title: Design and synthesis of conformationally constrained glucagon analogues.
abstract: Glucagon was systematically modified by forming lactam bridges within the central region of the molecule to give conformationally constrained cyclic analogues. Six cyclic glucagon analogues have been designed and synthesized. They are c[Asp(9),Lys(12)][Lys(17,18), Glu(21)]glucagon-NH(2) (1), c[Asp(9),Lys(12)]glucagon-NH(2) (2), c[Lys(12),Asp(15)]glucagon-NH(2) (3), c[Asp(15), Lys(18)]glucagon-NH(2) (4), [Lys(17)-c[Lys(18), Glu(21)]glucagon-NH(2) (5), and c[Lys(12),Asp(21)]glucagon-NH(2) (6). The receptor binding potencies and receptor second messenger activities were determined by radio-receptor binding assays and adenylate cyclase assays, respectively, using rat liver plasma membranes. Most interestingly, analogues 1, 2, 3, and 4 were antagonists of glucagon stimulated adenylate cyclase activity, whereas analogues 5 and 6 were partial agonists in the functional assay. All of the cyclic analogues were found to have reduced binding potencies relative to glucagon. The structural features that might be responsible for these effects were studied using circular dichroism spectroscopy and molecular modeling. These results demonstrated the significant modulations of both receptor binding affinity and transduction (adenylate cyclase activity) that can accompany regional conformational constraints even in larger polypeptide ligands. These studies suggest that the entire molecular conformation, including the flexible middle portion, is important for molecular recognition and transduction at the hepatic glucagon receptor.
authors.affiliation: Departments of Chemistry and Pharmaceutical Sciences, University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: D
authors.initials: D
authors.lastname: Trivedi
authors.forename: Y
authors.initials: Y
authors.lastname: Lin
authors.forename: J M
authors.initials: JM
authors.lastname: Ahn
authors.forename: M
authors.initials: M
authors.lastname: Siegel
authors.forename: N N
authors.initials: NN
authors.lastname: Mollova
authors.forename: K H
authors.initials: KH
authors.lastname: Schram
authors.forename: V J
authors.initials: VJ
authors.lastname: Hruby
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D015195:Drug Design; D005765:Gastrointestinal Agents / Q000138:chemical synthesis*; D005934:Glucagon / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D016339:Spectrometry, Mass, Fast Atom Bombardment
chemical_list: D005765:Gastrointestinal Agents; D005934:Glucagon