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Synthesis and Biological Assessment of Sulfonic Acid-Based Glucagon Antagonists
Bin Yang, Vasily M. Gelfanov and Richard DiMarchi
Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, U.S.A.
Abstract
The structure-activity relationship of glucagon has been studied with a particular emphasis on the identification
and refinement for selective receptor antagonism. While the C-terminal -helical region is believed to be
important for receptor recognition there are a set of N-terminal amino acids that collectively function in signal
transduction. Replacement of Asp9 with Glu9, in addition to the deletion of His1 yields a potent antagonist
[desHis1, Glu9]glucagon amide, which is purported to retain weak partial agonist activity [1].
Discussion
Glucagon
Peptide 1
Peptide 1 plus G
Peptide 2
Peptide 2 plus G
Peptide 3
Peptide 3 plus G
Peptide 4
Peptide 4 plus G
100
80
cAMP (of maximal %)
Sulfonic acid-based amino acids are structurally and electronically homologous to the more native carboxylic
acid containing amino acids. Sulfonic acid modified peptide can provide a specific character both in structure
and function to a biological peptide [2]. The dramatic biological significance exhibited by the subtle replacement
of Asp9 with Glu9 attracted our attention to explore the suitability of a set of sulfonic acid homologs for Asp and
Glu. Additional amino acid modifications to the native glucagon sequence were explored to further enhance the
biological, physical and synthetic aspects of a more optimal glucagon antagonist.
120
• Phe25 and Leu27 substitutions for Trp25 and Met27 in glucagon were previously reported to
increase the potency of the hormone [3]. Consequently, all the cysteic acid-based peptides were
prepared with Phe25 and Leu27 substitutions to facilitate peptide synthesis without formation of
adverse oxidative by-products. Our results demonstrate an increase in the relative binding
potency of the Phe25, Leu27 analog. Additionally, these amino acid changes were observed to
increase cAMP stimulation of the known glucagon antagonist [desHis1, Glu9]Glucagon amide
(peptide 1) rendering it a less effective antagonist [desHis1, Glu9, Phe25, Leu27 ]Glucagon amide
(peptide 2, Fig. 3).
60
40
• HomoCysteic acid substitution for Glu9 was tested in a set of peptides (peptide 4, 7 and 8)
20
0
-5
10
-4
10
-3
10
-2
10
Experimental Design and Results
1
10
2
10
3
10
4
10
5
10
6
10
Figure. 3
cAMP stimulation (solid) and inhibition of glucagon-induced cAMP release (short dash dot)
by peptides 1, 2, 3, and 4. The results shown are representative of two experiments.
120
Table 1 Receptor Binding , cAMP Stimulation & Inhibition of Glucagon-induced cAMP Release
Peptide
cAMP Stimulation
cAMP Inhibition
IC50(nM)
EC50(nM)
Maximum %a
IC50(nM)b
1.75±0.31
0.21±0.11
100
N/A
80
60
1
[desHis1, Glu9]Glucagon-NH2
36.90±0.32
65±37
38.5
1862±1234
2
[desHis1, Glu9, Phe25, Leu27]Glucagon-NH2
12.59±0.41
81±23
81.5
N/A*
3
[desHis1, Cys9(SO3-), Phe25, Leu27]Glucagon-NH2
74.82±0.38
312±31
100
N/A*
4
[desHis1, homoCys9(SO3-), Phe25, Leu27]Glucagon-NH2
13.90±0.37
430±45
85.2
N/A*
5
[desHis1, desPhe6, Glu9]Glucagon-NH2
128.47±7.53
1178±105
88.2
N/A*
6
[desHis1, Leu4, Glu9]Glucagon-NH2
36.88±0.03
318±112
54.7
102±52
7
[desHis1, desPhe6, homoCys9(SO3-), Phe25, Leu27]Glucagon-NH2
37.08±0.30
3212±368
9.3
9217 ±3176
Peptide (nM)
8
[desHis1, Leu4, homoCys9(SO3-), Phe25, Leu27]Glucagon-NH2
4456±1469
Figure. 4
cAMP stimulation (solid) and inhibition of glucagon-induced cAMP release (short dash dot)
by peptides 1, 5 and 7. The results shown are representative of two experiments.
170.0±47.50
1614±1132
27.2
• Leu4 was reported to increase the antagonistic potency of [desHis1, Glu9]Glucagon amide and
40
our results confirm this observation [5]. Surprisingly, the substitution of Glu9 with homocysteic
acid in [des-His1, Leu4, Glu9, Phe25, Leu27]Glucagon amide (peptide 6) yielded a weak mixed
agonist-antagonist with lower receptor binding activity (peptide 8, Fig. 2 and Fig. 5).
20
• In summary,
0
N/A* not antagonist; a The maximum activity as compared with glucagon; b In the presence of constant 0.25 nM glucagon (plus G).
