Design and Synthesis of Glucagon Partial Agonists and Antagonists

The hyperglycemia and ketosis of diabetes mellitus are generally associated with elevated levels of glucagon in the blood. This suggests that glucagon is a contributing factor in the metabolic abnormalities of diabetes mellitus. A glucagon-receptor antagonist might provide important evidence for glucagons's role in this disease. In this work we describe how we combined structural modifications that led to glucagon analogues with partial agonist activity to give glucagon analogues that can act as competitive antagonists of glucagon-stimulated adenylate cyclase activity. Using solid-phase synthesis methodology and preparative reverse-phase high-performance liquid chromatography, we synthesized the following seven glucagon analogues and obtained them in high purity: [D-Phe⁴,Tyr⁵,Arg¹²]glucagon (2); [D-Phe⁴,Tyr⁵,Lys¹⁷'¹⁸]glucagon (3); [Phe¹,Glu³,Lys^17,18]glucagon (4); [Glu³,Val⁵,Lys^17,18]glucagon (5); [Asp³,D-Phe⁴,Ser⁵,Lys^17,18]glucagon (6); I₄-[Asp³,D-Phe⁴,Ser⁵,Lys¹⁷'¹⁸]glucagon (7); [Pro³]glucagon (8). Purity was assessed by enzymatic total hydrolysis, by chymotryptic peptide mapping, and by reverse-phase high-performance liquid chromatography. The new analogues were tested for specific binding, for their effect on the adenylate cyclase activity in rat liver membranes, and for their effect on the blood glucose levels in normal rats relative to glucagon. Analogues showing no adenylate cyclase activity were examined for their ability to act as antagonists by displacing glucagon-stimulated adenylate cyclase dose-response curves to the right (higher concentrations). The binding potencies of the new analogues relative to glucagon (=100) were respectively 1.0 (2), 1.3 (3), 3.8 (4), 0.4 (5), 1.3 (6), 5.3 (7), and 3 (8). Glucagon analogues 3–5 and 8 were all weak partial agonists with EC₅₀ values of 500 (3), 250 (4), 1600 (5), and 395 nM (8), respectively. None of these analogues were able to fully stimulate in vitro adenylate cyclase activity relative to glucagon (=100), and they displayed partial agonist activities of 5 (3), 10 (4), 10 (5), and 40 (8), respectively, in this assay. All showed weak glycogenolytic activity in vivo. Glucagon analogues 2, 6, and 7 showed no adenylate cyclase activity at concentrations up to 10–100 μM in vitro, and none showed glycogenolytic activity in vivo at concentrations up to 2000 μg/kg. These latter compounds could act as glucagon antagonists in the in vitro liver plasma membrane adenylate cyclase system by shifting glucagon-stimulated adenylate cyclase dose-response curves to higher values. Glucagon antagonists such as those described here will be useful tools for investigating the mechanisms of glucagon action and for the further development of glucagon antagonists.