A Novel Glucagon Receptor Antagonist Inhibits Glucagon-Mediated Biological Effects Sajjad A. Qureshi, 1 Mari Rios Candelore, 1 Dan Xie, 1 Xiaodong Yang, 1 Laurie M. Tota, 1 Victor D.-H. Ding, 1 Zhihua Li, 1 Alka Bansal, 2 Corin Miller, 3 Sheila M. Cohen, 3 Guoqiang Jiang, 1 Ed Brady, 4 Richard Saperstein, 4 Joseph L. Duffy, 5 James R. Tata, 5 Kevin T. Chapman, 5 David E. Moller, 1 and Bei B. Zhang 1 Glucagon maintains glucose homeostasis during the fasting state by promoting hepatic gluconeogenesis and glycogenolysis. Hyperglucagonemia and/or an elevated glucagon-to-insulin ratio have been reported in diabetic patients and animals. Antagonizing the glucagon recep- tor is expected to result in reduced hepatic glucose overproduction, leading to overall glycemic control. Here we report the discovery and characterization of compound 1 (Cpd 1), a compound that inhibits binding of 125 I-labeled glucagon to the human glucagon receptor with a half-maximal inhibitory concentration value of 181  10 nmol/l. In CHO cells overexpressing the human glucagon receptor, Cpd 1 increased the half-maximal effect for glucagon stimulation of adenylyl cyclase with a K DB of 81  11 nmol/l. In addition, Cpd 1 blocked glucagon-mediated glycogenolysis in primary human hepatocytes. In contrast, a structurally related analog (Cpd 2) was not effective in blocking glucagon-mediated biological effects. Real-time measurement of glycogen synthesis and breakdown in perfused mouse liver showed that Cpd 1 is capable of blocking glucagon- induced glycogenolysis in a dosage-dependent manner. Finally, when dosed in humanized mice, Cpd 1 blocked the rise of glucose levels observed after intraperitoneal administration of exogenous glucagon. Taken together, these data suggest that Cpd 1 is a potent glucagon receptor antagonist that has the capability to block the effects of glucagon in vivo. Diabetes 53:3267–3273, 2004 G lucagon is a 29-amino acid polypeptide pro- duced in the pancreatic -cells and secreted in response to falling glucose levels during the fasting period (1). Glucagon increases glucose production by promoting glycogenolysis and gluconeogen- esis in the liver and attenuation of the ability of insulin to inhibit these processes (2). The combined action of gluca- gon and insulin is responsible for maintaining whole-body glucose homeostasis (3). Both increased glucagon secre- tion during the fasting state and the lack of insulin- mediated suppression of glucagon production in postprandial state contribute to the elevated glucagon levels associated with the hyperglycemia observed in the diabetic state (4,5). Therefore, reducing circulating gluca- gon levels and inhibiting glucagon-mediated biological effects in target tissues have long been considered as means of reducing hyperglycemia in diabetes. Studies using potent peptide antagonists have demonstrated sig- nificant blood glucose-lowering effects in diabetic animal models (6,7). Furthermore, it has been demonstrated that immunoneutralization of glucagon in diabetic animals effectively diminishes glucagon-stimulated hyperglycemia (8 –10). Although these reagents have shown promising results in animal models, their development for use in humans has not progressed because of limitations im- posed by the delivery methods necessary to achieve significant levels of exposure for the peptide agents. The glucagon receptor (GCGR) is a member of the family B of the seven transmembrane G-protein– coupled receptor (GPCR) superfamily (11). Other closely related members of the family include the receptors for glucagon- like peptide 1 and glucose-dependent insulinotropic pep- tide (GIP). Glucagon signals by binding to the receptor, which leads to activation of adenylyl cyclase and an increase in intracellular cAMP levels (12). In addition, the GCGR also couples to an intracellular Ca 2+ -mediated pathway (13). Activation of the GCGR results in increased glycogenolysis and gluconeogenesis, which are responsi- ble for increased hepatic glucose output (14,15). Given the key role of glucagon in elevating glycemia and owing to the success of finding small-molecule inhibitors for many receptors in the GPCR family (16,17), the GCGR is a clear target for the development of small-molecule antagonists. A number of antagonists with varying degree of potency and structures have been reported in recent From the 1 Department of Metabolic Disorder and Molecular Endocrinology, Merck Research Laboratories, Rahway, New Jersey; the 2 Department of Human-Animal Infectious Disease Research, Merck Research Laboratories, Rahway, New Jersey; the 3 Department of Image Research, Merck Research Laboratories, Rahway, New Jersey; the 4 Department of Pharmacology, Merck Research Laboratories, Rahway, New Jersey; and the 5 Department of Medic- inal Chemistry, Merck Research Laboratories, Rahway, New Jersey. Address correspondence and reprint requests to Dr. Sajjad A. Qureshi, RY80N-A62, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065. E-mail: sajjad_a_qureshi@merck.com. Received for publication 7 June 2004 and accepted in revised form 27 August 2004. S.A.Q. and M.R.C. contributed equally to this article. GCGR, glucagon receptor; GIP, glucose-dependent insulinotropic peptide; GPCR, G-protein– coupled receptor; HCM, hepatocyte culture medium; hGCGR, human GCGR; hGIPR, human glucose insulinotropic peptide recep- tor; HGP, hepatic glucose production; IMDM, Iscove’s modified Dulbecco’s medium; NMR, nuclear magnetic resonance. © 2004 by the American Diabetes Association. DIABETES, VOL. 53, DECEMBER 2004 3267