Molecular design of peptide therapeutics via N-terminal modification.

Glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, and glucagon are three naturally occurring peptide hormones that mediate glucoregulation. Several agonists representing appropriately modified native ligands have been developed to maximize metabolic benefits with reduced side-effects and many have entered the clinic as type 2 diabetes and obesity therapeutics. In this work, we describe strategies for improving the stability of the peptide ligands by making them refractory to dipeptidyl peptidase-4 catalyzed hydrolysis and inactivation. We describe a series of alkylations with variations in size, shape, charge, polarity, and stereochemistry that are able to engender full activity at the receptor(s) while simultaneously resisting enzyme-mediated degradation. Utilizing this strategy, we offer a novel method of modulating receptor activity and fine-tuning pharmacology without a change in peptide sequence.

Corrination mitigates peptide aggregation as exemplified for Glucagon.

Pharmaceutical development of glucagon for use in acute hypoglycemia has proved challenging, due in large part to poor solubility, poor stability and aggregate formation. Herein, we describe highly soluble, low aggregating, glucagon conjugates generated through use of the commercially available vitamin B12 precursor dicyanocobinamide ('corrination'), which retain full stimulatory action at the human glucagon receptor. The modified glucagon analogs were tested in a chemical stability assay in 50 mM phosphate buffer and the percentage of original concentration retained was determined after two weeks of incubation at 37° C. Aggregate formation assays were also performed after 48 h of agitation at 37°C using a thioflavin (ThT) fluorescence-based assay. All corrinated compounds retained original concentration to a higher degree than glucagon controls and showed markedly decreased aggregation compared to their respective noncorrinated analogues. Based on the statistically significant increase in chemical stability coupled with the notably decreased tendency to form aggregates, analogues 2 and its corrinated conjugate 5 were used for a functional assay study performed after agitation at 37°C for 24-hr after which agonism was measured at the human glucagon receptor using a cAMP FRET assay. Corrinated 5 exhibited a 6.6-fold increased potency relative to glucagon, which was shown to have a 165-fold reduction in potency. The relative potency of 5 was also improved compared to that of 2 with EC50 values of 5.5 nM and 9.6 nM for 5 and 2, respectively. In conclusion, corrination of peptides mitigates aggregation, presenting a compound with prolonged stability and agonism as demonstrated for glucagon.

De novo design of high-affinity binders of bioactive helical peptides.

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

Native Glucagon Amyloids Catalyze Key Metabolic Reactions.

Glucagon is a prominent peptide hormone, playing central roles in the regulation of glucose blood-level and lipid metabolism. Formation of glucagon amyloid fibrils has been previously reported, although no biological functions of such fibrils are known. Here, we demonstrate that glucagon amyloid fibrils catalyze biologically important reactions, including esterolysis, lipid hydrolysis, and dephosphorylation. In particular, we found that glucagon fibrils catalyze dephosphorylation of adenosine triphosphate (ATP), a core metabolic reaction in cell biology. Comparative analysis of several glucagon variants allowed mapping the catalytic activity to an enzymatic pocket-like triad formed at the glucagon fibril surface, comprising the histidyl-serine domain at the N-terminus of the peptide. This study may point to previously unknown physiological roles and pathological consequences of glucagon fibrillation and supports the hypothesis that catalytic activities of native amyloid fibrils play functional roles in human physiology and disease.

Glucose-Fueled Peptide Assembly: Glucagon Delivery via Enzymatic Actuation.

