The glucagon-like peptide-1 receptor (GLP-1R), which belongs to the class B1 G protein-coupled receptor (GPCR) family, is an important target for treatment of metabolic disorders, including type 2 diabetes and obesity. The growing interest in GLP-1R-based therapies is driven by the development of various functional agonists as well as the huge commercial market. Thus, understanding the structural details of ligand-induced signaling are important for developing improved GLP-1R drugs. Here, we investigated the conformational dynamics of the receptor in complex with a selection of prototypical functional agonists, including CHU-128 (small molecule-biased), danuglipron (small molecule balanced), and Peptide 19 (peptide balanced), which exhibit unique, distinct binding modes and induced helix packing. Furthermore, our all-atom molecular dynamics (MD) simulations revealed atomic feature how different those ligands led to signaling pathway preference. Our findings offer valuable insights into the mechanistic principle of GLP-1R activation, which are helpful for the rational design of next-generation GLP-1R drug molecules.
G protein-coupled receptors (GPCRs) are important mediators of extracellular signals across the cell’s plasma membrane, conveying external stimuli to intracellular effectors that regulate various cellular processes. The activation process of GPCRs is driven by an internal network of interacting residues, which coordinates conformational transitions between the functional states of the receptor. Structural elucidation of receptor–ligand complexes has provided valuable insights into important interaction networks that trigger specific signaling pathways. Recent advances in the determination of GPCR structures have significantly enhanced our understanding of receptor functions and fostered the structure-based drug discovery of GPCR drugs. Yet, experimentally determined GPCR structures often fail to capture the full inherent flexibility of GPCRs, as they typically represent a very limited number of static snapshots of the receptor’s large dynamic conformational landscape in the activation process. To better understand the receptors’ functional transitions and conformational dynamics, the static structures of the receptor in specific activation states serve as a constitutive basis for in silico simulation of the entire GPCR activation process.
In recent years, glucagon-like peptide 1 (GLP-1) receptor have gained considerable attention as a target for the clinical treatment of metabolic disorders, particularly of type 2 diabetes (T2D) and obesity. GLP-1R belongs the secretin-like family of class B GPCRs, which is predominantly expressed in pancreatic β-cells. It controls both insulin and glucagon secretion in glucose-dependent manner. Activation of GLP-1R induces insulin secretion, inhibits glucagon secretion, and slows gastric emptying, thus contributing to the central regulation of blood glucose levels. Dysfunctional GLP-1R activation disrupts metabolic homeostasis, contributing to the pathogenesis of metabolic diseases. However, the administration of GLP-1R agonists (GLP-1RAs) in T2D patients has been shown to improve glycemic control.
Over the past few decades, numerous GLP-1RAs, such as semaglutide and liraglutide, have been developed, featuring intrinsic modification of the native GLP-1 peptide to imitate the pharmacological profile of GLP-1, while providing resistance to degradation by the enzyme dipeptidyl peptidase-4. Interestingly, these molecules have demonstrated extended therapeutic benefits, including potential applications in cardiovascular diseases and neurodegenerative diseases, as well as in non-alcoholic fatty liver disease. Despite their efficacy, some GLP-1RAs exhibit reduced therapeutic effects due to undesired receptor trafficking, attenuating the glucoregulatory responses by desensitization and downregulation caused by β-arrestin recruitment. To overcome this limitation, the development of G s-biased agonists has been pursued; they enhance insulin release in cell-based experiments by selectively amplifying cAMP signaling. Further evidence has been delivered showing that the administration of G s-biased agonists is more effective than cAMP and β-arrestin 2 signaling balanced agonists in regulation glucose homeostasis and bodyweight control in mice. However, additional clinical studies are essential to fully evaluate the long-term implications of selective signaling modulation.
Furthermore, peptide GLP-1RAs are typically administrated via injection, which leads to challenge of patient compliance, gastrointestinal side effects, and production costs. Consequently, the development of oral small-molecule agonists has emerged as an alternative approach, offering advantages such as improved patient compliance and bioavailability through oral administration. These developments have prompted further investigation into the elucidation of experimental structure of activate state GLP-1R-G s complexes with or without orthosteric agonists, providing crucial insights into the activation mechanisms induced by different ligands.
Despite significant progress in understanding the activation mechanisms of GLP-1R, structural insights into ligand-induced modulation by balanced and biased agonists remain limited. In this study, we aim to provide molecular insights into the different functional agonists binding to GLP-1R. Specifically, we investigate three ligands Peptide 19, danuglipron, and CHU-128 using all-atom model molecular dynamics (MD) simulations. They show distinct functional features: (1) Peptide 19 is an analog of gastric inhibitory polypeptide (GIP) exhibiting high potency for cAMP production at both GLP-1R and GIPR, but with notable partial agonism toward β-arrestin 2 recruitment. (2) Danuglipron is a small molecule identified from high-throughput screening and shows potency comparable to GLP-1 the canonical GLP-1R peptide in in vitro studies, inducing both cAMP and β-arrestin signaling. In a phase 2 trial, the administration of danuglipron improved metabolic function, although gastrointestinal side effects were observed. (3) CHU-128 is a small molecule that exhibits strong bias toward the cAMP pathway and distinct binding mode profile compared to danuglipron. All mentioned ligands induce unique conformational changes in the receptor, resulting in distinct packing arrangement of the 7TM helical bundle that influences the downstream protein sensitivity to the intracellular binding pocket.
Using all-atom MD simulations of the ligand–GLP-1R–G s complex, we explore how orthosteric-bound binders induce the transduction of signals across the receptor to activate specific or impartial intracellular signaling pathways. Furthermore, we perform complementary simulations with peptide-free GLP-1R to gain deeper insights into the receptor conformational dynamics, particularly in the extracellular domain, which is critical for agonist binding and activation. Our simulations reveal that the ligand binding mode is directly correlated to the signaling pathway preference. Importantly, the limitations of small molecules in simultaneously managing the extracellular domain (ECD), transmembrane region, and core vestibule reflect their distinct advantages in conducting GLP-1R activation.
To understand how the conformation, orientation, and binding mode of ligands with distinct pharmacological profiles influence the receptor’s signaling pathway, we performed all-atom MD simulations for high-resolution structures of GLP-1R bound to different orthosteric ligands: (i) CHU-128 is a biased nonpeptide agonist that induces a receptor conformation that favors G protein activation over β-arrestin recruitment signaling; (ii) danuglipron and (iii) Peptide 19 are a balanced nonpeptide and peptide agonist, respectively, which induce both cAMP production and β-arrestin recruitment pathways. All these structures were simulated in complex with the G s protein α-subunit (G α) only. To visualize the potential mechanism that promotes the receptor to transit from one set of conformational states populations to another one, we performed an additional ligand-free MD simulation using a GLP-1R–Peptide 19 complex by removing the ligand and G α. Three independent replicas’ simulations of 500 ns duration were performed for each above-mentioned system.
(i) The location of each ligand bound to the receptor. Static structural analyses of ligand–receptor complexes indicate that danuglipron inserts deeply in the receptor’s binding cavity closing the binding pocket, whereas CHU-128 adopts a planar binding pose above the helical transmembrane bundle, close to the extracellular domain (ECD), extracellular loop 1 (ECL1), and ECL2 liberating regions around TM6-TM7. The position and binding modes of small-molecule agonists involve different structural modulations associated with distinct pharmacology profiles. Peptide agonists are integrally buried with a longitudinal orientation in the receptor’s binding vestibule, forming a helical structure within the transmembrane segments. Either balanced or biased small-molecule agonists form a limited number of interactions with the core vestibule, suggesting that the extracellular
