The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism.

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a ß-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.

Chemokine CXCL12 drives pericyte accumulation and airway remodeling in allergic airway disease.

BACKGROUND: Airway remodeling is a significant contributor to impaired lung function in chronic allergic airway disease. Currently, no therapy exists that is capable of targeting these structural changes and the consequent loss of function. In the context of chronic allergic inflammation, pericytes have been shown to uncouple from the pulmonary microvasculature, migrate to areas of inflammation, and significantly contribute to airway wall remodeling and lung dysfunction. This study aimed to elucidate the mechanism by which pulmonary pericytes accumulate in the airway wall in a model of chronic allergic airway inflammation. METHODS: Mice were subjected to a protocol of chronic airway inflammation driven by the common environmental aeroallergen house dust mite. Phenotypic changes to lung pericytes were assessed by flow cytometry and immunostaining, and the functional capacity of these cells was evaluated using in vitro migration assays. The molecular mechanisms driving these processes were targeted pharmacologically in vivo and in vitro. RESULTS: Pericytes demonstrated increased CXCR4 expression in response to chronic allergic inflammation and migrated more readily to its cognate chemokine, CXCL12. This increase in migratory capacity was accompanied by pericyte accumulation in the airway wall, increased smooth muscle thickness, and symptoms of respiratory distress. Pericyte uncoupling from pulmonary vessels and subsequent migration to the airway wall were abrogated following topical treatment with the CXCL12 neutraligand LIT-927. CONCLUSION: These results provide new insight into the role of the CXCL12/CXCR4 signaling axis in promoting pulmonary pericyte accumulation and airway remodeling and validate a novel target to address tissue remodeling associated with chronic inflammation.

Functional characterisation of G protein-coupled receptors.

Characterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either directly monitoring ligand itself with techniques such as fluorescence polarisation or indirectly via resonance energy transfer (fluorescence/Forster resonance energy transfer, FRET and bioluminescence resonance energy transfer, BRET). Label free techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are also increasingly being used. For GPCRs, the main measure of receptor activation is to investigate the association of the G protein with the receptor. The chief assay measures the receptor-stimulated binding of GTP or a suitable analogue to the receptor. The direct association of the G protein with the receptor has been investigated via resonance energy techniques. These have also been used to measure ligand-induced conformational changes within the receptor; a variety of experimental techniques are available to incorporate suitable donors and acceptors within the receptor.

The Structure of the CGRP and Related Receptors.

The canonical CGRP receptor is a complex between calcitonin receptor-like receptor (CLR), a family B G-protein-coupled receptor (GPCR) and receptor activity-modifying protein 1 (RAMP1). A third protein, receptor component protein (RCP) is needed for coupling to Gs. CGRP can interact with other RAMP-receptor complexes, particularly the AMY1 receptor formed between the calcitonin receptor (CTR) and RAMP1. Crystal structures are available for the binding of CGRP27-37 [D31,P34,F35] to the extracellular domain (ECD) of CLR and RAMP1; these show that extreme C-terminal amide of CGRP interacts with W84 of RAMP1 but the rest of the analogue interacts with CLR. Comparison with the crystal structure of a fragment of the allied peptide adrenomedullin bound to the ECD of CLR/RAMP2 confirms the importance of the interaction of the ligand C-terminus and the RAMP in determining pharmacology specificity, although the RAMPs probably also have allosteric actions. A cryo-electron microscope structure of calcitonin bound to the full-length CTR associated with Gs gives important clues as to the structure of the complete receptor and suggests that the N-terminus of CGRP makes contact with His5.40b, high on TM5 of CLR. However, it is currently not known how the RAMPs interact with the TM bundle of any GPCR. Major challenges remain in understanding how the ECD and TM domains work together to determine ligand specificity, and how G-proteins influence this and the role of RCP. It seems likely that allosteric mechanisms are particularly important as are the dynamics of the receptors.

Photoaffinity Cross-Linking and Unnatural Amino Acid Mutagenesis Reveal Insights into Calcitonin Gene-Related Peptide Binding to the Calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein 1 (CLR/RAMP1) Complex.

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.

Improving virtual screening of G protein-coupled receptors via ligand-directed modeling.