Nature achieves remarkable function from the formation of transient, nonequilibrium materials realized through continuous energy input. The role of enzymes in catalyzing chemical transformations to drive such processes, often as part of stimuli-directed signaling, governs both material formation and lifetime. Inspired by the intricate nonequilibrium nanostructures of the living world, this work seeks to create transient materials in the presence of a consumable glucose stimulus under enzymatic control of glucose oxidase. Compared to traditional glucose-responsive materials, which typically engineer degradation to release insulin under high-glucose conditions, the transient nanofibrillar hydrogel materials here are stabilized in the presence of glucose but destabilized under conditions of limited glucose to release encapsulated glucagon. In the context of blood glucose control, glucagon offers a key antagonist to insulin in responding to hypoglycemia by signaling the release of glucose stored in tissues so as to restore normal blood glucose levels. Accordingly, these materials are evaluated in a prophylactic capacity in diabetic mice to release glucagon in response to a sudden drop in blood glucose brought on by an insulin overdose. Delivery of glucagon using glucose-fueled nanofibrillar hydrogels succeeds in limiting the onset and severity of hypoglycemia in mice. This general strategy points to a new paradigm in glucose-responsive materials, leveraging glucose as a stabilizing cue for responsive glucagon delivery in combating hypoglycemia. Moreover, compared to most fundamental reports achieving nonequilibrium and/or fueled classes of materials, the present work offers a rare functional example using a disease-relevant fuel to drive deployment of a therapeutic.

Mutational Landscape of the Proglucagon-Derived Peptides.

Strong efforts have been placed on understanding the physiological roles and therapeutic potential of the proglucagon peptide hormones including glucagon, GLP-1 and GLP-2. However, little is known about the extent and magnitude of variability in the amino acid composition of the proglucagon precursor and its mature peptides. Here, we identified 184 unique missense variants in the human proglucagon gene GCG obtained from exome and whole-genome sequencing of more than 450,000 individuals across diverse sub-populations. This provides an unprecedented source of population-wide genetic variation data on missense mutations and insights into the evolutionary constraint spectrum of proglucagon-derived peptides. We show that the stereotypical peptides glucagon, GLP-1 and GLP-2 display fewer evolutionary alterations and are more likely to be functionally affected by genetic variation compared to the rest of the gene products. Elucidating the spectrum of genetic variations and estimating the impact of how a peptide variant may influence human physiology and pathophysiology through changes in ligand binding and/or receptor signalling, are vital and serve as the first important step in understanding variability in glucose homeostasis, amino acid metabolism, intestinal epithelial growth, bone strength, appetite regulation, and other key physiological parameters controlled by these hormones.

Optimization of Truncated Glucagon Peptides to Achieve Selective, High Potency, Full Antagonists.

Antagonism of glucagon's biological action is a proven strategy for decreasing glucose in diabetic animals and patients. To achieve full, potent, and selective suppression, we chemically optimized N-terminally truncated glucagon fragments for the identification and establishment of the minimum sequence peptide, [Glu9]glucagon(6-29) amide (11) as a full antagonist in cellular signaling and receptor binding (IC50 = 36 nM). Substitution of Phe6 with l-3-phenyllactic acid (Pla) produced [Pla6, Glu9]glucagon(6-29) amide (21), resulting in a 3-fold improvement in receptor binding (IC50 = 12 nM) and enhanced antagonist potency. Further substitution of Glu9 and Asn28 with aspartic acid yielded [Pla6, Asp28]glucagon amide (26), which demonstrated a further increase in inhibitory potency (IC50 = 9 nM), and improved aqueous solubility. Peptide 26 and a palmitoylated analogue, [Pla6, Lys10(γGluγGlu-C16), Asp28]glucagon(6-29) amide (31), displayed sustained duration in vivo action that successfully reversed glucagon-induced glucose elevation in mice.

Glucagon Prevents Cytotoxicity Induced by Methylglyoxal in a Rat Neuronal Cell Line Model.

