A Facile Procedure for One-Pot Stable Conjugation of Two Proglucagon Cysteine-Containing Peptide Analogs.

Optimization of peptides for therapeutic purposes often includes chemical conjugation or modification with substituents that serve to broaden pharmacology or improve pharmacokinetics. We report a convenient and rapid procedure for one-pot, site-specific conjugation of two cysteine-containing peptides that utilizes a bivalent linker comprising maleimide and iodoacetyl functional groups. Following maleimide-mediated peptide conjugation the linker was converted from an unstable thiosuccinimide to a stable thioether bond suitable for biological study by mild aqueous hydrolysis. The procedure is exemplified by peptide-peptide, peptide-small molecule, and peptide-fatty acid conjugations. The method provides a facile approach to search for enhanced biological outcomes through additive and sustained peptide pharmacology unencumbered by the prospect of chemical rearrangement in the course of biological study.

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.

Hepatic Glucagon Receptor Signaling Enhances Insulin-Stimulated Glucose Disposal in Rodents.

Glucagon receptor (GCGR) agonists cause hyperglycemia but also weight loss. However, GCG-like peptide 1 receptor (GLP1R)/GCGR mixed agonists do not exhibit the diabetogenic effects often attributed to GCGR activity. Thus, we sought to investigate the effect of glucagon agonism on insulin action and glucose homeostasis. Acute GCGR agonism induced immediate hyperglycemia, followed by improved glucose tolerance and enhanced glucose-stimulated insulin secretion. Moreover, acute GCGR agonism improved insulin tolerance in a dose-dependent manner in both lean and obese mice. Improved insulin tolerance was independent of GLP1R, FGF21, and hepatic glycogenolysis. Moreover, we observed increased glucose infusion rate, disposal, uptake, and suppressed endogenous glucose production during euglycemic clamps. Mice treated with insulin and GCGR agonist had enhanced phosphorylation of hepatic AKT at Ser473; this effect was reproduced in isolated mouse primary hepatocytes and resulted in increased AKT kinase activity. These data reveal that GCGR agonism enhances glucose tolerance, in part, by augmenting insulin action, with implications for the use of GCGR agonism in therapeutic strategies for diabetes.

Three-chain insulin analogs demonstrate the importance of insulin secondary structure to bioactivity.

This report describes the chemical synthesis and biological characterization of novel three-chain insulin analogs with a destabilized secondary structure. The analogs, obtained by chemical synthesis via a single-chain precursor and selective enzymatic digestion, were used to investigate the role of the highly conserved 'insulin fold'. Biological characterization through in vitro biochemical signaling showed extremely low activity at each insulin receptor when compared with native insulin. We conclude that the 'insulin fold' is a structural foundation that supports insulin biological action.

A glucagon analog chemically stabilized for immediate treatment of life-threatening hypoglycemia.

For more than half a century glucagon has been used as a critical care medicine in the treatment of life-threatening hypoglycemia. It is commercially supplied as a lyophilized powder intended to be solubilized in dilute aqueous hydrochloric acid immediately prior to administration. We have envisioned a "ready-to-use" glucagon as a drug of more immediate and likely use. Through a series of iterative changes in the native sequence we have identified glucagon analogs of appreciably enhanced aqueous solubility at physiological pH, and of chemical stability suitable for routine medicinal use. The superior biophysical properties were achieved in part through adjustment of the isoelectric point by use of a C-terminal Asp-Glu dipeptide. The native glutamines at positions 3, 20 and 24 as well as the methionine at 27 were substituted with amino acids of enhanced chemical stability, as directed by a full alanine scan of the native hormone. Of utmost additional importance was the dramatically enhanced stability of the peptide when Ser16 was substituted with alpha,aminoisobutyric acid (Aib), a substitution that stabilizes peptide secondary structure. The collective set of changes yield glucagon analogs of comparable in vitro and in vivo biological character to native hormone but with biophysical properties much more suitable for clinical use.

GLP-1/glucagon coagonism restores leptin responsiveness in obese mice chronically maintained on an obesogenic diet.

