Characterization of a novel, non-peptidyl antagonist of the human glucagonreceptor

We have identified a series of potent, orally bioavailable, non-peptidyl, t riarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyri dyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrrole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon t o the human glucagon receptor with an IC50 = 3.7 +/- 3.4 nM (n = 7) but doe s not inhibit binding of labeled glucagon-like peptide to the highly homolo gous human glucagon-like peptide receptor at concentrations up to 10 mu M. The binding affinity of L-168,049 for the human glucagon receptor is decrea sed 24-fold by the inclusion of divalent cations (5 mM), L-168,049 increase s the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases t he maximal glucagon stimulation observed, with a K-b (concentration of anta gonist that shifts the agonist dose-response 2-fold) of 25 nM. These data s uggest that L-168,049 is a noncompetitive antagonist of glucagon action. In clusion 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 transmem brane domain residues, phenylalanine 184 in transmembrane domain 2 and tyro sine 239 in transmembrane domain 3, for which substitution by alanine reduc es the affinity of L-168,049 46- and 4.5-fold, respectively. These mutation s do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.