Although diabetic polyneuropathy (DPN) is a frequent diabetic complication, no effective therapeutic approach has been established. Glucagon is a crucial hormone for glucose homeostasis but has pleiotropic effects, including neuroprotective effects in the central nervous system. However, the importance of glucagon in the peripheral nervous system (PNS) has not been clarified. Here, we hypothesized that glucagon might have a neuroprotective function in the PNS. The immortalized rat dorsal root ganglion (DRG) neuronal cell line 50B11 was treated with methylglyoxal (MG) to mimic an in vitro DPN model. The cells were cultured with or without glucagon or MG. Neurotoxicity, survival, apoptosis, neurite projection, cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) were examined. Glucagon had no cytotoxicity and rescued the cells from neurotoxicity. Cell survival was increased by glucagon. The ratio of apoptotic cells, which was increased by MG, was reduced by glucagon. Neurite outgrowth was accelerated in glucagon-treated cells. Cyclic AMP and PKA accumulated in the cells after glucagon stimulation. In conclusion, glucagon protected the DRG neuronal cells from MG-induced cellular stress. The cAMP/PKA pathway may have significant roles in those protective effects of glucagon. Glucagon may be a potential target for the treatment of DPN.

Novel glucagon- and OXM-based peptides acting through glucagon and GLP-1 receptors with body weight reduction and anti-diabetic properties.

Oxyntomodulin (OXM) is an endogenous gastrointestinal hormone, which activates both the Glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR). However, OXM has shortcomings including poor GLP-1R agonism to control glycemia, short half-life and others. Inspired from the sequence relationship between OXM and glucagon, in this study, we introduced different C-terminus residues of GLP-1, exenatide and OXM to glucagon to get a series of hybrid peptides with enhanced GLP-1R activation. The formed glucagon-exenatide hybrid peptide shows higher GLP-1R activation properties than OXM. Then the peptides based on the glucagon-exenatide hybrid peptide were coupled with fatty acid side chains to prolong their half-lives. As a result, the most potent compound 16a could stimulate insulin secretion and maintain blood glucose in normal level for ~42.6 h in diabetic mice. 16a exhibited reduced HbA1c level in diabetic mice, lowered body weight significantly in obesity mice on chronic treatment assay. 16a, combined efficient GCGR/GLP-1R activity, is potential as novel treatment for obesity and diabetes. This finding provides new insights into balancing GLP-1/GCGR potency of glucagon-exenatide hybrid peptide and is helpful for discovery of novel anti-diabetic and bodyweight-reducing drugs.

Selection and progression of unimolecular agonists at the GIP, GLP-1, and glucagon receptors as drug candidates.

The continued global growth in the prevalence of obesity coupled with the limited number of efficacious and safe treatment options elevates the importance of innovative pharmaceutical approaches. Combinatorial strategies that harness the metabolic benefits of multiple hormonal mechanisms have emerged at the preclinical and more recently clinical stages of drug development. A priority has been anti-obesity unimolecular peptides that function as balanced, high potency poly-agonists at two or all the cellular receptors for the endocrine hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon. This report reviews recent progress in this area, with emphasis on what the initial clinical results demonstrate and what remains to be addressed.

Cellular and sub-cellular localisation of oxyntomodulin-like immunoreactivity in enteroendocrine cells of human, mouse, pig and rat.

We use a monoclonal antibody against the C-terminal of oxyntomodulin (OXM) to investigate enteroendocrine cells (EEC) in mouse, rat, human and pig. This antibody has cross-reactivity with the OXM precursor, glicentin (Gli) but does not recognise glucagon. The antibody stained EEC in the jejunum and colon of each species. We compared OXM/Gli immunoreactivity with that revealed by antibodies against structurally related peptides, GLP-1 and glucagon and against GIP and PYY that are predicted to be in some EEC that express OXM/Gli. We used super-resolution to locate immunoreactive vesicles. In the pancreas, OXM/Gli was in glucagon cells but was located in separate storage vesicles to glucagon. In jejunal EEC, OXM/Gli and GIP were in many of the same cells but often in separate vesicles, whereas PYY and OXM/Gli were commonly colocalised in the same storage vesicles of colonic EEC. When binding of anti-GLP-1 to the structurally related GIP was removed by absorption with GIP peptide, GLP-1 and OXM/Gli immunoreactivities were contained in the same population of EEC in the intestine. We conclude that anti-OXM/Gli is a more reliable marker than anti-GLP-1 for EEC expressing preproglucagon products. Storage vesicles that were immunoreactive for OXM/Gli were almost always immunoreactive for GLP-1. OXM concentrations, measured by ELISA, were highest in the distal ileum and colon. Lesser concentrations were found in more proximal parts of small intestine and pancreas. Very little was in the stomach. In EEC containing GIP and OXM/Gli, these hormones are packaged in different secretory vesicles. Separate packaging also occurred for OXM and glucagon, whereas OXM/Gli and PYY and OXM/Gli and GLP-1 were commonly contained together in secretory vesicles.

