Characterization of a Novel, Non-peptidyl Antagonist of the Human Glucagon Receptor

Abstract

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...

Small-molecule agonists for the glucagon-like peptide 1 receptor

“…Many of the identified small-molecule agonists were discovered through random or directed functional highthroughput screening of large chemical libraries, which also would identify agonists that do not necessarily bind to the peptide-binding site, the orthosteric site. Peptides are thought to bind to the extracellular portion of the receptor, whereas small molecules may bind more deeply in the transmembrane region (31,32). Several GPCR agonists that affect the receptor response to the natural ligands via a noncompetitive site or allosteric site have during recent years been found for receptor families A and C, but so far not for family B.…”

“…To date, no small-molecule agonists have been described in the B family. Small-molecule antagonists have been described for two members of this family, the glucagon receptor and the CRF receptor (27)(28)(29). The GLP-1 peptide and several of the other peptide hormone ligands in this receptor family do not have a well defined secondary structure.…”

“…Today, most small-molecule ligands have been identified in binding assays. This approach has not proven useful within this class of receptors, at least for the identification of agonists (28). We have used a functional screening assay to identify nonpeptide agonists for the human GLP-1 receptor.…”

A Novel Glucagon Receptor Antagonist Inhibits Glucagon-Mediated Biological Effects

“…Chinese hamster ovary (CHO) cells expressing the cloned human GCGR (CHO-hGCGR) have been previously described (20,21). The CHO-hGIPR cell line was produced by transfecting human glucose insulinotropic peptide receptor (hGIPR) cDNA (kindly provided by T. Usdin, National Institutes of Health, Bethesda, MD) into CHO cells.…”

Ligand-gated Ion Channels

“…Selective antagonists L168049 (Cascieri et al, 1999), des-His 1 -[Glu 9 ]glucagon amide (Post et al, 1993), BAY27-9955 (Petersen and Sullivan, 2001), xNNC92-1687 (Madsen et al, 1998) Exendin-(9-39) (Thorens et al, 1993) (Schally and Varga, 1999), JV-1-38 (Schally and Varga, 1999) [(CH2NH) 4,5 ]secretin (Kim et al, 1993) The glucagon receptor has been reported to interact with receptor activity modifying proteins (RAMPs), specifically RAMP2, in heterologous expression systems (Christopoulos et al, 2003), although the physiological significance of this has yet to be established. …”