Synthesis of a possible glucagon inhibitor by semisynthesis of a glucagon analogue of [HARG¹²]-glucagon and [DES-HIS¹][HARG¹²]-glucagon
PublisherThe University of Arizona.
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Degree GrantorUniversity of Arizona
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Design and synthesis of conformationally constrained glucagon analogues to study the conformational features important for glucagon bioactivity.Lin, Ying.; Hruby, Victor J.; Bates, Robert B.; Mash, Eugene A., Jr.; Law, John H.; Hadley, Mac E. (The University of Arizona., 1993)We have synthesized ten glucagon analogues that are either conformationally constrained systematically in the middle portion of the molecule, or modified from the known superagonist analogue [Lys¹⁷,¹⁸, Glu²¹]glucagon to study the structure-activity relationships of glucagon. The analogues were prepared using the solid-phase peptide synthesis method. Cyclizations were accomplished by forming the side chain lactam (amide) bridges on the resin. All peptide analogues were cleaved from the solid support, deprotected by the low-high HF procedure, and purified by a combination of gel filtration chromatography and dialysis followed by reverse-phase high performance liquid chromatography. A new characterization method for cyclic glucagon analogues using fast atom bombardment mass spectrometry with endoproteinase Asp-N peptide mapping has been developed that has provided unequivocal confirmation of the presence and site of the rings as well as the amino acid compositions. Receptor binding and adenylate cyclase activity assays and circular dichroism spectroscopy have been used to reveal the role of the structure and conformation of the middle portion of the molecule. The effects of the modification of the 17, 18 and 21 positions on the superagonist activity have also been examined. Several key features of the peptide backbone conformation responsible for binding and transduction have been further studied by theoretical calculations and computer modeling (energy minimization) using the Sybyl program.
Search for bioactive conformation of glucagon and development of potent glucagon antagonistsHruby, Victor J.; Ahn, Jung-Mo (The University of Arizona., 2000)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.
Structure-activity relationship analysis: Developing glucagon agonists and antagonists for studies of glucagon action in normal and diabetic statesHruby, Victor J.; Azizeh, Bassem Yousef (The University of Arizona., 1996)Several glucagon analogues containing substitutions in the N-terminal region, in particular residues 1, 5, 6, 9 and 10 (histidine, threonine, phenylalanine, aspartic acid and tyrosine, respectively), were synthesized. In addition four β-methylphenylalanine isomers were introduced at position ten to assess the role of these topographical modifications on hormone activity, and to study the effect of constraint and biased conformational preferences of the side chain moieties on biological activity. All the analogues were synthesized by solid-phase methodology, purified to homogeneity by reverse-phase high-performance liquid chromatography, and characterized by electrospray mass spectroscopy, amino acid analysis and thin layer chromatography. Following characterization they were analyzed using rat liver plasma membranes for receptor-binding affinity as well as their ability to stimulate adenylate cyclase. Structure-activity relationship analysis provided critical information about the conformational, chemical and structural properties of amino acid residues required for receptor recognition and signal transduction in the glucagon sequence. His¹ was confirmed to operate along with Asp⁹ for the activation and binding to the glucagon receptor. These new findings should permit the design of more pure and potent glucagon receptor antagonists by focusing on the role of Phe⁶ and other residues in the N-terminal region. A newly developed assay for examining low levels of cAMP accumulation in response to glucagon antagonists, agonists and partial agonists was developed. Previously reported glucagon receptor antagonists had partial agonist activity in rat hepatocytes. This assay system, in conjunction with binding and adenylate cyclase studies in both hepatocytes and liver plasma membranes, redefines the major characteristics of pure glucagon antagonists. The most potent glucagon receptor antagonist [des-His¹, des-Phe⁶, Glu⁹]glucagon-NH₂ was studied using conformational analysis and 2D NMR techniques to analyze the secondary structure of the analogue. Proton resonance assignments using COSY, NOESY and TOCSY in d₆-DMSO were made.