High throughput detection of deamidation using S-(5'-adenosyl)-l-homocysteine hydrolase and a fluorogenic reagent.

Deamidation of asparagine (Asn) residues is one of the most common chemical degradation pathways observed in proteins. This reaction must be understood and controlled in therapeutic drug candidates, as chemical changes can affect their efficacy and safety. The analytical tools available for detection of deamidation reaction products, such as isoaspartic acid residues, are either chromatographic or electrophoretic, and require MS detection for absolute identification of peaks. High-throughput measurement of protein degradation has typically been limited to probing the target's physical state using spectroscopic techniques. Here, we describe a high throughput assay for isoaspartate residues using fluorescent detection in a microtiter plate format. The method allows for fast detection of protein deamidation in a cost-efficient manner. The method can be employed even if the target peptide or protein contains free Cys residues. The technique appears to be selective, linear, and accurate.

Clinical implications of current cardiovascular outcome trials with sodium glucose cotransporter-2 (SGLT2) inhibitors.

The final goal in the management of patients with type 2 diabetes (T2D) is reduction in cardiovascular (CV) complications and total mortality. Various factors including hyperglycemia contribute to these complications and mortality directly and indirectly. In recent years, large-scale CV outcome trials with new antidiabetic medications, such as dipeptidyl peptidase-4 (DPP4) inhibitors, glucagon-like peptide-1 (GLP1) receptor agonists, and sodium glucose cotransporter-2 (SGLT2) inhibitors, have been completed. Most clinical trials with DPP4 inhibitors have shown no inferiority compared with placebo treatments in terms of CV safety. However, they did not show benefits in terms of adverse CV events or mortality. CV outcome trials with GLP1 receptor agonists showed inconsistent results: lixisenatide did not show benefits in preventing major adverse CV events. In contrast, liraglutide and semaglutide (longer acting GLP1 receptor agonists) proved to be superior in terms of alleviating CV morbidity and mortality. Two large-scale CV outcome trials with SGLT2 inhibitors showed significant results: empagliflozin proved to be superior in preventing CV and all-cause mortality, and canagliflozin proved to be superior in preventing CV mortality but not all-cause mortality. So far, controlling cardiometabolic risk factors such as hemodynamic changes and weight loss by SGLT2 inhibitors are suggested to be the main mechanisms for these results. However, the risk-benefit profile for these new drugs will need further elucidation, and more studies are warranted to reveal the possible mechanisms. It will also be important to confirm these results from other ongoing trials with SGLT2 inhibitors.

Peptide degradation and the role of DPP-4 inhibitors in the treatment of type 2 diabetes.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are now a widely used, safe and efficacious class of antidiabetic drugs, which were developed prospectively using a rational drug design approach based on a thorough understanding of the endocrinology and degradation of glucagon-like peptide-1 (GLP-1). GLP-1 is an intestinal hormone with potent insulinotropic and glucagonostatic effects and can normalise blood glucose levels in patients with type 2 diabetes, but the native peptide is not therapeutically useful because of its inherent metabolic instability. Using the GLP-1/DPP-4 system and type 2 diabetes as an example, this review summarises how knowledge of a peptide's biological effects coupled with an understanding of the pathways involved in its metabolic clearance can be exploited in a rational, step-by-step manner to develop a therapeutic agent, which is effective and well tolerated, and any side effects are minor and largely predictable. Other peptides with metabolic effects which can also be degraded by DPP-4 will be reviewed, and their potential role as additional mediators of the effects of DPP-4 inhibitors will be assessed.

