Structure-function studies on positions 17, 18, and 21 replacement analogues of glucagon: the importance of charged residues and salt bridges in glucagon biological activity.
We have designed and synthesized eight compounds 2-9 which incorporate various amino acid residues in positions 17, 18, and 21 of the glucagon molecule: 2, [Lys17]glucagon amide; 3, [Lys18]glucagon amide; 4, [Nle17,Lys18,Glu21]glucagon amide; 5, [Orn17,18, Glu21]glucagon amide; 6, [d-Arg17]glucagon; 7, [d-Arg18]glucagon; 8, [d-Phe17]glucagon; and 9, [d-Phe18]glucagon. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have binding affinity IC50 values (in nM) of 0.7, 4.1, 1.0, 2.0, 5.0, 25.0, 43.0, and 32.0, respectively. When these compounds were tested for their ability to stimulate adenylate cyclase (AC) activity, they were found to be full or partial agonists having maximum stimulation values of 100, 100, 100, 100, 87, 78, 94, and 100%, respectively. On the basis of the X-ray crystal structure of [Lys17,18,Glu21]glucagon amide reported here, the ability to form a salt bridge between Lys18 and Glu21 is probably key to their increased binding and second messenger activities. Among the eight analogues synthesized here, only analogue 4 preserves the ability to form a salt bridge between Lys18 and Glu21. However, since these modifications are minor they do not seem to change the amphiphilic character of the C-terminus, allowing these analogues to reach 78-100% stimulation in the adenylate cyclase assay. Biological data from analogues 6-9 supports the idea that position 18 of glucagon may influence binding only, while position 17 may influence both receptor recognition and transduction.
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.
N S
NS
Sturm
Y
Y
Lin
S K
SK
Burley
J L
JL
Krstenansky
J M
JM
Ahn
B Y
BY
Azizeh
D
D
Trivedi
V J
VJ
Hruby
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D005934:Glucagon / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008099:Liver / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008969:Molecular Sequence Data; D015394:Molecular Structure; D051381:Rats; D017207:Rats, Sprague-Dawley; D018027:Receptors, Glucagon / Q000378:metabolism
D018027:Receptors, Glucagon; D001224:Aspartic Acid; D005934:Glucagon; D001120:Arginine; D000262:Adenylyl Cyclases