New glucagon analogues with conformational restrictions and altered amphiphilicity: Effects on binding, adenylate cyclase and glycogenolytic activities

In an effort to obtain highly potent glucagon antagonists, we have investigated glucagon (1) structure-function relationships utilizing the following design principles: (1) structural changes known to lead to partial agonist activities; (2) conformational restrictions; (3) changes in the conformational probabilities of the primary sequence; and (4) increased amphiphilicity. In this report we present the total synthesis, purification, receptor binding, adenylate cyclase activity, in vivo glycogenolytic activity and CD spectrum of the following four glucagon analogues: [Ahx^17,18]glucagon (2), [D-Phe⁴,Tyr⁵, 3,5-diiodo- Tyr¹⁰,Arg¹²,Lys^17,18,Glu²¹]glucagon (3), [Asp⁹,Lys^17,18,Glu²¹]glucagon 4, and [Glu¹⁵,Lys^17,18]glucagon 5. Compound 2 binds exclusively to the high affinity receptor and compound 3 was a highly potent antagonist with respect to adenylate cyclase activity. Analog 4 showed distinct biphasic binding (IC₅₀ 5.6 nM and 630 nM), with only the low affinity binding leading to adenylate cyclase activity. Furthermore in analogue 5 receptor binding and adenylate cyclase activity were dissociated by a factor of 5. The results are consistent with a multistep binding mechanism in which glucagon interacts first nonspecifically with the anisotropic interphase of the cell membrane, followed by a conformational transition which occurs in the sequences 10-14 and 15-18 when the membrane bound peptide binds to its receptor.