Development of potent glucagon antagonists: structure-activity relationship study of glycine at position 4

We examined the functional role of glycine at position 4 in the potent gluc agon antagonist [desHis(1), Glu(9)]glucagon amide, by substituting the L- a nd D-enantiomers of alanine and leucine for Gly(4) in this antagonist. The methyl and isobutyl sidechain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of t he N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that th e Gly4 residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide, may have resulte d from the strengthened hydrophobic patch in the N-terminal region and/or t he increased propensity for a helical conformation due to the replacement o f alanine and leucine for glycine. Thus, as a result of the increased recep tor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis(1), Glu(9)]glucagon a mide. These potent glucagon antagonists have among the highest pA(2) values of any glucagon analogs reported to date.