Analysis of aggregation profile of glucagon using SEC-HPLC and FFF-MALS methods.

Recently, the first generic glucagon for injection was approved for the treatment of severe hypoglycemia. Unlike its brand name recombinant glucagon, the generic glucagon is synthetic. Since glucagon has a high propensity to form aggregates in solution, it is essential to assess the aggregation profile of the synthetic glucagon compared to the recombinant glucagon. In this study, two robust separation methods, size-exclusion chromatography (SEC-HPLC) and field-flow fractionation coupled with a multi-angle light scattering detector (FFF-MALS), were employed to characterize generic and brand glucagon aggregation in six lots (three newly released, three expired). The presence of aggregation in samples was determined from the generated chromatograms and analyzed. The study showed that both products have comparable aggregation profiles. The SEC-HPLC demonstrated that in both glucagon versions, the expired lots had a higher percentage of dimers than the newly released lots, but even at expiration, the amount was negligible (∼0.1%). The FFF-MALS method did not detect any dimers or higher molecular weight aggregates. Further evaluation of the detection limit found that FFF-MALS was unable to detect aggregates at amounts lower than 0.5% of total glucagon. The negligible amounts of dimer detected in the generic and brand glucagon indicate that both versions are physically stable and are not prone to aggregation under clinically relevant conditions.

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.

Dynamic membrane interaction and amyloid fibril formation of glucagon, melittin and human calcitonin.

Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic. It is important to reveal the glucagon-membrane interaction to understand activity and cytotoxicity of glucagon and glucagon oligomers. In this review, first glucagon-membrane interactions are described as morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in acidic and neutral conditions as compared to the case of melittin. Second, fibril formation by glucagon in acidic solution is discussed in light of morphological and structural changes. Third, kinetic analysis of glucagon fibril formation was performed using a two-step autocatalytic reaction mechanism, as investigated in the case of human calcitonin. The first step is a nuclear formation, and the second step is an autocatalytic fibril elongation. Forth, fibril formation of glucagon inside glucagon-DMPC bilayers in neutral solution under near physiological condition is described.

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.

Discovery of Novel Allosteric Modulators Targeting an Extra-Helical Binding Site of GLP-1R Using Structure- and Ligand-Based Virtual Screening.

Allosteric modulators have emerged with many potential pharmacological advantages as they do not compete the binding of agonist or antagonist to the orthosteric sites but ultimately affect downstream signaling. To identify allosteric modulators targeting an extra-helical binding site of the glucagon-like peptide-1 receptor (GLP-1R) within the membrane environment, the following two computational approaches were applied: structure-based virtual screening with consideration of lipid contacts and ligand-based virtual screening with the maintenance of specific allosteric pocket residue interactions. Verified by radiolabeled ligand binding and cAMP accumulation experiments, two negative allosteric modulators and seven positive allosteric modulators were discovered using structure-based and ligand-based virtual screening methods, respectively. The computational approach presented here could possibly be used to discover allosteric modulators of other G protein-coupled receptors.

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.

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.

Structurally Based Design of Glucagon Mutants That Inhibit Fibril Formation.

The peptide hormone glucagon is prescribed as a pharmaceutical compound to treat diabetic hypoglycemia. However, at the acidic pH where it is highly soluble, glucagon rapidly aggregates into inactive and cytotoxic amyloid fibrils. The recently determined high-resolution structure of these fibrils revealed various stabilizing molecular interactions. On the basis of this structure, we have now designed four arginine mutants of glucagon that resist fibrillization at pharmaceutical concentrations for weeks. An S2R, T29R double mutant and a T29R single mutant remove a hydrogen-bonding interaction in the wild-type fibril, whereas a Y13R, A19R double mutant and a Y13R mutant remove a cation-π interaction. 1H solution nuclear magnetic resonance spectra and ultraviolet absorbance data indicate that these mutants remain soluble in pH 2 buffer under quiescent conditions at concentrations of ≤4 mg/mL for weeks. Under stressed conditions with high salt concentrations and agitation, these mutants fibrillize significantly more slowly than the wild type. The S2R, T29R mutant and the T29R mutant exhibit a mixture of random coil and α-helical conformations, while the Y13R mutant is completely random coil. The mutation sites are chosen to be uninvolved in strong interactions with the glucagon receptor in the active structure of the peptide. Therefore, these arginine mutants of glucagon are promising alternative compounds for treating hypoglycemia.

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.

Dual self-regulated delivery of insulin and glucagon by a hybrid patch.

Reduced ß-cell function and insulin deficiency are hallmarks of diabetes mellitus, which is often accompanied by the malfunction of glucagon-secreting α-cells. While insulin therapy has been developed to treat insulin deficiency, the on-demand supplementation of glucagon for acute hypoglycemia treatment remains inadequate. Here, we describe a transdermal patch that mimics the inherent counterregulatory effects of ß-cells and α-cells for blood glucose management by dynamically releasing insulin or glucagon. The two modules share a copolymerized matrix but comprise different ratios of the key monomers to be "dually responsive" to both hyper- and hypoglycemic conditions. In a type 1 diabetic mouse model, the hybrid patch effectively controls hyperglycemia while minimizing the occurrence of hypoglycemia in the setting of insulin therapy with simulated delayed meal or insulin overdose.