We recently reported restoration of leptin responsiveness in diet-induced obese (DIO) mice using a pharmacologically optimized, polyethylene-glycolated (PEG)-leptin analog in combination with exendin-4 or FGF21. However, the return of leptin action required discontinuation of high-fat diet (HFD) exposure. Here we assess whether a single peptide possessing balanced coagonism at the glucagon-like peptide 1 (GLP-1) and glucagon receptors can restore leptin responsiveness in DIO mice maintained on a HFD. DIO mice were treated with PEG-GLP-1/glucagon (30 nmol/kg every fourth day) to induce an ∼15% body weight loss, upon which they were randomized to continue PEG-GLP-1/glucagon therapy or reassigned to receive supplemental daily PEG-leptin (185 nmol/kg/day). The addition of PEG-leptin to PEG-GLP-1/glucagon resulted in an ∼18% greater weight loss as compared with PEG-GLP-1/glucagon alone and was accompanied by further decreases in food intake and improved glucose and lipid metabolism. The beneficial effect of PEG-leptin supplementation occurred after an initial body weight loss similar to what we previously reported following reduced dietary fat along with PEG-leptin and exendin-4 or FGF21 cotreatment. In summary, we report that GLP-1/glucagon coagonism restores leptin responsiveness in mice maintained on a HFD, thus emphasizing the translational value of this polypharmacotherapy for the treatment of obesity and diabetes.

Direct control of brown adipose tissue thermogenesis by central nervous system glucagon-like peptide-1 receptor signaling.

We studied interscapular brown adipose tissue (iBAT) activity in wild-type (WT) and glucagon-like peptide 1 receptor (GLP-1R)-deficient mice after the administration of the proglucagon-derived peptides (PGDPs) glucagon-like peptide (GLP-1), glucagon (GCG), and oxyntomodulin (OXM) directly into the brain. Intracerebroventricular injection of PGDPs reduces body weight and increases iBAT thermogenesis. This was independent of changes in feeding and insulin responsiveness but correlated with increased activity of sympathetic fibers innervating brown adipose tissue (BAT). Despite being a GCG receptor agonist, OXM requires GLP-1R activation to induce iBAT thermogenesis. The increase in thermogenesis in WT mice correlates with increased expression of genes upregulated by adrenergic signaling and required for iBAT thermogenesis, including PGC1a and UCP-1. In spite of the increase in iBAT thermogenesis induced by GLP-1R activation in WT mice, Glp1r(-/-) mice exhibit a normal response to cold exposure, demonstrating that endogenous GLP-1R signaling is not essential for appropriate thermogenic response after cold exposure. Our data suggest that the increase in BAT thermogenesis may be an additional mechanism whereby pharmacological GLP-1R activation controls energy balance.

Optimization of the native glucagon sequence for medicinal purposes.

BACKGROUND: Glucagon is a life-saving medication used in the treatment of hypoglycemia. It possesses poor solubility in aqueous buffers at or near physiological pH values. At low and high pH, at which the peptide can be formulated to concentrations of a milligram or more per milliliter, the chemical integrity of the hormone is limited, as evidenced by the formation of multiple degradation-related peptides. Consequently, the commercial preparation is provided as a lyophilized solid with an acidic diluent and directions for rendering it soluble at the time of use. Any unused material is recommended for disposal immediately after initial use. METHODS: A set of glucagon analogs was prepared by solid-phase peptide synthesis to explore the identification of a glucagon analog with enhanced solubility and chemical stability at physiological pH. The physical properties of the peptide analogs were studied by solubility determination, high-performance chromatography, and mass spectral analysis. The biochemical properties were determined in engineered human embryonic kidney cell line 293 (HEK293) cells that overexpressed either the human glucagon or glucagon-like peptide-1 (GLP-1) receptors linked to a luciferase reporter gene. RESULTS: We observed the previously characterized formation of glucagon degradation products upon incubation of the peptide in dilute acid for extended periods or elevated temperature. Lowering the isoelectric point of the hormone through the substitution of asparagine-28 with aspartic acid significantly increased the solubility at physiological pH. Similarly, the C-terminal extension (Cex) of the hormone with an exendin-based, 10-residue, C-terminal sequence yielded a peptide of dramatically enhanced solubility. These two glucagon analogs, D28 and Cex, maintained high potency and selectivity for the glucagon receptor relative to GLP-1 receptor. CONCLUSIONS: Glucagon presents unique structural challenges to the identification of an analog of high biological activity and selectivity that also possesses sufficient aqueous solubility and stability such that it might be developed as a ready-to-use medicine. The glucagon analogs D28 and Cex demonstrated all of the chemical, physical, and biochemical properties supportive of further study as potential clinical candidates for treatment of hypoglycemia.