily administration of OXM-7 produced significant weight loss, normalized lipid metabolism, and improved glucose control. In DIO-nonalcoholic steatohepatitis (NASH) mice, OXM-7 treatment significantly reversed hepatic steatosis, and reduced serum and hepatic lipid levels. These preclinical data suggest the therapeutic potential of OXM-7 as a novel anti-diabetic, anti-steatotic and/or anti-obesity agent.

BI 456906: Discovery and preclinical pharmacology of a novel GCGR/GLP-1R dual agonist with robust anti-obesity efficacy.

OBJECTIVE: Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. METHODS: BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. RESULTS: BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906's superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. CONCLUSIONS: BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.

S3-2, a novel long-lasting oxyntomodulin derivative, exerts improvement on diabesity and renal injury through activating GLP-1 and glucagon receptors.

AIMS: To prolong the short lifespan of oxyntomodulin (OXM) for treating obesity and diabetes, we designed a novel fused OXM analog, containing an albumin-binding sequence, a protease cleavable tetrapeptide, and a mutated OXM. MAIN METHODS: We screened two albumin-binding sequences (S3 and S6) to construct OXM derivatives, termed S3-2 (with two cysteines) and S6-0 (without cysteine). After peptides were synthesized, isothermal titration calorimetry (ITC) was applied to assess binding-affinity for HSA. Further in vivo acute efficacies evaluation and candidate selection were performed in diabetic db/db mice via oral glucose tolerance test (OGTT) and glucose-lowering duration test. Chronic efficacy test of selected candidate was also performed in diabetic mice. RESULTS: Firstly, S3-2 and S6-0 with purity over 99% were prepared. ITC measurements demonstrated that S3-2 and S6-0 associate with HSA with high-affinity (Kd = 12.81 ± 1.11 nM and 26.98 ± 2.39 nM, respectively). Then hypoglycemic efficacies showed deoxidation S3-2 (S3-2re) showed longer hypoglycemic duration than the oxidation one (S3-2ox), and better blood glucose level (BGL) control effect than S6-0. OGTTs in diabetic mice revealed the glucose-lowering efficacies of S3-2re were similar to Liraglutide. The protracted antidiabetic effects of S3-2re were further confirmed by multiple OGTTs in db/db mice. Furthermore, twice weekly injection of S3-2re to db/db mice achieved beneficial effects on body weight gain, glucose tolerance, postprandial BGL and obesity. Moreover, S3-2 produces significantly protective effects on the impaired renal functions of the diabetic mice. CONCLUSION: S3-2re exhibits outstanding therapeutical potential as a candidate drug for treating the obesity and diabetes.

Novel peptidic dual GLP-1/glucagon receptor agonist alleviates diabetes and diabetic complications in combination with low-intensity ultrasound.

OBJECTIVE: To design and evaluate a novel oxyntomodulin (OXM) derivative with albumin-binding helix domain and dual GLP-1 receptor (GLP-1R) and glucagon receptor (GcgR) activation activity to achieve metabolize improvement on the diabetes-related complication. MATERIALS AND METHODS: Mutation (D-Ser2) on OXM was performed and then different helix albumin-binding domains were fused to the mutated OXM via a thrombin-cleavable linker to generate seven fusion peptides, named LM01-LM07. Seven LM peptides were synthesized and screened via in vitro receptor activation test, albumin binding measurement and protease cleavage assay to select potent candidate peptide for further in vivo study. Moreover, acute and chronic efficacy studies were conducted to evaluate the efficacy of selected candidate using db/db mice. RESULTS: LM06, as selected OXM derivative, exhibited higher albumin-binding affinity, sustained-release efficiency and balanced activation activities on both GLP-1R and GcgR compared with other ones. Moreover, LM06 was demonstrated with improved hypoglycemic and insulinotropic abilities in receptor-deficient mice via activating GLP-1R. In addition, prolonged anti-diabetic efficacies of LM06 were demonstrated via hypoglycemic duration assay and OGTT in db/db mice. Further pharmacokinetic test of LM06 in both rats and monkeys identified improved half-life and other metabolic characteristics. Nevertheless, 8-week subcutaneously dosed LM06 in db/db mice achieved prominent efficacies on glucostasis, weight-lowering, pancreatic function and adipogenesis via activating GLP-1R and GcgR. Moreover, LM06 also could accelerate diabetic skin wound closure in combination with low-intensity ultrasound. CONCLUSIONS: LM06, as a long-acting dual GLP-1R/GcgR agonist, exerts potential as a once-weekly therapeutic candidate against diabetes-related complication in combination with low-intensity ultrasound.

Conjugation of a peptide to an antibody engineered with free cysteines dramatically improves half-life and activity.

The long circulating half-life and inherently bivalent architecture of IgGs provide an ideal vehicle for presenting otherwise short-lived G-protein-coupled receptor agonists in a format that enables avidity-driven enhancement of potency. Here, we describe the site-specific conjugation of a dual agonist peptide (an oxyntomodulin variant engineered for potency and in vivo stability) to the complementarity-determining regions (CDRs) of an immunologically silent IgG4. A cysteine-containing heavy chain CDR3 variant was identified that provided clean conjugation to a bromoacetylated peptide without interference from any of the endogenous mAb cysteine residues. The resulting mAb-peptide homodimer has high potency at both target receptors (glucagon receptor, GCGR, and glucagon-like peptide 1 receptor, GLP-1R) driven by an increase in receptor avidity provided by the spatially defined presentation of the peptides. Interestingly, the avidity effects are different at the two target receptors. A single dose of the long-acting peptide conjugate robustly inhibited food intake and decreased body weight in insulin resistant diet-induced obese mice, in addition to ameliorating glucose intolerance. Inhibition of food intake and decrease in body weight was also seen in overweight cynomolgus monkeys. The weight loss resulting from dosing with the bivalently conjugated dual agonist was significantly greater than for the monomeric analog, clearly demonstrating translation of the measured in vitro avidity to in vivo pharmacology.

A novel long-acting oxyntomodulin analogue eliminates diabetes and obesity in mice.

Oxyntomodulin (OXM) was identified as a glucagon (GCG) receptor (GCGR) and glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) dual agonist to suppress appetite, increase energy expenditure, and induce body weight loss in obese humans. However, the activities of native OXM to activate GCGR and GLP-1R in vitro were much weaker than the natural ligands. To address this gap, structural modifications were adopted and novel OXM analogues were obtained through chimeric peptide sequence design. One specific analogue with enhanced and balanced GCGR/GLP-1R activations was chemically conjugated with polyethylene glycol (PEG) to achieve sustained release in vivo. This PEGylated analogue was further explored pharmacologically in db/db and diet-induced obese (DIO) mice models. Chronic weekly administration significantly induced hypoglycemic effects and body weight loss with dose dependency, along with normalized adiposity, lipid metabolism, and liver steatosis. Based on its profiles in vitro and in vivo, the analogue has the great potential to develop as a novel anti-diabetic and/or anti-obese candidate. As observed more insulin stimulation and improved insulin resistance, it may be also explored for the treatment of nonalcoholic steatohepatitis (NASH) in the future.