-5
10
120
120
120
100
Specific Binding (% )
100
Glucagon
Peptide 1
Peptide 5
Peptide 6
Peptide 7
Peptide 8
140
Glucagon
[F25, L27]G
Peptide 1
Peptide 2
Peptide 3
Peptide 4
80
60
40
80
80
60
40
20
-4
10
-3
10
-2
10
-1
0
10
10
1
10
2
10
3
10
4
10
5
10
6
10
Glucagon
Peptide 1
Peptide 1 plus G
Peptide 6
Peptide 6 plus G
Peptide 8
Peptide 8 plus G
100
cAMP (of maximal %)
140
References
40
20
0
0
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
10
Peptide(nM)
Figure. 1
Displacement of [125I]glucagon from human glucagon receptors by peptides 1,
2, 3 and 4. The results shown are representative of two experiments
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
10
Peptide(nM)
Figure. 2
Displacement of [125I]glucagon from human glucagon receptors by
peptides 5, 6, 7 and 8. The results shown are representative of two experiments
the effectiveness of antagonism was a function of the specific N-terminal
sequence in each of the glucagon analogs studied. The single deletion of the N-terminal histidine
was insufficient to render the Glu9 glucagon-based analogs pure antagonists. The previously
reported three antagonists (peptide 1, 5 and 6) that exhibited no cAMP stimulation activity in rat
liver membranes [1, 4 and 5] were demonstrated to have appreciable cAMP activity that varied
between 38100% of maximal in these engineered cells that over-express the glucagon receptor.
We found that homocysteic acid can function as a substitute for Glu9 in glucagon structurefunction relationships, although the correlation is not simple with a number of unexpected
findings. Substitution of Glu9 with homocysteic acid in peptides 2 and peptide 6 failed to produce
significant antagonist properties, while this same modification rendered peptide 5 conversion
from agonist to a highly specific antagonist with modest agonist activity (peptide 7). We conclude
that the differences in performance provides opportunity to identify more optimal antagonists
through further structure-function study.
60
20
0
• des-Phe6 as an additional modification to the standard antagonist [desHis1, Glu9]Glucagon
amide yielded a weak agonist analog devoid of any apparent antagonistic properties (peptide 5,
Fig. 4). This result is inconsistent with the previous report [4] and warrants additional study.
Interestingly, the substitution of Glu9 with homocysteic acid in [des-His1, des-Phe6, Glu9, Phe25,
Leu27]Glucagon amide (peptide 5) yielded a relatively pure antagonist (peptide 7, Fig. 4) with
higher receptor binding than the analogous Glu9-based peptide 5. Peptide 7 was the purest and
most effective antagonist among the set of analogs that we investigated. Peptide 7 was identified
to have the lowest level of cAMP stimulation and blocked glucagon-induced cAMP release to the
fullest extent (less than 10% of maximal) at a concentration of 50M (Fig. 4).
Glucagon
Peptide 1
Peptide 1 plus G
Peptide 5
[Peptide 5 plus G
Peptide 7
Peptide 7 plus G
100
cAMP (of maximal %)
Receptor Binding
Glucagon
Specific Binding (%)
0
10
Peptide (nM)
Synthesis of Glucagon Analogs: Peptide amines were generally synthesized with a Rink resin using Fmoc/tBu
chemistry. Sulfonic acid modified peptides devoid of Trp and Met residues were synthesized by oxidation of the
corresponding cysteine and homocysteine containing peptides in CH3COOH/HCOOH/H2O2.
NO
-1
10
related to previously reported glucagon antagonists, and yielded fully efficacious and highly
potent peptide ligands (Figs 1, 2). These three analogs demonstrated a variable level of cAMP
stimulation release that varied between 985% of the maximal release. The differences were
clearly a function of the additional N-terminal modifications, such as des-Phe6 (peptide 7, Fig. 4)
and Leu4 (peptide 8, Fig. 5). Similar to the native Asp9, substitution of Glu9 in peptide 2 with the
homologous cysteic acid yielded a full agonist (peptide 3, Fig. 3) without any apparent antagonist
property.
-5
10
-4
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
10
5
10
6
1. Unson, C. G., Gurzenda, E. M. and Merrifield, R. B. Peptides, 10, 1171-1177 (1989).
2. Arendt, A., et al. Protein and Peptide Letters. 7, 359-364 (2000).
3. Murphy, W. A., Coy, D. H. and Lance, V. A. Peptides. 7, 69-74 (1986).
4. Azizeh, B. Y. et al. Peptides. 18, 633-641 (1997).
5. Ahn, J. M. et al. J. Pept. Res. 58, 151-158 (2001).
10
Peptide (nM)
Figure. 5
cAMP stimulation (solid) and inhibition of glucagon-induced cAMP release (short dash dot)
by peptides 1, 6 and 8. The results shown are representative of two experiments.
Acknowledgements
We would like to thank David L. Smiley, Jay L. Levy , Tasia M. Pyburn and
Jonathan A. Karty for their assistance in completion of this work.