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Binding of the glucagon peptide to the glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting; thus GCGR plays an important role in glucose homeostasis. Here we report the crystal structure of the seven transmembrane helical domain of human GCGR at 3.4 Å resolution, complemented by extensive site-specific mutagenesis, and a hybrid model of glucagon bound to GCGR to understand the molecular recognition of the receptor for its native ligand. Beyond the shared seven transmembrane fold, the GCGR transmembrane domain deviates from class A G-protein-coupled receptors with a large ligand-binding pocket and the first transmembrane helix having a ‘stalk’ region that extends three alpha-helical turns above the plane of the membrane. The stalk positions the extracellular domain (∼12 kilodaltons) relative to the membrane to form the glucagon-binding site that captures the peptide and facilitates the insertion of glucagon’s amino terminus into the seven transmembrane domain. The X-ray crystal structure of the human glucagon receptor, a potential drug target for type 2 diabetes, offers a structural basis for molecular recognition by class B G-protein-coupled receptors. Two class B human GPCR receptors G-protein-coupled receptors (GPCRs) are membrane proteins that act as sensors for a broad range of extracellular signals, including photons, ions, small organic molecules and even entire proteins. Approximately a third of known drugs target GPCRs. Until now all the published structures of GPCRs have been from class A GPCRs. In this issue of Nature two groups independently report the crystal structures of two receptors of the B family, the second largest of four family divisions based on primary sequence and pharmacology. Hollenstein et al . solved the structure of human corticotropin-releasing factor receptor 1. This GPCR binds to corticotropin-releasing hormone, a potent mediator of endocrine, autonomic, behavioral and immune responses to stress. In all known class A GPCRs, the ligand-binding sites are close to the extracellular boundaries of the receptors; in this GPCR, the antagonist (CP-376395) binds in a hydrophobic pocket located in the cytoplasmic half of the V-shaped receptor. Siu et al . solved the X-ray crystal structure of the human glucagon receptor. This GPCR binds to the glucagon peptide, which triggers the release of glucose from the liver, making it a potential drug target for type 2 diabetes. The structure reveals a larger ligand-binding pocket than that seen in class A GPCRs.

During recent years combining GLP-1 and glucagon receptor agonism with the purpose of achieving superior weight loss and metabolic control compared to GLP-1 alone has received much attention. The superior efficacy has been shown by several in preclinical models but has been difficult to reproduce in humans. In this paper, we present the pre-clinical evaluation of NN1177, a long-acting GLP-1/glucagon receptor co-agonist previously tested in clinical trials. To further investigate the contribution from the respective receptors, two other co-agonists (NN1151, NN1359) with different GLP-1-to-glucagon receptor ratios were evaluated in parallel. In the process of characterizing NN1177, species differences and pitfalls in traditional pre-clinical evaluation methods were identified, highlighting the translational challenges in predicting the optimal receptor balance in humans. In diet-induced obese (DIO) mice, NN1177 induced a dose-dependent body weight loss, primarily due to loss of fat mass, and improvement in glucose tolerance. In DIO rats, NN1177 induced a comparable total body weight reduction, which was in contrast mainly caused by loss of lean mass, and glucose tolerance was impaired. Furthermore, despite long half-lives of the three co-agonists, glucose control during steady state was seen to depend on compound exposure at time of evaluation. When evaluated at higher compound exposure, glucose tolerance was similarly improved for all three co-agonists, independent of receptor balance. However, at lower compound exposure, glucose tolerance was gradually impaired with higher glucagon receptor preference. In addition, glucose tolerance was found to depend on study duration where the effect of glucagon on glucose control became more evident with time. To conclude, the pharmacodynamic effects at a given GLP-1-to-glucagon ratio differs between species, depends on compound exposure and study length, complicating the identification of an optimally balanced clinical candidate. The present findings could partly explain the low number of clinical successes for this dual agonism.

