Search for bioactive conformation of glucagon and development of potent glucagon antagonists

Abstract

In pursuit of the working model of how glucagon interacts with the glucagon receptor and how glucagon antagonists exert their different activities, 42 glucagon analogues were designed and synthesized. An attempt to determine the minimum sequence for binding affinity of glucagon analogues was carried out and resulted in several potent truncated glucagon antagonists with substantial binding affinity, such as phenylbutyryl-glucagon(10-29) amide. Furthermore, a new method for determining the bioactive conformations of peptide hormones has been designed. In a positional cyclization scanning study, several conformationally constrained glucagon analogues containing disulfide or lactam bridges were synthesized, and the biological assay results showed that the alpha-helical conformation is required for the maximal receptor recognition. This study resulted in two superpotent glucagon analogues, c[Lys⁵, Glu⁹]glucagon amide and c[Lys¹⁷, Glu²¹]glucagon amide, which have picomolar binding affinities. A structure-activity relationship study of glycine at position 4 was performed to determine the importance of flexibility in the N-terminal region of glucagon. Four glucagon analogues were designed and synthesized, and all showed extremely potent antagonistic activity with improved binding affinity. Also, the potent glucagon antagonist [desHis¹, desPhe⁶, Glu⁹] glucagon amide was synthesized on a large scale (ca. 1.5 g), and the effect of the glucagon antagonist on diabetic ketoacidosis was studied in vivo in alloxan-induced diabetic dogs. The glucagon antagonist clearly showed its effectiveness in controlling serum bicarbonate concentration, while the control experiment with saline demonstrated increased diabetic ketoacidosis. This study clearly showed the possibility of using glucagon antagonists as therapeutic agents for the treatment of diabetic ketoacidosis. The conformation of the potent glucagon antagonist [desHis¹, desPhe⁶, Glu⁹] glucagon amide was studied using 2D NMR spectroscopy, and deuterated dodecylphosphocholine micelles were utilized to imitate the membrane environment. In this investigation, TOCSY, DQF-COSY, and NOESY spectra of the glucagon antagonist in a deuterated DPC micelle solution were acquired at pH 6.0 and 37°C. Restrained molecular dynamics (simulated annealing) using 332 distance restraints and 16 torsion angle restraints resulted in a conformation which displayed a similar C-terminal conformation, but a distinctly different N-terminal region, as compared to the conformation of glucagon. The newly discovered salt bridge between Ser² and Glu⁹ presumably resulted from the increased flexibility of the N-terminal region by the deletion of Phe⁶ and substitution of Glu⁹, which may shed light on how small changes in the sequence of peptides can significantly modify the conformation.