Characterization of a novel, non-peptidyl antagonist of the human glucagon receptor

J Biol Chem. 1999 Mar 26;274(13):8694-7. doi: 10.1074/jbc.274.13.8694.

Abstract

We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)p yrr ole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC50 = 3. 7 +/- 3.4 nM (n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 microM. The binding affinity of L-168,049 for the human glucagon receptor is decreased 24-fold by the inclusion of divalent cations (5 mM). L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases the maximal glucagon stimulation observed, with a Kb (concentration of antagonist that shifts the agonist dose-response 2-fold) of 25 nM. These data suggest that L-168,049 is a noncompetitive antagonist of glucagon action. Inclusion of L-168, 049 increases the rate of dissociation of labeled glucagon from the receptor 4-fold, confirming that the compound is a noncompetitive glucagon antagonist. In addition, we have identified two putative transmembrane domain residues, phenylalanine 184 in transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for which substitution by alanine reduces the affinity of L-168,049 46- and 4. 5-fold, respectively. These mutations do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Animals
  • CHO Cells
  • Cations, Divalent / pharmacology
  • Cricetinae
  • Enzyme Activation / drug effects
  • Glucagon-Like Peptide-1 Receptor
  • Glucagon-Like Peptides
  • Hormone Antagonists / chemistry
  • Hormone Antagonists / pharmacology
  • Humans
  • Molecular Structure
  • Mutation
  • Peptides / metabolism*
  • Protein Binding / drug effects
  • Pyridines / pharmacology*
  • Pyrroles / pharmacology*
  • Receptors, Glucagon / antagonists & inhibitors*
  • Receptors, Glucagon / genetics
  • Receptors, Glucagon / metabolism*

Substances

  • Cations, Divalent
  • GLP1R protein, human
  • Glucagon-Like Peptide-1 Receptor
  • Hormone Antagonists
  • L 168049
  • Peptides
  • Pyridines
  • Pyrroles
  • Receptors, Glucagon
  • Glucagon-Like Peptides
  • glucagon-like-immunoreactivity
  • Adenylyl Cyclases