Design and synthesis of conformationally constrained glucagon analogues

Glucagon was systematically modified by forming lactam bridges within the c entral region of the molecule to give conformationally constrained cyclic a nalogues. Six cyclic glucagon analogues have been designed and synthesized. They are C[Asp(9),Lys(12)][Lys(17,18),Glu(21)]glucagon-NH2 (1), c[Asp(9),L ys(12)]glucagon-NH2 (2), c[Lys(12),Asp(15)]glucagon-NH2 (3), c[Asp(15),Lys( 18)]glucagon-NH2 (4), [Lys(17)-c[Lys(18),Glu(21)]glucagon-NH2 (5), and c[Ly s(12),Asp(21)]glucagon-NH2 (6). The receptor binding potencies and receptor second messenger activities were determined by radio-receptor binding assa ys and adenylate cyclase assays, respectively, using rat liver plasma membr anes. Most interestingly, analogues 1, 2, 3, and 4 were antagonists of gluc agon 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 structura l features that might be responsible for these effects mere studied using c ircular dichroism spectroscopy and molecular modeling. These results demons trated the significant modulations of both receptor binding affinity and tr ansduction (adenylate cyclase activity) that can accompany regional conform ational constraints even in larger polypeptide ligands. These studies sugge st that the entire molecular conformation, including the flexible middle po rtion, is important for molecular recognition and transduction at the hepat ic glucagon receptor.