Structural basis of Gs and Gi recognition by the human glucagon receptor.

Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1 These two structures adopt a similar open binding cavity to accommodate Gs and Gi1 The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.

31P and 13C solid-state NMR analysis of morphological changes of phospholipid bilayers containing glucagon during fibril formation of glucagon under neutral condition.

Glucagon is a 29 amino acid peptide hormone secreted by pancreatic α-cells that interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibrillar aggregates that are cytotoxic due to their activation of apoptotic signaling pathways. To understand the glucagon-membrane interactions, morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in neutral solution were investigated by observing 31P NMR spectra. First, lipid bilayers with a DMPC/glucagon molar ratio of 50/1 were observed. One day after preparing the DMPC/glucagon lipid bilayer sample, lipid bilayers were disrupted below the phase transition temperature (Tc). Membrane disruption was reduced 2 days after preparation due to the reduction of glucagon-DMPC interaction, and subsequently increased by 4 days and was reduced again by 7 days. TEM measurements showed that small ellipsoidal intermediates of glucagon were observed inside the small size of lipid bilayer after 4 days, while fibrils grew inside lipid bilayer after 19 days. These results indicate that morphological changes in DMPC/glucagon lipid bilayers are correlated with the evolution of glucagon aggregate state. Particularly, fibril intermediate shows a strong glucagon lipid bilayer interaction. We further investigated the structure and kinetics of glucagon fibril formation inside the DMPC lipid bilayer in a neutral solution using 13C solid-state NMR spectroscopy. α-Helical structures were observed around Gly4 and Ala19 in the monomeric form, which changed to ß-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k1), and the second step is an autocatalytic heterogeneous fibrillation process (k2).

Engineering Xaa-Pro dipeptidyl aminopeptidase for specific cleavage of glucagon and glucagon-like peptide 1 from fusion proteins.

N-terminal extensions ("tags") have proven valuable for producing peptides using high throughput recombinant expression technologies. However, the applicability is hampered by the limited options for specific and efficient proteases to release the fully native sequence without additional amino acids in the N-terminal. Here we describe the Escherichia coli (E. coli) expression, purification and characterization of engineered variants of Xaa-Pro dipeptidyl aminopeptidase (Xaa-Pro-DAP) derived from Lactococcus lactis for cleavage of Gly-Pro dipeptide extension in the N-terminal of glucagon and glucagon-like peptide 1 (GLP-1(7-37)). By single amino acid substitution in the Xaa-Pro-DAP protease, significantly higher product yields were achieved. The combination of HRV14 3C protease and engineered Xaa-Pro-DAP is suggested for obtaining native N-terminal of peptides.

Revisiting the Pharmacological Value of Glucagon: An Editorial for the Special Issue "The Biology and Pharmacology of Glucagon".

In 1921, a Canadian research team led by Frederick Banting and John Macleod succeeded in the isolation of insulin from pancreatic homogenate [...].

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.

Structural Refinement of Glucagon for Therapeutic Use.

Glucagon counters insulin's effects on glucose metabolism and serves as a rescue medicine in the treatment of hypoglycemia. Acute hypoglycemia, a common occurrence in insulin-dependent diabetes, is the central obstacle to correcting high blood glucose, a primary cause of long-term microvascular complications. As a result, there has been a resurgence of interest in improved glucagon therapy, including nonconventional liquid formulations, alternative routes of administration, and novel analogs with optimized biophysical properties. These options collectively minimize the complexity of glucagon delivery and enable its application in ways not feasible with conventional emergency rescue kits. These advances have indirectly promoted the integrated use of glucagon agonism with other hormones in a manner that runs counter to the long-standing pursuit of glucagon antagonism. This review summarizes novel approaches to glucagon optimization, methods with potential application to the broader family of therapeutic peptides, where biophysical challenges may be encountered.

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.

The peptide hormone glucagon forms amyloid fibrils with two coexisting ß-strand conformations.

Glucagon and insulin maintain blood glucose homeostasis and are used to treat hypoglycemia and hyperglycemia, respectively, in patients with diabetes. Whereas insulin is stable for weeks in its solution formulation, glucagon fibrillizes rapidly at the acidic pH required for solubility and is therefore formulated as a lyophilized powder that is reconstituted in an acidic solution immediately before use. Here we use solid-state NMR to determine the atomic-resolution structure of fibrils of synthetic human glucagon grown at pharmaceutically relevant low pH. Unexpectedly, two sets of chemical shifts are observed, indicating the coexistence of two ß-strand conformations. The two conformations have distinct water accessibilities and intermolecular contacts, indicating that they alternate and hydrogen bond in an antiparallel fashion along the fibril axis. Two antiparallel ß-sheets assemble with symmetric homodimer cross sections. This amyloid structure is stabilized by numerous aromatic, cation-π, polar and hydrophobic interactions, suggesting mutagenesis approaches to inhibit fibrillization could improve this important drug.