Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets

Abstract

Objective: The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor.

Research design and methods: Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo.

Results: Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment.

Conclusions: These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

FIG. 1.

The chemical structures of two pyrimidine-based GLP-1 receptor agonists, compound A (A) and compound B (B) are depicted.

FIG. 2.

Low molecular weight GLP-1 receptor agonists activate GLP-1 receptor signaling. A: Compound A and compound B induced GLP-1 receptor-mediated signaling in HEK293 cells coexpressing the human GLP-1 receptor and a 3x-cAMP response element-luciferase reporter, but were not active in cells lacking the GLP-1 receptor. In the human GLP-1 receptor HEK293 cells, the EC₅₀ values for compounds A and B were 1.6 μmol/l and 0.66 μmol/l, respectively; data are presented as percentages of stimulation of a maximum concentration of human GLP-1. B: The competitive GLP-1 receptor peptide antagonist exendin (9-39) blunted GLP-1 peptide activity but did not reduce compound B-induced signaling. C: Compound B was active in HEK293 cells expressing a modified form of the GLP-1 receptor lacking the NH₂-terminal ECD (Δ–ECD-GLP-1 receptor); the native GLP-1 peptide had no effect in cells expressing the Δ–ECD-GLP-1 receptor. D: Compound B was not active in HEK293 cells expressing the glucagon-like peptide-2 receptor (GLP-2R), glucose-dependent insulinotropic polypeptide receptor (GIP-R), glucagon receptor (GCG-R), or parathyroid hormone receptor (PTH-R).

FIG. 3.

Compound B increases glucose-dependent insulin secretion from SD rat islets. A: Insulin concentrations from static cultures of SD rat islets incubated in media containing low glucose (2.8 mmol/l) and either GLP-1 (100 nmol/l), compound B (3 μmol/l), or the sulfonylurea glibenclamide (5 μmol/l). B: Insulin concentrations from static cultures of SD rat islets incubated in media containing high glucose (11.2 mmol/l) and either GLP-1 (100 nmol/l) or various concentrations of compound B (0.3–10 μmol/l). All islet treatments were performed for 90 min. Results are expressed as mean ± SEM, *P < 0.05.

FIG. 4.

GLP-1 and compound B increase GLP-1 receptor signaling in an additive manner. A: Combination treatment of the GLP-1 peptide and compound B increases the intracellular concentration of cAMP in HEK293 cells expressing the human GLP-1 receptor. Treatment times for the cAMP assays were 20 min. B: Insulin concentrations from static cultures of SD rat islets incubated in media containing high glucose (11.2 mmol/l) and compound B plus increasing concentrations of GLP-1. Islet treatments were performed for 90 min. Results are expressed as mean ± SEM, *P < 0.05.

FIG. 5.

Compound B increases plasma insulin levels in the SD rat IVGTT model. Time course of plasma (A) glucose and (B) insulin concentrations in fasted, anesthetized animals treated with either vehicle (■), GLP-1 (×) (10 μg/kg), or compound B (○) (dosed at 10 mg/kg) immediately before intravenous administration of a glucose bolus (0.5 g/kg). Results are expressed as mean ± SEM. C: AUC of the insulin excursion curves for vehicle versus the GLP-1 or compound B treatment groups, *P < 0.05.

FIG. 6.

Compound B increases plasma insulin levels in the SD rat hyperglycemic clamp model. A: Intravenous dosing with vehicle (■) or 10 mg/kg compound B (○) occurred immediately before intravenous infusion of glucose. Glucose levels measured from venous blood every 5 min are shown. Results are expressed as mean ± SEM. B: Blood glucose concentrations of ∼250 mg/dl were maintained throughout the experiment by varying the glucose infusion rates. C: Time course of plasma insulin concentrations in fasted animals treated with either vehicle (■) or compound B (○). Results are expressed as mean ± SEM. D: AUC_{20–60 min} of the insulin secretion for vehicle (filled bar) versus compound B-treated (open bar) animals, *P < 0.05.

FIG. 7.

Compound B enhances insulin secretion in normal and diabetic human islets. A: Insulin concentrations from static cultures of normal human islets incubated in media containing high glucose (11.2 mmol/l) and either GLP-1 (100 nmol/l) or various concentrations of compound B (1–10 μmol/l). The treatments were performed for 90 min, and results are expressed as mean ± SEM, *P < 0.05. In perifusion experiments, insulin concentrations from reaction chambers containing 20 human islets from (B) normal or (C) diabetic individuals perifused with media containing either vehicle (■) or compound B (○) (3 or 10 μmol/l) in low glucose (3.3 mmol/l) for 40 min followed by high glucose (16.7 mmol/l) for an additional 35 min. AUC of the insulin excursion for vehicle versus compound B-treated (B) normal and (C) diabetic human islets, *P < 0.05.