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 2ϩ 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.

Optical Control of Insulin Secretion Using an Incretin Switch

“…This phenomenon is well‐reported for the GLP‐1R15, 18 and forms the basis of intense research efforts, since drug side effects may stem from presently unknown signaling interactions 19. The GLP‐1R is coupled to multiple pathways (e.g., cAMP, PKA, Epac2, ERK, and β‐arrestin), however, orthosteric ligands can provoke different receptor conformations to engage distinct signals 15. 18 This is best exemplified by responses to oxyntomodulin, which is biased for cAMP over ERK when compared to GLP‐1 7–36 15.…”

“…While the former two require complex pharmacokinetic studies, the latter can be examined by using functional in vitro assays. To determine the relative potency and specificity of LirAzo versus Lira, concentration–response curves were recorded for cAMP generation in CHO cells expressing the GLP‐1R (CHO‐GLP‐1R) 15. The half maximal effective concentration (EC 50 ) values for cis ‐ LirAzo (EC 50 =262.0 n m ) and trans ‐ LirAzo (EC 50 =993.6 n m ) were only slightly higher than for Lira (EC 50 =98.9 n m ) and GLP‐1 (EC 50 =20.3 n m ; Figure 2 d and Table S4).…”

“…The GLP‐1R is coupled to multiple pathways (e.g., cAMP, PKA, Epac2, ERK, and β‐arrestin), however, orthosteric ligands can provoke different receptor conformations to engage distinct signals 15. 18 This is best exemplified by responses to oxyntomodulin, which is biased for cAMP over ERK when compared to GLP‐1 7–36 15. 18 Such divergent effects likely arise from interactions with specific conserved polar residues 20…”

The Effective Application of Biased Signaling to New Drug Discovery

“…Because allosteric molecules are by nature permissive in that they may allow the interaction of the receptor with the natural agonist, there is a probability that the allosteric ligand will change the quality of the natural agonist signaling-that is, it will produce a bias for the natural agonist effect. Such bias has been noted for negative allosteric molecules in the selective blockade of NK2 receptors (Maillet et al, 2007), prostaglandin D2 receptors (Mathiesen et al, 2005), and calcium-sensing receptors (Davey et al, 2012;Cook et al, 2015) and positive allosteric modulators for glucagon-like peptide 1 (GLP-1) receptors (Koole et al, 2010) and mGlutamic acid 5 receptors (Bradley et al, 2011). The possibility of producing induced-bias in natural signaling can be viewed as a potential positive aspect of allosteric modulation of drug effect but also should be seen as an added consideration in the development of allosteric molecules.…”

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

“…2C), demonstrating distinct consequences of dimerization across pathways. Oxyntomodulin is an endogenous agonist of the GLP-1R that exhibits signal bias relative to that of GLP-1 peptides for at least cAMP versus pERK (34), whereas exendin-4 is clinically used as a GLP-1 mimetic (35). Disruption of dimerization had similar effect on the responses to all three peptides (Fig.…”