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The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target for the treatment of type 2 diabetes due to its role in glucose homeostasis. Despite the availability of peptide-based GLP-1R drugs for treatment of this disease, there is great interest in developing small molecules that can be administered orally. The GLP-1R system is complex, with multiple endogenous and clinically used peptide ligands that exhibit different signaling biases at this receptor. This study revealed that small molecule ligands acting at this receptor are differentially biased to peptide ligands and also from each other with respect to the signaling pathways that they activate. Furthermore, allosteric small molecule ligands were also able to induce bias in signaling mediated by orthosteric ligands. This was dependent on both the orthosteric and allosteric ligand as no two allosteric-orthosteric ligand pairs could induce the same signaling profile. We highlight the need to profile compounds across multiple signaling pathways and in combination with multiple orthosteric ligands in systems such as the GLP-1R where more than one endogenous ligand exists. In the context of pleiotropical coupling of receptors and the interplay of multiple pathways leading to physiologic responses, profiling of small molecules in this manner may lead to a better understanding of the physiologic consequences of biased signaling at this receptor. This could enable the design and development of improved therapeutics that have the ability to fine-tune receptor signaling, leading to beneficial therapeutic outcomes while reducing side effect profiles.

Identifying novel mechanisms to enhance glucagon-like peptide-1 (GLP-1) receptor signaling may enable nascent medicinal chemistry strategies with the aim of developing new orally available therapeutic agents for the treatment of type 2 diabetes mellitus. Therefore, we tested the hypothesis that selectively modulating the low-affinity GLP-1 receptor agonist, oxyntomodulin, would improve the insulin secretory properties of this naturally occurring hormone to provide a rationale for pursuing an unexplored therapeutic approach. Signal transduction and competition binding studies were used to investigate oxyntomodulin activity on the GLP-1 receptor in the presence of the small molecule GLP-1 receptor modulator, 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP). In vivo, the intravenous glucose tolerance test characterized oxyntomodulin-induced insulin secretion in animals administered the small molecule. BETP increased oxyntomodulin binding affinity for the GLP-1 receptor and enhanced oxyntomodulin-mediated GLP-1 receptor signaling as measured by activation of the α subunit of heterotrimeric G protein and cAMP accumulation. In addition, oxyntomodulin-induced insulin secretion was enhanced in the presence of the compound. BETP was pharmacologically characterized to induce biased signaling by oxyntomodulin. These studies demonstrate that small molecules targeting the GLP-1 receptor can increase binding and receptor activation of the endogenous peptide oxyntomodulin. The biased signaling engendered by BETP suggests that GLP-1 receptor mobilization of cAMP is the critical insulinotropic signaling event. Because of the unique metabolic properties of oxyntomodulin, identifying molecules that enhance its activity should be pursued to assess the efficacy and safety of this novel mechanism.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a key drug target class. Recently, allosteric drugs that can co-bind with and modulate the activity of the endogenous ligand(s) for the receptor have become a major focus of the pharmaceutical and biotechnology industry for the development of novel GPCR therapeutic agents. This class of drugs has distinct properties compared with drugs targeting the endogenous (orthosteric) ligand-binding site that include the ability to sculpt cellular signaling and to respond differently in the presence of discrete orthosteric ligands, a behavior termed "probe dependence." Here, using cell signaling assays combined with ex vivo and in vivo studies of insulin secretion, we demonstrate that allosteric ligands can cause marked potentiation of previously "inert" metabolic products of neurotransmitters and peptide hormones, a novel consequence of the phenomenon of probe dependence. Indeed, at the muscarinic M(2) receptor and glucagon-like peptide 1 (GLP-1) receptor, allosteric potentiation of the metabolites, choline and GLP-1(9-36)NH(2), respectively, was ~100-fold and up to 200-fold greater than that seen with the physiological signaling molecules acetylcholine and GLP-1(7-36)NH(2). Modulation of GLP-1(9-36)NH(2) was also demonstrated in ex vivo and in vivo assays of insulin secretion. This work opens up new avenues for allosteric drug discovery by directly targeting modulation of metabolites, but it also identifies a behavior that could contribute to unexpected clinical outcomes if interaction of allosteric drugs with metabolites is not part of their preclinical assessment.