A novel GLP-1/xenin hybrid peptide improves glucose homeostasis, circulating lipids and restores GIP sensitivity in high fat fed mice.

Combined modulation of peptide hormone receptors including, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and xenin, have established benefits for the treatment of diabetes. The present study has assessed the biological actions and therapeutic efficacy of a novel exendin-4/xenin-8-Gln hybrid peptide, both alone and in combination with the GIP receptor agonist (DAla2)GIP. Exendin-4/xenin-8-Gln was enzymatically stable and exhibited enhanced insulin secretory actions when compared to its parent peptides. Exendin-4/xenin-8-Gln also possessed ability to potentiate the in vitro actions of GIP. Acute administration of exendin-4/xenin-8-Gln in mice induced appetite suppressive effects, as well as significant and protracted glucose-lowering and insulin secretory actions. Twice daily administration of exendin-4/xenin-8-Gln, alone or in combination with (DAla2)GIP, for 21-days significantly reduced non-fasting glucose and increased circulating insulin levels in high fat fed mice. In addition, all exendin-4/xenin-8-Gln treated mice displayed improved glucose tolerance, insulin sensitivity and metabolic responses to GIP. Combination therapy with (DAla2)GIP did not result in any obvious further benefits. Metabolic improvements in all treatment groups were accompanied by reduced pancreatic beta-cell area and insulin content, suggesting reduced insulin demand. Interestingly, body weight, food intake, circulating glucagon, metabolic rate and amylase activity were unaltered by the treatment regimens. However, all treatment groups, barring (DAla2)GIP alone, exhibited marked reductions in total- and LDL-cholesterol. Furthermore, exendin-4 therapy also reduced circulating triacylglycerol. This study highlights the positive antidiabetic effects of exendin-4/xenin-8-Gln, and suggests that combined modulation of GLP-1 and xenin related signalling pathways represents an exciting treatment option for type 2 diabetes.

Stable Liquid Glucagon: Beyond Emergency Hypoglycemia Rescue.

Glycemic control is the mainstay of preventing diabetes complications at the expense of increased risk of hypoglycemia. Severe hypoglycemia negatively impacts the quality of life of patients with type 1 diabetes and can lead to morbidity and mortality. Currently available glucagon emergency kits are effective at treating hypoglycemia when correctly used, however use is complicated especially by untrained persons. Better formulations and devices for glucagon treatment of hypoglycemia are needed, specifically stable liquid glucagon. Out of the scope of this review, other potential uses of stable liquid glucagon include congenital hyperinsulinism, post-bariatric surgery hypoglycemia, and insulinoma induced hypoglycemia. In the 35 years since Food and Drug Administration (FDA) approval of the first liquid stable human recombinant insulin, we continue to wait for the glucagon counterpart. For mild hypoglycemia, a commercially available liquid stable glucagon would enable more widespread implementation of mini-dose glucagon use as well as glucagon in dual hormone closed-loop systems. This review focuses on the current and upcoming pharmaceutical uses of glucagon in the treatment of type 1 diabetes with an outlook on stable liquid glucagon preparations that will hopefully be available for use in patients in the near future.

Evolution of the glucagon-like system across fish.