G protein-coupled receptors (GPCRs) play crucial roles in cell physiology and pathophysiology. There is increasing interest in using structural information for virtual screening (VS) of libraries and for structure-based drug design to identify novel agonist or antagonist leads. However, the sparse availability of experimentally determined GPCR/ligand complex structures with diverse ligands impedes the application of structure-based drug design (SBDD) programs directed to identifying new molecules with a select pharmacology. In this study, we apply ligand-directed modeling (LDM) to available GPCR X-ray structures to improve VS performance and selectivity towards molecules of specific pharmacological profile. The described method refines a GPCR binding pocket conformation using a single known ligand for that GPCR. The LDM method is a computationally efficient, iterative workflow consisting of protein sampling and ligand docking. We developed an extensive benchmark comparing LDM-refined binding pockets to GPCR X-ray crystal structures across seven different GPCRs bound to a range of ligands of different chemotypes and pharmacological profiles. LDM-refined models showed improvement in VS performance over origin X-ray crystal structures in 21 out of 24 cases. In all cases, the LDM-refined models had superior performance in enriching for the chemotype of the refinement ligand. This likely contributes to the LDM success in all cases of inhibitor-bound to agonist-bound binding pocket refinement, a key task for GPCR SBDD programs. Indeed, agonist ligands are required for a plethora of GPCRs for therapeutic intervention, however GPCR X-ray structures are mostly restricted to their inactive inhibitor-bound state.

Relative Antagonism of Mutants of the CGRP Receptor Extracellular Loop 2 Domain (ECL2) Using a Truncated Competitive Antagonist (CGRP8-37): Evidence for the Dual Involvement of ECL2 in the Two-Domain Binding Model.

The second extracellular loop (ECL2) of the G protein-coupled receptor (GPCR) family is important for ligand interaction and drug discovery. ECL2 of the family B cardioprotective calcitonin gene-related peptide (CGRP) receptor is required for cell signaling. Family B GPCR ligands have two regions; the N-terminus mediates receptor activation, and the remainder confers high-affinity binding. Comparing antagonism of CGRP8-37 at a number of point mutations of ECL2 of the CGRP receptor, we show that the ECL2 potentially facilitates interaction with up to the 18 N-terminal residues of CGRP. This has implications for understanding family B GPCR activation and for drug design at the CGRP receptor.

A Hydrogen-Bonded Polar Network in the Core of the Glucagon-Like Peptide-1 Receptor Is a Fulcrum for Biased Agonism: Lessons from Class B Crystal Structures.

The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiologic significance of this is yet to be elucidated. Previous work has implicated R2.60(190), N3.43(240), Q7.49(394), and H6.52(363) as key residues involved in peptide-mediated biased agonism, with R2.60(190), N3.43(240), and Q7.49(394) predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict additional key molecular interactions with these amino acids. We have introduced E6.53(364)A, N3.43(240)Q, Q7.49(394)N, and N3.43(240)Q/Q7.49(394)N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium, or extracellular signal-regulated kinase (ERK) signaling. A polar interaction between E6.53(364) and R2.60(190) was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49(394), but not R2.60(190)/E6.53(364) was critical for calcium mobilization for all three peptides. Mutation of N3.43(240) and Q7.49(394) had differential effects on individual peptides, providing evidence for molecular differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.

Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations.

Recently, the concept of ligand-directed signaling--the ability of different ligands of an individual receptor to promote distinct patterns of cellular response--has gained much traction in the field of drug discovery, with the potential to sculpt biological response to favor therapeutically beneficial signaling pathways over those leading to harmful effects. However, there is limited understanding of the mechanistic basis underlying biased signaling. The glucagon-like peptide-1 receptor is a major target for treatment of type-2 diabetes and is subject to ligand-directed signaling. Here, we demonstrate the importance of polar transmembrane residues conserved within family B G protein-coupled receptors, not only for protein folding and expression, but also in controlling activation transition, ligand-biased, and pathway-biased signaling. Distinct clusters of polar residues were important for receptor activation and signal preference, globally changing the profile of receptor response to distinct peptide ligands, including endogenous ligands glucagon-like peptide-1, oxyntomodulin, and the clinically used mimetic exendin-4.

Differential impact of amino acid substitutions on critical residues of the human glucagon-like peptide-1 receptor involved in peptide activity and small-molecule allostery.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor system is highly complex, able to be activated by both endogenous [GLP-1(1-36)NH2, GLP-1(1-37), GLP-1(7-36)NH2, GLP-1(7-37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptide-mediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca(2+) mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.

Classifying tetraspanins: A universal system for numbering residues and a proposal for naming structural motifs and subfamilies.

