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 glucagon 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 METHODSCharacterization 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...

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