Allosteric ligands of the glucagon-like peptide 1 receptor (GLP-1R) differentially modulate endogenous and exogenous peptide responses in a pathway-selective manner: implications for drug screening

Abstract

The glucagon-like peptide-1 (GLP-1) receptor is a key regulator of insulin secretion and a major therapeutic target for treatment of diabetes. However, GLP-1 receptor function is complex, with multiple endogenous peptides that can interact with the receptor, including full-length (1-37) and truncated (7-37) forms of GLP-1 that can each exist in an amidated form and the related peptide oxyntomodulin. We have investigated two GLP-1 receptor allosteric modulators, Novo Nordisk compound 2 (6,7-dichloro2-methylsulfonyl-3-tert-butylaminoquinoxaline) and quercetin, and their ability to modify binding and signaling (cAMP formation, intracellular Ca(2+) mobilization, and extracellular signal-regulated kinase 1/2 phosphorylation) of each of the naturally occurring endogenous peptide agonists, as well as the clinically used peptide mimetic exendin-4. We identified and quantified stimulus bias across multiple endogenous peptides, with response profiles for truncated GLP-1 peptides distinct from those of either the full-length GLP-1 peptides or oxyntomodulin, the first demonstration of such behavior at the GLP-1 receptor. Compound 2 selectively augmented cAMP signaling but did so in a peptide-agonist dependent manner having greatest effect on oxyntomodulin, weaker effect on truncated GLP-1 peptides, and negligible effect on other peptide responses; these effects were principally driven by parallel changes in peptide agonist affinity. In contrast, quercetin selectively modulated calcium signaling but with effects only on truncated GLP-1 peptides or exendin and not oxyntomodulin or full-length peptides. These data have significant implications for how GLP-1 receptor targeted drugs are screened and developed, whereas the allosterically driven, agonist-selective, stimulus bias highlights the potential for distinct clinical efficacy depending on the properties of individual drugs.

Fig. 1

Structure and binding interactions elicited by allosteric modulators of the human GLP-1R. A, structures of the human GLP-1R small-molecule allosteric modulators used in this study. B, characterization of the inhibition binding profiles of compound 2 and quercetin at the human GLP-1R in relation to the endogenous peptide agonist GLP-1(7–36)NH₂ using ¹²⁵I-exendin(9–39) as the radioligand and membranes prepared from FlpInCHO cells stably expressing the human GLP-1R. Data are normalized to total binding and are analyzed with an allosteric modulator titration curve as defined in eqs. 2 and 3, assuming non-depletion (compound 2 and quercetin) or a competitive inhibition model [GLP-1(7–36)NH₂]. All values are mean ± S.E.M. of 6 to 12 independent experiments conducted in duplicate. Nonspecific binding, measured in the presence of 10⁻⁶ M exendin-4, ranged from 25 to 30% of total binding (dotted line in B). B, bound radioligand; B_o, binding in the absence of peptide ligand (total binding).

Fig. 2

Characterization of the inhibition binding of varying concentrations of compound 2 in the presence of GLP-1(7–36)NH₂ (A), exendin-4 (B), GLP-1(1–36)NH₂ (C), or oxyntomodulin using membranes prepared from FlpInCHO cells stably expressing the human GLP-1R (D). Data are normalized to total radioligand binding and are analyzed with a one-site competition plus allosteric modulator curve as defined in eqs. 2 and 3, assuming nondepletion. All values are mean ± S.E.M. of four to six independent experiments conducted in duplicate. Nonspecific binding, measured in the presence of 10⁻⁶ M exendin-4, ranged from 25 to 30% of total binding. B, bound radioligand; B_o, binding in the absence of peptide ligand (total binding).

Fig. 3

Characterization of the interaction between compound 2 and GLP-1(7–36)NH₂ (A), exendin-4 (B), GLP-1(1–36)NH₂ (C), or oxyntomodulin (D) in a cAMP accumulation assay using FlpInCHO cells stably expressing the human GLP-1R. E, compound 2 alone. Data are normalized to maximal peptide response and analyzed with an operational model of allosterism as defined in eq. 4. All values are mean ± S.E.M. of three to eight independent experiments conducted in duplicate.

Fig. 4

Characterization of the interaction between quercetin and GLP-1(7–36)NH₂ (A), exendin-4 (B), GLP-1(7–37) (C), or oxyntomodulin in an intracellular Ca²⁺ mobilization assay using FlpInCHO cells stably expressing the human GLP-1R (D). E, GLP-1(1–36)NH₂, GLP-1(1–37), or quercetin alone. Data are normalized to the maximal response elicited by 100 μM ATP and analyzed with a three-parameter logistic curve as defined in eq. 1. All values are mean ± S.E.M. of four to eight independent experiments conducted in duplicate. Statistical significance of changes in E_max in the presence of quercetin in comparison to the E_max of the peptide alone were determined by one-way analysis of variance and Dunnett’s post test and are indicated with an asterisk (*, p < 0.05).

Fig. 5

Characterization of the interaction between compound 2 and GLP-1(7–36)NH₂ (A), exendin-4 (B), GLP-1(7–37) (C) GLP-1(1–36)NH₂ (D), oxyntomodulin (E), or GLP-1(1–37) (F) in an ERK 1/2 phosphorylation assay using FlpInCHO cells stably expressing the human GLP-1R. G, compound 2 alone. Data are normalized to maximal peptide response and analyzed with a three-parameter logistic curve as defined in eq. 1. All values are mean ± S.E.M. of three independent experiments conducted in duplicate.