All tetraspanins have four transmembrane domains (TMs). The large extracellular loop (LEL) that connects the third and fourth TMs contains multiple secondary structures together with the family's signature Cys-Cys-Gly motif. These intriguing membrane proteins are involved in diverse and incompletely understood cellular processes including cell adhesion, tissue differentiation, immune cell maturation and host-parasite interactions. Here we present a classification system that accurately describes the position of each amino acid within its primary sequence based on both sequence and topological conservation of the TMs and LEL. This builds on the numbering systems that have been used in the G protein-coupled receptor (GPCR) field for nearly three decades and which have aided the understanding of GPCR structure/activity relationships and ligand interactions. The high-resolution structures of the tetraspanins CD81, CD9, CD53 and Tspan15 were used to validate the structural relevance of our new tetraspanin classification system. Modelling of all tetraspanin LELs highlighted flexibility in LEL disulfide bonding across the family and suggests that the structural arrangement of tetraspanin LELs is more complex than previously thought. We therefore propose a new subfamily naming system that addresses this added complexity and facilitates the systematic classification of human tetraspanins, shedding light on all structural motifs within the family. We anticipate that our universal tetraspanin classification system will enable progress in defining how sequence and structure inform function.

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function.

The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical family B G protein-coupled receptor that exhibits physiologically important pleiotropic coupling and ligand-dependent signal bias. In our accompanying article (Koole, C., Wootten, D., Simms, J., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2012) J. Biol. Chem. 287, 3642-3658), we demonstrate, through alanine-scanning mutagenesis, a key role for extracellular loop (ECL) 2 of the receptor in propagating activation transition mediated by GLP-1 peptides that occurs in a peptide- and pathway-dependent manner for cAMP formation, intracellular (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). In this study, we examine the effect of ECL2 mutations on the binding and signaling of the peptide mimetics, exendin-4 and oxyntomodulin, as well as small molecule allosteric agonist 6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2). Lys-288, Cys-296, Trp-297, and Asn-300 were globally important for peptide signaling and also had critical roles in governing signal bias of the receptor. Peptide-specific effects on relative efficacy and signal bias were most commonly observed for residues 301-305, although R299A mutation also caused significantly different effects for individual peptides. Met-303 was more important for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Globally, ECL2 mutation was more detrimental to exendin-4-mediated Ca(2+)i release than GLP-1(7-36)-NH(2), providing additional evidence for subtle differences in receptor activation by these two peptides. Unlike peptide activation of the GLP-1R, ECL2 mutations had only limited impact on compound 2 mediated cAMP and pERK responses, consistent with this ligand having a distinct mechanism for receptor activation. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition of the receptor by peptide agonists.

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation.

The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.

Structure-function studies of muscarinic acetylcholine receptors.

There has been great interest in the structure-function relationships of the muscarinic acetylcholine receptors (mAChRs) because these prototypical Family A/class 1 G protein-coupled receptors (GPCRs) are attractive therapeutic targets for both peripheral and central nervous system disorders. A multitude of drugs that act at the mAChRs have been identified over the years, but many of these show minimal selectivity for any one of the five mAChR subtypes over the others, which has hampered their development into therapeutics due to adverse side effects. The lack of drug specificity is primarily due to high sequence similarity in this family of receptor, especially in the orthosteric binding pocket. Thus, there remains an ongoing need for a molecular understanding of how mAChRs bind their ligands, and how selectivity in binding and activation can be achieved. Unfortunately, there remains a paucity of solved high-resolution structures of GPCRs, including the mAChRs, and thus most of our knowledge of structure-function mechanisms related to this receptor family to date has been obtained indirectly through approaches such as mutagenesis. Nonetheless, such studies have revealed a wealth of information that has led to novel insights and may be used to guide future rational drug design campaigns.

RAMPs and CGRP receptors.

Receptor activity modifying protein 1 (RAMP1) forms a complex with calcitonin receptor-like receptor (CLR) to produce the receptor for calcitonin gene-related peptide (CGRP). RAMP1 has two main roles. It facilitates the cell-surface expression of CLR. It is also essential for the binding of CGRP to the receptor. It seems likely that Y66, F93, H97 and F101, amongst other residues, form a binding site for CLR. These cluster together on the same face of the extracellular portion of RAMP1, probably close to where it enters the plasma membrane. Residues at the other end of RAMP1 are most likely to be involved in CGRP recognition, although it is currently unclear how they do this. Within this area, W74 is important for the binding of the nonpeptide antagonist, BIBN4096BS, although it does not seem to be involved in the binding of CGRP itself. It has been shown that there is an epitope within residues 23-60 of CLR that are essential for RAMP recognition. Under some circumstances, changes in the expression of RAMP1 can alter the sensitivity of cells to CGRP, demonstrating that regulation of its levels may be of physiological or pathophysiological importance.

RAMPs as drug targets.