The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen–deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors. The crystal structure of the full-length human glucagon receptor reveals the essential role of the 12-residue ‘stalk’ segment and an extracellular loop in the regulation of ligand binding and receptor activation. Full-length class B GPCR structures The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) belong to the class B G-protein-coupled receptor family and have opposing physiological roles in glucose homeostasis and insulin release. As such, they are important in regulating metabolism and appetite and offer significant treatment possibilities for type 2 diabetes. However, as yet, no full-length structures of these receptors have been solved. Three papers in this issue of Nature report the structure of GLP-1R. Ray Stevens and colleagues describe the crystal structure of the human GLP-1R transmembrane domain in an inactive state in complex with negative allosteric modulators. Fiona Marshall and colleagues describe the active-state full-length receptor in complex with truncated peptide agonists, which have potent activity in mice on oral administration. Georgios Skiniotis, Brian Kobilka and colleagues describe the cryo-electron microscopy structure of an unmodified GLP-1R in complex with its endogenous peptide ligand, GLP-1, and the heterotrimeric G protein. Finally, in a fourth paper in this week's issue of Nature , Beili Wu and colleagues report the crystal structure of the full-length GCGR in an inactive conformation. Taken together, these studies provide key insights into the activation and signalling mechanisms of class B receptors and provide therapeutic opportunities for targeting this receptor family.

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.

Transport of biologically active molecules across tight epithelial barriers is a major challenge preventing therapeutic peptides from oral drug delivery. Here, we identify a set of synthetic glycosphingolipids that harness the endogenous process of intracellular lipid-sorting to enable mucosal absorption of the incretin hormone GLP-1. Peptide cargoes covalently fused to glycosphingolipids with ceramide domains containing C6:0 or smaller fatty acids were transported with 20-100-fold greater efficiency across epithelial barriers in vitro and in vivo. This was explained by structure-function of the ceramide domain in intracellular sorting and by the affinity of the glycosphingolipid species for insertion into and retention in cell membranes. In mice, GLP-1 fused to short-chain glycosphingolipids was rapidly and systemically absorbed after gastric gavage to affect glucose tolerance with serum bioavailability comparable to intraperitoneal injection of GLP-1 alone. This is unprecedented for mucosal absorption of therapeutic peptides, and defines a technology with many other clinical applications. To work properly, drugs need to be absorbed efficiently into the body. Medications that are injected directly into the bloodstream are often quickly transported to the organs or tissues they target. But injections are not always convenient, and many patients would instead prefer to swallow a pill or tablet. If a drug is swallowed, however, it must first be absorbed through the gut before it can enter the bloodstream. The lining of the gut consists of tightly linked layers of cells that readily take up small molecules, such as water and simple nutrients, but exclude almost all larger ones. Since several important types of drugs are large or poorly absorbed molecules, such as proteins, finding methods to help them cross the gut barrier is a major part of drug development. Originally from bacteria, cholera toxin is an example of a large, naturally occurring protein that does cross the gut lining. To do this, the toxin specifically attaches onto GM1, a type of lipid molecule that is found on the outer surface of gut cells, and hijacks the system that moves this lipid within cells. Previous studies identified several key features of GM1’s structure that enable this movement; and, in 2014, researchers tested GM1 as a ‘carrier’ to help the gut to absorb large therapeutic molecules. This approach was successful in cells grown in the laboratory, but not when the drugs were fed to animals. To overcome this issue, Garcia-Castillo, Chinnapen et al. – who include some of the researchers involved in the earlier studies – set out to further boost GM1’s ability to transport drugs across the gut lining. First several hybrid molecules were made, consisting of different structures of GM1 (the ‘carrier’) fused to a reporter peptide (the ‘cargo’). Laboratory experiments with human intestinal cells and dog kidney cells, both of which form tightly-linked layers much like the actual lining of the gut, revealed specific structural variations of the GM1-derived carrier that transported the cargo across the cell barrier more efficiently. Garcia-Castillo, Chinnapen et al. went on to test the efficiency of these carriers further by switching the reporter cargo to a therapeutic hormone called GLP-1. This hormone is used to treat people with type II diabetes but is currently given via an injection. The same structural variants of GM1 that enhanced delivery of the reporter cargo also worked for the larger GLP-1 hormone. Garcia-Castillo, Chinnapen et al. then fed the GM1-GLP-1 fusions to mice, and measured the amount of GLP-1 hormone absorbed into the blood. Crucially, the mice fed GM1-GLP-1 molecules absorbed the drug just as well as mice injected with the GLP-1 that is normally given to diabetes patients. Together these findings represent a major contribution to the pharmaceutical toolbox. They may also ultimately lead to more drugs that can be given as a patient-friendly pill or tablet, readily cross the gut barrier and achieve widespread drug delivery around the body.

