Development of Potent Truncated Glucagon Antagonists

Abstract

In pursuit of truncated glucagon analogues that can interact with the glucagon receptor with substantial binding affinity, 23 truncated glucagon analogues have been designed and synthesized. These truncated analogues consist of several fragments of glucagon with 11 or 12 amino acid residues (1-4), conformationally constrained analogues containing the sequence of the middle region of glucagon (5-15), and truncated analogues containing the sequence of the C-terminal region (16-23). Biological assays of these analogues showed that the truncated glucagon analogues with the sequence of the C-terminal region possess significantly better binding affinity compared to the truncated analogues with the sequence of the middle region, and these analogues (17-23) demonstrated potent antagonistic activity (pA(2) values between 6.5 and 7.5). On the basis of these results, it can be suggested that glucagon interacts with its receptor with two hydrophobic patches located in the middle and the C-terminal regions of glucagon, and both hydrophobic patches are necessary for significant receptor recognition. These two hydrophobic binding motifs, located in two different regions of glucagon, appear to be the reason why the earlier attempts to obtain truncated analogues with good binding affinity did not result in any success. Long peptide hormones such as glucagon seem to require more than one binding pocket on the receptors for maximal interaction.

Structure of the human glucagon class B G-protein-coupled receptor

“…4d, Supplementary Table 5), which likely distorts the α-helical conformation of the stalk region. The stalk may also function as part of the binding site for the previously proposed middle hinge region of glucagon 27 . Interestingly, an α-helical conformation in this region of GLP1R in complex with the GLP1 peptide was recently proposed based on crosslinking data between receptor and peptide residues 12 .…”

Insights into the structure of class B GPCRs

“…The extended stalk region (residues 125–136 in GCGR), which links the ECD and 7TM domain, may be involved in peptide ligand binding and help define the orientation of the ECD with respect to the TMD (Figure 4a). The α-helical structure of the stalk is stabilized by intrahelical interactions in the GCGR crystal structure (Glu 133–Lys 136) and GCGR-glucagon model (Glu127–Gln131 and Glu129–Lys132) and fits a GCGR-glucagon binding mode model supported by GCGR mutagenesis [11,29–35] and glucagon structure-activity relationship [36–38] studies in which: i) a hydrophobic region in the C-terminus of glucagon (residues Phe22, Val23, Trp25, and Met27) binds the ECD (as observed in the GLP-1 peptide bound GLP-1 ECD crystal structure [39]), ii) a second hydrophobic region in the middle region of glucagon (residues Tyr10, Tyr13, and Leu14 [38]) interacts with the stalk and extracellular loop 1, and iii) the N-terminus of glucagon (residues 1–6) interacts with the TMD (Figures 2 and 4). The helical structure of the stalk is further supported by a reduced glucagon affinity to a Ala135Pro mutant, which probably distorts the α-helical conformation [11].…”

Max Bergmann award lecture:Macromolecular medicinal chemistry as applied to metabolic diseases

“…As a synthetic target, glucagon served an important role and a controversial synthetic entity in the development of solid‐phase peptide chemistry . Paradoxically, the primary medicinal interest in the hormone has resided in advancement of antagonists for treatment of hyperglycemia . Glucagon as an emergency drug is highly compromised by its limited aqueous solubility, pronounced chemical instability, and further worsened by a strong tendency to self‐aggregate.…”

“…[29,30] Paradoxically, the primary medicinal interest in the hormone has resided in advancement of antagonists for treatment of hyperglycemia. [31,32] Glucagon as an emergency drug is highly compromised by its limited aqueous solubility, pronounced chemical instability, and further worsened by a strong tendency to self-aggregate. The drug is currently supplied as a crystalline powder to be solubilized in dilute hydrochloric acid for singular, immediate use at time of compromised consciousness due to severe hypoglycemia.…”