The receptor activity-modifying protein (RAMP) family of membrane proteins regulates G protein-coupled receptor (GPCR) function in several ways. RAMPs can alter their pharmacology and signalling as well as the trafficking of these receptors to and from the cell surface. Accordingly, RAMPs may be exploited as drug targets, offering new opportunities for regulating the function of therapeutically relevant RAMP-interacting GPCRs. For example, several small molecule antagonists of RAMP1/ calcitonin receptor-like receptor complexes, which block the actions of the neuropeptide calcitonin gene-related peptide are in development for the treatment of migraine headache.

Membrane Protein Production in the Yeast P. pastoris.

The first crystal structures of recombinant mammalian membrane proteins were solved using high-quality protein that had been produced in yeast cells. One of these, the rat Kv1.2 voltage-gated potassium channel, was synthesized in Pichia pastoris. Since then, this yeast species has remained a consistently popular choice of host for synthesizing eukaryotic membrane proteins because it is quick, easy, and cheap to culture and is capable of posttranslational modification. Very recent structures of recombinant membrane proteins produced in P. pastoris include a series of X-ray crystallography structures of the human vitamin K epoxide reductase and a cryo-electron microscopy structure of the TMEM206 proton-activated chloride channel from pufferfish. P. pastoris has also been used to structurally and functionally characterize a range of membrane proteins including tetraspanins, aquaporins, and G protein-coupled receptors. This chapter provides an overview of the methodological approaches underpinning these successes.

Polymorphism and ligand dependent changes in human glucagon-like peptide-1 receptor (GLP-1R) function: allosteric rescue of loss of function mutation.

The glucagon-like peptide-1 receptor (GLP-1R) is a key physiological regulator of insulin secretion and a major therapeutic target for the treatment of type II diabetes. However, regulation of GLP-1R function is complex with multiple endogenous peptides that interact with the receptor, including full-length (1-37) and truncated (7-37) forms of GLP-1 that can exist in an amidated form (GLP-1(1-36)NH2 and GLP-1(7-36)NH2) and the related peptide oxyntomodulin. In addition, the GLP-1R possesses exogenous agonists, including exendin-4, and the allosteric modulator, compound 2 (6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline). The complexity of this ligand-receptor system is further increased by the presence of several single nucleotide polymorphisms (SNPs) that are distributed across the receptor. We have investigated 10 GLP-1R SNPs, which were characterized in three physiologically relevant signaling pathways (cAMP accumulation, extracellular signal-regulated kinase 1/2 phosphorylation, and intracellular Ca²âº mobilization); ligand binding and cell surface receptor expression were also determined. We demonstrate both ligand- and pathway-specific effects for multiple SNPs, with the most dramatic effect observed for the Met¹49 receptor variant. At the Met¹49 variant, there was selective loss of peptide-induced responses across all pathways examined, but preservation of response to the small molecule compound 2. In contrast, at the Cys³³³ variant, peptide responses were preserved but there was attenuated response to compound 2. Strikingly, the loss of peptide function at the Met¹49 receptor variant could be allosterically rescued by compound 2, providing proof-of-principle evidence that allosteric drugs could be used to treat patients with this loss of function variant.

Modulation of the glucagon-like peptide-1 receptor signaling by naturally occurring and synthetic flavonoids.

The glucagon-like peptide 1 receptor (GLP-1R) is a promising target for the treatment of type II diabetes mellitus because of its role in metabolic homeostasis. In recent years, difficulties with peptide therapies have driven the search for small-molecule compounds to modulate the activity of this receptor. We recently identified quercetin, a naturally occurring flavonoid, as a probe-dependent, pathway-selective allosteric modulator of GLP-1R-mediated signaling. Using Chinese hamster ovary cells expressing the human GLP-1R, we have now extended this work to identify the structural requirements of flavonoids to modify GLP-1R binding and signaling (cAMP formation and intracellular Ca(2+) mobilization) of each of the GLP-1R endogenous agonists, as well as the clinically used exogenous peptide mimetic exendin-4. This study identified a chemical series of hydroxyl flavonols with the ability to selectively augment calcium (Ca(2+)) signaling in a peptide agonist-specific manner, with effects only on truncated GLP-1 peptides [GLP-1(7-36)NH(2) and GLP-1(7-37)] and exendin-4, but not on oxyntomodulin or full-length GLP-1 peptides [GLP-1(1-36)NH(2) and GLP-1(1-37)]. In addition, the 3-hydroxyl group on the flavone backbone (i.e., a flavonol) was essential for this activity, however insufficient on its own, to produce the allosteric effects. In contrast to hydroxyl flavonols, catechin had no effect on peptide-mediated Ca(2+) signaling but negatively modulated peptide-mediated cAMP formation in a probe-dependent manner. These data represent a detailed examination of the action of different flavonoids on peptide agonists at the GLP-1R and may aid in the development of future small molecule compounds targeted at this receptor.