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

Abstract

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists.

Fig. 1.

Structure of compound 1 and effects on the cloned human GLP-1 receptor and the closely related GIP, GLP-2, and glucagon receptors, all expressed in BHK cells. (a) cAMP functional assay by using either the cloned human GLP-1, GLP-2, glucagon, or GIP receptors. The EC₅₀ value for GLP-1 and compound 1 was 23 pM and 1.4 μM, respectively. (b) Binding assay for the cloned human GLP-1 receptor. Both assays were carried out by using plasma membranes prepared from BHK cells expressing the different cloned human receptors. Data are from one of three to five identical experiments, in each experiment all concentrations were tested in triplicate.

Fig. 2.

Structure of compound 2 and mechanistic functional data (cAMP) by using the cloned human GLP-1 receptor. (a) Activation of the GLP-1 receptor by GLP-1 and compound 2. (b) Antagonism of forskolin-induced cAMP by using the cloned human glucagon receptor. (c) Dose–response curves of GLP-1 and compound 2 and GLP-1 receptor antagonist exendin (9–39) added to fixed half-maximal concentrations of either GLP-1 or compound 2, respectively. (d) Potentiation of GLP-1 activity by compound 2. Dose–response curves for GLP-1 in the absence or presence of three different fixed concentrations of compound 2. For a and b, data are from one of three identical experiments with samples in triplicate. For c and d, data from 3 identical experiments were pooled and normalized.

Fig. 3.

Saturation plot and Scatchard analysis for GLP-1 radioligand binding to the cloned human GLP-1 receptor, in the absence or presence of compound 2. (a) Saturation plot with GLP-1 + 100 nM compound 2 and GLP-1 alone. (b) Scatchard plot of data from a. ¹²⁵I-GLP-1 (7–36)amide (80 kBq/pmol) was dissolved in buffer and added in amounts ranging from 600,000 cpm down to ≈10,000 cpm per well. Data are from one of two identical experiments with samples in triplicate.

Fig. 4.

Insulin secretion from islets isolated from normal and GLP-1 receptor knockout mice. (a) Islets from CD1 wild-type mice. After a preperifusion the concentration of glucose was 3 mM from 0–60 min and 10 mM after 60 min. At 10 min, 100, and 1,000 nM compound 2, 100 nM GLP-1 or glucose alone was added and removed again at 70 min. Data are shown as mean ± SEM for three groups of 30 islets. Asterisks indicate a significant difference between GLP-1 and the 1,000 nM compound 2 vs. glucose alone. (b) Islets from CD1 GLP-1 receptor knockout mice. After the preperifusion, the concentration of glucose was 3 mM from 0 to 30 min and 10 mM after 30 min. At 10 min, 1,000 nM compound 2 and 100 nM GLP-1, 10 μM imidazoline control compound NNC77-0074, or vehicle were added and removed again at 60 min. Groups of islets were perifused the day after isolation. Before perifusate collection was started, all groups of islets were perifused with 3 mM glucose for 30 min to establish stable basal rates of release. Data are shown as mean ± SEM for three groups of 30 islets. Asterisks indicate a significant difference between imidazoline vs. all other groups.

Fig. 5.

Insulin release from perfused rat pancreas. (a) compound 2 (10 μM) was administered at t = 20–40 min with 10 mM glucose or with 4 mM glucose. (b) compound 2 (10 μM) was administered at t = 40–55 min, with 7 mM glucose and with 20 pM GLP-1 or without GLP-1. Data are presented as mean ± SD of two to three experiments.