Characterization of a Novel, Non-peptidyl Antagonist of the Human Glucagon Receptor
Journal of Biological Chemistry
We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrrole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC 50 ؍ 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
... centrations up to 10 M. 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 EC 50 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 K b (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 46and 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. Glucagon is a 29-amino acid peptide that is an important counter-regulatory hormone in the control of glucose homeostasis (1). Glucagon secretion from the endocrine pancreas induces an increase in hepatic glycogenolysis and gluconeogenesis, and it attenuates the ability of insulin to inhibit these processes. As such, the overall rates of hepatic glucose synthesis and glycogen metabolism are controlled by the systemic ratio of insulin and glucagon (2, 3). Therefore, glucagon antagonists have the potential to improve hepatic insulin sensitivity and to be effective hypoglycemic agents. Peptidyl glucagon antagonists and their hypoglycemic activity were first described over 15 years ago, and an extensive exploration of the structure/activity relationships of these glu-cagon analogs has been reported (4 -6). The hepatic receptor for glucagon was cloned recently (7, 8), confirming that it is a member of the seven-transmembrane domain, G-protein-coupled receptor superfamily. This receptor superfamily has a binding pocket for small-molecule ligands within the transmembrane domain that has made it possible to identify nonpeptidyl antagonists for many receptor families in which the endogenous ligands are small peptides or proteins (9). Thus, we initiated an effort to identify non-peptidyl, orally active antagonists for the human glucagon receptor. Collins et al. (10) have described a dichloroquinoxaline glucagon antagonist with weak affinity (IC 50 ϭ 4 M) for the rat glucagon receptor. However, there have been no subsequent reports in the patent or scientific literature describing the development of potent antagonists from this series. Our initial screening efforts identified a series of triarylimidazole and triarylpyrrole compounds with significant binding affinity for the human glucagon receptor, and efforts to evaluate the structure-activity relationships of this series have lead to the identification of potent glucagon antagonists (11). In the present article, we describe the identification and characterization of a potent glucagon antagonist from this series. MATERIALS AND METHODS Characterization of Binding Affinity and Functional Activity-Stable CHO 1 cell lines or COS cells transiently expressing the human glucagon receptor were prepared as described previously (8, 12). Antagonist binding affinity was assessed by measuring inhibition of radiolabeled glucagon binding to CHO cell membranes. Briefly, 125 I-glucagon (58 pM) binding to the membrane preparation was measured in 20 mM Tris, pH 7.4, containing 1 mM dithiothreitol, 5 g/ml leupeptin, 10 g/ml benzamidine, 40 g/ml bacitracin, 5 g/ml soybean trypsin inhibitor, and 3 M o-phenanthroline Ϯ 1 M glucagon for 1 h at room temperature. Bound cpm were recovered by filtration using a Tomtec harvester and quantified in a ␥-scintillation counter. The ability of compound to inhibit glucagon-stimulated adenylyl cyclase was assessed as described previously (12) . Briefly, cells were harvested from monolayers with enzyme-free cell dissociation solution (Specialty Media, Inc.) and were pelleted at 500 ϫ g. The cells were resuspended at 100,000 cells/100 l in 75 mM Tris-HCl, pH 7.4, containing 250 mM sucrose, 12.5 mM MgCl 2 , 1.5 mM EDTA, 0.1 mM Ro-20-1724 (Biomol, Inc.), leupeptin (5 g/ml), benzamidine (10 g/ml), bacitracin (40 g/ml), soybean trypsin inhibitor (5 g/ml), and 0.02% bovine serum albumin. Cells were incubated for 30 min at 22°C with increasing peptide concentrations in the presence or absence of antagonist followed by lysis by boiling. Lysates were analyzed for cAMP content versus a nonacetylated cAMP standard curve using the Amersham cAMP radioimmunoassay scintillation proximity assay kit. Data were analyzed using Packard TopCount with RIASmart and GraphPad Prism software.