Topographical amino acid substitution in position 10 of glucagon leads to antagonists/partial agonists with greater binding differences.

The role of position 10 in the β-turn region of glucagon was investigated by substituting chiral constrained amino acids and other modifications in the N-terminal region. A series of glucagon analogues have been designed and synthesized by incorporating β-methylphenylalanine isomers (2S,3S, 2S,3R, 2R,3R, and 2R,3S) at position 10 in order to explore the structural and topographical requirements of the glucagon receptor, and, in addition, utilizing previous studies which indicated that antagonism could be enhanced by modifications (des-His 1 , Glu 9 ) and a bulky group at position 5. The structures of the new analogues are as follows : [des-His 1 ,-Tyr 5 ,Glu 9 ]glucagon-NH 2 (II), [des-His 1 ,Tyr 5 ,Glu 9 ,Phe 10 ]glucagon-NH2 (III), [des-His 1 ,Tyr 5 ,Glu 9 ,-Ala 10 ]gucagon-NH 2 (IV), [des-His 1 ,Tyr 5 ,Glu 9 ,(2S,3R)-β-MePhe 10 ]glucagon-NH2 (V), [des-His 1 ,-Tyr 5 ,Glu 9 ,(2S,3S)-β-MePhe 10 ]glucagon-NH 2 (VI), [des-His 1 ,Tyr 5 ,Glu 9 ,D-Tyr 10 ]glucagon-NH 2 (VII), [des-His 1 ,Tyr 5 ,Glu 9 ,D-Phe 10 ]glucagon-NH2 (VIII), [des-His 1 ,Tyr 5 ,Glu 9 ,D-Ala 10 ]glucagon-NH 2 (IX), [des-His 1 ,Tyr 5 ,Glu 9 ,(2R,3R)-β-MePhe 10 ]glucagon-NH 2 (X), and [des-His 1 ,Tyr 5 ,Glu 9 ,(2R,3S)-β-MePhe 10 ]glucagon-NH 2 (XI). These analogues led to dramatically different changes in in vitro binding affinities for glucagon receptors. Their receptor binding potencies IC 50 values (nM) are 2.3 (II), 4.1 (III), 395.0 (IV), 10.0 (V), 170.0 (VI), 74.0 (VII), 34.5 (VIII), 510.0 (IX), 120.0 (X), and 180.0 (XI). Analogues II, III, V, VI, and XI were found to be weak partial agonists/partial antagonists with maximum stimulation between 5%-9%, while the other compounds (IV and VII-X) were antagonists unable to activate the adenylate cyclase system even at concentrations as high as 10 -5 M. In competition experiments, all of the analogues caused a right shift of the glucagon-stimulated adenylate cyclase dose-response curve. The pA 2 values were 6.60 (II), 6.85 (III), 6.20 (IV), 6.20 (V), 6.10 (VI), 6.50 (VII), 6.20 (VIII), 5.85 (IX), 6.20 (X), and 6.00 (XI). Putative topographical requirements of the glucagon receptor for the aromatic side chain conformation in position 10 of glucagon antagonists are discussed.