Crystal structures of the human GLP-1 receptor in complex with two negative allosteric modulators reveal a common binding pocket, and, together with mutagenesis and modelling studies, further our understanding of the receptor activation mechanism.Author: Please check the wording of the following statement, which will appear online only. Full-length class B GPCR structures The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) belong to the class B G-protein-coupled receptor family and have opposing physiological roles in glucose homeostasis and insulin release. As such, they are important in regulating metabolism and appetite and offer significant treatment possibilities for type 2 diabetes. However, as yet, no full-length structures of these receptors have been solved. Three papers in this issue of Nature report the structure of GLP-1R. Ray Stevens and colleagues describe the crystal structure of the human GLP-1R transmembrane domain in an inactive state in complex with negative allosteric modulators. Fiona Marshall and colleagues describe the active-state full-length receptor in complex with truncated peptide agonists, which have potent activity in mice on oral administration. Georgios Skiniotis, Brian Kobilka and colleagues describe the cryo-electron microscopy structure of an unmodified GLP-1R in complex with its endogenous peptide ligand, GLP-1, and the heterotrimeric G protein. Finally, in a fourth paper in this week's issue of Nature , Beili Wu and colleagues report the crystal structure of the full-length GCGR in an inactive conformation. Taken together, these studies provide key insights into the activation and signalling mechanisms of class B receptors and provide therapeutic opportunities for targeting this receptor family. The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis 1 . The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner 2 . Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR 3 and located outside helices V–VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs 4 , 5 , 6 . Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling.

The inappropriate overproduction of glucose by the liver is one of the key contributors to the hyperglycaemia of the diabetic state, and thus is a logical site of intervention for novel anti-diabetic approaches. Metformin is the only currently marketed anti-hyperglycaemic drug whose action is attributed largely to its having inhibitory effects on hepatic glucose production, but its molecular site and mechanism(s) of action remain unknown, whereas the liver acting PPARalpha agonists have their effects primarily on lipid metabolism. This review therefore rather focuses on candidate molecular targets within the liver for anti-hyperglycaemic therapy, and describes potential rate-controlling receptors and enzymes within the glucose producing pathways (glycogenolysis and gluconeogenesis). Most focus is directed towards inhibitors of the enzymes glucose-6-phosphatase, fructose-1,6-bisphosphatase and glycogen phosphorylase, and towards glucagon receptor antagonists, as these appear to be the most advanced in preclinical and clinical development, although progress with other potential targets is also described. Evidence of the anti-diabetic potential of such agents from animal studies is presented, and the relative merits of each approach are reviewed and compared. It is likely that such agents will become important additions to the therapeutic approaches to combat diabetes.

The crystal structure of the full-length glucagon receptor in complex with a glucagon analogue NNC1702 reveals how the peptide ligand interacts with its target and shows the conformational changes required for receptor activation. Structure of a glucagon complex The glucagon receptor is a class B G-protein-coupled receptor with an important role in glucose homeostasis. Activation of this receptor triggers the release of glucose, making it an important target for the treatment of type 2 diabetes. Beili Wu and colleagues now report the crystal structure of the full-length glucagon receptor bound to a glucagon analogue and a partial agonist, NNC1702. The 3.0 Å resolution of the structure provides key insights into how the peptide ligand interacts with its target and the conformational changes involved in the initial stages of activation. The structural data is reinforced by using double electron–electron resonance (DEER) spectroscopy, which demonstrates the extent of rearrangement that is involved in accommodating the glucagon analogue. Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 . Previous work 9 , 10 , 11 has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved 12 , 13 , 14 . These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR–NNC0640–mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation—which requires conformational changes of the stalk, first extracellular loop and TMD—that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

Since the Cadbury Report of 1992 several Member States of the European Union have provided national codes on corporate governance or best practice, and the European Commission in its Company Law Action Plan has declared its intention to see these coordinated and converge. The five Nordic countries that are all members of either the EU or EEA have passed such codes relatively late. Based on these Nordic Codes, this paper takes a critical look at what this venture into European soft law or best practices is about, and points out the differences in the Nordic approach to this kind of company law regulation that are visible in the Codes and may be at odds with the intended convergence. [PUBLICATION ABSTRACT]