In fishes, including the jawless lampreys, the most ancient lineage of extant vertebrates, plasma glucose levels are highly variable and regulation is more relaxed than in mammals. The regulation of glucose and lipid in fishes in common with mammals involves members of the glucagon (GCG)-like family of gastrointestinal peptides. In mammals, four peptides GCG, glucagon-like peptide 1 and 2 (GLP1 and GLP2) and glucose-dependent insulinotropic peptide (GIP) that activate four specific receptors exist. However, in lamprey and other fishes the glucagon-like family evolved differently and they retained additional gene family members (glucagon-related peptide, gcrp and its receptor, gcrpr) that are absent from mammals. In the present study, we analysed the evolution of the glucagon-like system in fish and characterized gene expression of the family members in the European sea bass (Dicentrarchus labrax) a teleost fish. Phylogenetic analysis revealed that multiple receptors and peptides of the glucagon-like family emerged early during the vertebrate radiation and evolved via lineage specific events. Synteny analysis suggested that family member gene loss is likely to be the result of a single gene deletion event. Lamprey was the only fish where a putative glp1r persisted and the presence of the receptor gene in the genomes of the elephant shark and coelacanth remains unresolved. In the coelacanth and elephant shark, unique proglucagon genes were acquired which in the former only encoded Gcg and Glp2 and in the latter, shared a similar structure to the teleost proglucagon gene but possessed an extra exon coding for Glp-like peptide that was most similar to Glp2. The variable tissue distribution of the gene transcripts encoding the ligands and receptors of the glucagon-like system in an advanced teleost, the European sea bass, suggested that, as occurs in mammals, they have acquired distinct functions. Statistically significant (p < .05) down-regulation of teleost proglucagon a in sea bass with modified plasma glucose levels confirmed the link between these peptides and metabolism. The tissue distribution of members of the glucagon-like system in sea bass and human suggests that evolution of the brain-gut-peptide regulatory loop diverged between teleosts and mammals despite the overall conservation and similarity of glucagon-like family members.

Rebirth of the Incretin Concept: Its conception and early development.

This paper describes the resurrection of the Incretin Concept in the early 1960s. It began with the more or less simultaneous discovery by three groups working independently in London. Dupre demonstrated that secretin given intravenously with glucose increased its rate of disappearance from the blood, McIntyre and co-workers established that hyperglycaemia evoked by oral glucose stimulated more insulin secretion than comparable hyperglycaemia produced by intravenous glucose and Marks and Samols established the insulinotropic properties of glucagon. The concept evolved with the discovery by Samols and co-workers that oral glucose stimulated the release of immunoreactive glucagon-like substances from the gut mucosa and the subsequent isolation of glucagon immunoreactive compounds, most notably oxyntomodulin and glicentin, and of gastic inhibitory polypetide (GIP). It concluded with the isolation and characterisation of glucagon-like peptide 1 (7-36) amide.

A novel glucagon-like peptide-1/glucagon receptor dual agonist exhibits weight-lowering and diabetes-protective effects.

Glucagon has plenty of effects via a specific glucagon receptor(GCGR) like elevating the blood glucose, improving fatty acids metabolism, energy expenditure and increasing lipolysis in adipose tissue. The most important role of glucagon is to regulate the blood glucose, but the emergent possibilities of hyperglycaemia is exist. Glucagon could also slightly activate glucagon-like peptide-1 receptor(GLP-1R), which lead to blood glucose lowering effect. This study aims to erase the likelihood of hyperglycaemia and to remain the inherent catabolic effects through improving GLP-1R activation and deteriorating GCGR activation so as to lower the bodyweight and show diabetes-protective effects. Firstly, twelve cysteine modified GLP-1/GCGR dual agonists were synthesized (1-12). Then, the GLP-1R/GCGR mediated activation and biological activity in normal ICR mice were comprehensively performed. Compounds substituted by cysteine at positions 22, 23 and 25 in glucagon were observed to be better regulators of the body weight and blood glucose. To prolong the half-lives of derivatives, various fatty side chain maleimides were modified to optimal glucagon analogues. Laurate maleimide conjugate 4d was the most potent. Administration of 1000 nmol/kg 4d once every two days for a month normalized adiposity and glucose tolerance in diet-induced obese (DIO) mice. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were observed. These studies suggest that compound 4d behaves well in lowering body weight and maintaining energy expenditure without a chance of hyperglycaemia, 4d has strong clinical potential as an efficient GLP-1/GCGR agonist in the prevention and treatment of obesity and dyslipidemia.