Synthetic linear and cyclic glucagon antagonists.
The synthesis and biological activities of seven new glucagon analogues are reported. The design of compounds 2-5 is based on potent antagonists recently reported from this laboratory, where we have focused on modifications in the N-terminal region. In this report we have concentrated specifically on modifications to histidine-1. In addition we have prepared two cyclic compounds 7 and 8, related to a linear in vivo antagonist [Glu9]glucagon, reported by Merrifield (Unson et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4083-4087). The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue (S)-5,6,7,8-tetrahydro-5-oxoimidazo(1,5-c)pyrimidine-7-carboxylic acid (Toc), desaminohistidine (dHis) and 3-(4-nitrobenzyl)histidine. The structures of the new compounds are as follows. [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (2); [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon amide (3); [3-(4-nitrobenzyl)His1,D-Phe4,Tyr5,Arg12,Lys17,18,G lu21]glucagon (4); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (5); [dHis1,Glu9]glucagon (6); (desHis1)[Glu9,Lys12]glucagon amide (7); (desHis1)-[Glu9,Lys12,Asp15]glucagon amide (8). The binding potencies of the linear analogues, as expressed a percentage of glucagon binding, are 2.6 (2), 0.13 (3), 0.8 (4), 0.8 (5), 2.2 (6). Both cyclic analogues 7 and 8 show biphasic binding curves. The IC50 values for 7 at the high and low affinity sites are 1.5 and 167 nM, respectively (IC50 of glucagon = 1.3 nM). The IC50 values for 8 at the high and low affinity sites are 4.7 and 3451 nM, respectively. The cyclic analogues are characterized by fast atom bombardment mass spectrometry of endoproteinase ASP-N digests. The specificity of the enzyme used in these studies enables differentiation of isomers of the cyclic glucagon analogues which differ only in the position of cyclic amide bond. Analogues 2, 3 and 5-8 are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase (AC) system. Analogue 4 is a partial agonist (5.7% compared to glucagon) of AC. Introduction of unusual amino acids which do not contain a primary alpha-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.
Department of Chemistry, College of Pharmacy, University of Arizona, Tucson.
R
R
Dharanipragada
D
D
Trivedi
A
A
Bannister
M
M
Siegel
D
D
Tourwe
N
N
Mollova
K
K
Schram
V J
VJ
Hruby
D000595:Amino Acid Sequence; D000818:Animals; D005934:Glucagon / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011964:Receptors, Gastrointestinal Hormone / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D018027:Receptors, Glucagon; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013329:Structure-Activity Relationship
D010455:Peptides; D010456:Peptides, Cyclic; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D005934:Glucagon