Synthetic linear and cyclic glucagon antagonists

R. Dharanipragada, D. Trivedi, A. Bannister, M. Siegel, D. Tourwe, N. Mollova, K. Schram, Victor J Hruby

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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,Glu21] 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), 0.12 (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 α-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.

Original languageEnglish (US)
Pages (from-to)68-77
Number of pages10
JournalInternational Journal of Peptide and Protein Research
Volume42
Issue number1
StatePublished - 1993

Fingerprint

Glucagon
Amides
Glucagon Receptors
Inhibitory Concentration 50
Histidine
Adenylyl Cyclases
Fast Atom Bombardment Mass Spectrometry
Aminopeptidases
Carboxylic Acids
Bioactivity
Isomers
Mass spectrometry
Substitution reactions

Keywords

  • Conformational constraints
  • Enzymatic digestion
  • FAB mapping
  • Glucagon analogues
  • Glucagon antagonists
  • Structure-activity relationships
  • Unusual amino acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dharanipragada, R., Trivedi, D., Bannister, A., Siegel, M., Tourwe, D., Mollova, N., ... Hruby, V. J. (1993). Synthetic linear and cyclic glucagon antagonists. International Journal of Peptide and Protein Research, 42(1), 68-77.

Synthetic linear and cyclic glucagon antagonists. / Dharanipragada, R.; Trivedi, D.; Bannister, A.; Siegel, M.; Tourwe, D.; Mollova, N.; Schram, K.; Hruby, Victor J.

In: International Journal of Peptide and Protein Research, Vol. 42, No. 1, 1993, p. 68-77.

Research output: Contribution to journalArticle

Dharanipragada, R, Trivedi, D, Bannister, A, Siegel, M, Tourwe, D, Mollova, N, Schram, K & Hruby, VJ 1993, 'Synthetic linear and cyclic glucagon antagonists', International Journal of Peptide and Protein Research, vol. 42, no. 1, pp. 68-77.
Dharanipragada R, Trivedi D, Bannister A, Siegel M, Tourwe D, Mollova N et al. Synthetic linear and cyclic glucagon antagonists. International Journal of Peptide and Protein Research. 1993;42(1):68-77.
Dharanipragada, R. ; Trivedi, D. ; Bannister, A. ; Siegel, M. ; Tourwe, D. ; Mollova, N. ; Schram, K. ; Hruby, Victor J. / Synthetic linear and cyclic glucagon antagonists. In: International Journal of Peptide and Protein Research. 1993 ; Vol. 42, No. 1. pp. 68-77.
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TY - JOUR

T1 - Synthetic linear and cyclic glucagon antagonists

AU - Dharanipragada, R.

AU - Trivedi, D.

AU - Bannister, A.

AU - Siegel, M.

AU - Tourwe, D.

AU - Mollova, N.

AU - Schram, K.

AU - Hruby, Victor J

PY - 1993

Y1 - 1993

N2 - 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,Glu21] 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), 0.12 (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 α-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.

AB - 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,Glu21] 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), 0.12 (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 α-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.

KW - Conformational constraints

KW - Enzymatic digestion

KW - FAB mapping

KW - Glucagon analogues

KW - Glucagon antagonists

KW - Structure-activity relationships

KW - Unusual amino acids

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