Impact of secretin receptor homo-dimerization on natural ligand binding.

Class B G protein-coupled receptors can form dimeric complexes important for high potency biological effects. Here, we apply pharmacological, biochemical, and biophysical techniques to cells and membranes expressing the prototypic secretin receptor (SecR) to gain insights into secretin binding to homo-dimeric and monomeric SecR. Spatial proximity between peptide and receptor residues, probed by disulfide bond formation, demonstrates that the secretin N-terminus moves from adjacent to extracellular loop 3 (ECL3) at wild type SecR toward ECL2 in non-dimerizing mutants. Analysis of fluorescent secretin analogs demonstrates stable engagement of the secretin C-terminal region within the receptor extracellular domain (ECD) for both dimeric and monomeric receptors, while the mid-region exhibits lower mobility while docked at the monomer. Moreover, decoupling of G protein interaction reduces mobility of the peptide mid-region at wild type receptor to levels similar to the mutant, whereas it has no further impact on the monomer. These data support a model of peptide engagement whereby the ability of SecR to dimerize promotes higher conformational dynamics of the peptide-bound receptor ECD and ECLs that likely facilitates more efficient G protein recruitment and activation, consistent with the higher observed functional potency of secretin at wild type SecR relative to the monomeric mutant receptor.

Mechanism of Action and Structure-Activity Relationships of Tetracyclic Small Molecules Acting as Universal Positive Allosteric Modulators of the Cholecystokinin Receptor.

As part of an ongoing effort to develop a drug targeting the type 1 cholecystokinin receptor (CCK1R) to help prevent and/or treat obesity, we recently performed a high throughput screening effort of small molecules seeking candidates that enhanced the action of the natural agonist, CCK, thus acting as positive allosteric modulators without exhibiting intrinsic agonist action. Such probes would be expected to act in a temporally finite way to enhance CCK action to induce satiety during and after a meal and potentially even modulate activity at the CCK1R in a high cholesterol environment present in some obese patients. The current work focuses on the best scaffold, representing tetracyclic molecules identified through high throughput screening we previously reported. Extensive characterization of the two top "hits" from the previous effort demonstrated them to fulfill the desired pharmacologic profile. We undertook analog-by-catalog expansion of this scaffold using 65 commercially available analogs. In this effort, we were able to eliminate an off-target effect observed for this scaffold while retaining its activity as a positive allosteric modulator of CCK1R in both normal and high cholesterol membrane environments. These insights should be useful in the rational medicinal chemical enhancement of this scaffold and in the future development of candidates to advance to pre-clinical proof-of-concept and to clinical trials.

Targeting VIP and PACAP Receptor Signaling: New Insights into Designing Drugs for the PACAP Subfamily of Receptors.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Vasoactive Intestinal Peptide (VIP) are neuropeptides involved in a diverse array of physiological and pathological processes through activating the PACAP subfamily of class B1 G protein-coupled receptors (GPCRs): VIP receptor 1 (VPAC1R), VIP receptor 2 (VPAC2R), and PACAP type I receptor (PAC1R). VIP and PACAP share nearly 70% amino acid sequence identity, while their receptors PAC1R, VPAC1R, and VPAC2R share 60% homology in the transmembrane regions of the receptor. PACAP binds with high affinity to all three receptors, while VIP binds with high affinity to VPAC1R and VPAC2R, and has a thousand-fold lower affinity for PAC1R compared to PACAP. Due to the wide distribution of VIP and PACAP receptors in the body, potential therapeutic applications of drugs targeting these receptors, as well as expected undesired side effects, are numerous. Designing selective therapeutics targeting these receptors remains challenging due to their structural similarities. This review discusses recent discoveries on the molecular mechanisms involved in the selectivity and signaling of the PACAP subfamily of receptors, and future considerations for therapeutic targeting.

Screening for positive allosteric modulators of cholecystokinin type 1 receptor potentially useful for management of obesity.

Obesity has become a prevailing health burden globally and particularly in the US. It is associated with many health problems, including cardiovascular disease, diabetes and poorer mental health. Hence, there is a high demand to find safe and effective therapeutics for sustainable weight loss. Cholecystokinin (CCK) has been implicated as one of the first gastrointestinal hormones to reduce overeating and suppress appetite by activating the type 1 cholecystokinin receptor (CCK1R). Several drug development campaigns have focused on finding CCK1R-specific agonists, which showed promising efficacy for reducing meal size and weight, but fell short on FDA approval, likely due to side effects associated with potent, long-lasting activation of CCK1Rs. Positive allosteric modulators (PAMs) without inherent agonist activity have been proposed to overcome the shortcomings of traditional, orthosteric agonists and restore CCK1R signaling in failing physiologic systems. However, drug discovery campaigns searching for such novel acting CCK1R agents remain limited. Here we report a high-throughput screening effort and the establishment of a testing funnel, which led to the identification of novel CCK1R modulators. We utilized IP-One accumulation to develop robust functional equilibrium assays tailored to either detect PAMs, agonists or non-specific activators. In addition, we established the CCK1R multiplex PAM assay as a novel method to evaluate functional selectivity capable of recording CCK1R-induced cAMP accumulation and ß-arrestin recruitment in the same well. This selection and arrangement of methods enabled the discovery of three scaffolds, which we characterized and validated in an array of functional and binding assays. We found two hits incorporating a tetracyclic scaffold that significantly enhanced CCK signaling at CCK1Rs without intrinsically activating CCK1Rs in an overexpressing system. Our results demonstrate that a well-thought-out testing funnel can identify small molecules with a distinct pharmacological profile and provides an important milestone for the development of novel potential treatments of obesity.

Secretin Amino-Terminal Structure-Activity Relationships and Complementary Mutagenesis at the Site of Docking to the Secretin Receptor.

Class B1 G protein-coupled receptors are activated by peptides, with amino-terminal regions critical for biologic activity. Although high resolution structures exist, understanding of key features of the peptide activation domain that drive signaling is limited. In the secretin receptor (SecR) structure, interactions are observed between peptide residues His1 and Ser2 and seventh transmembrane segment (TM7) receptor residue E373. We interrogated these interactions using systematic structure-activity analysis of peptide and receptor. His1 was critical for binding and cAMP responses, but its orientation was not critical, and substitution could independently modify affinity and efficacy. Ser2 was also critical, with all substitutions reducing peptide affinity and functional responses proportionally. Mutation of E373 to conserved acidic Asp (E373D), uncharged polar Gln (E373Q), or charge-reversed basic Arg (E373R) did not alter receptor expression, with all exhibiting secretin-dependent cAMP accumulation. All position 373 mutants displayed reduced binding affinities and cAMP potencies for many peptide analogs, although relative effects of position 1 peptides were similar whereas position 2 peptides exhibited substantial differences. The peptide including basic Lys in position 2 was active at SecR having acidic Glu in position 373 and at E373D while exhibiting minimal activity at those receptors in which an acidic residue is absent in this position (E373Q and E373R). In contrast, the peptide including acidic Glu in position 2 was equipotent with secretin at E373R while being much less potent than secretin at wild-type SecR and E373D. These data support functional importance of a charge-charge interaction between the amino-terminal region of secretin and the top of TM7. SIGNIFICANCE STATEMENT: This work refines our molecular understanding of the activation mechanisms of class B1 G protein-coupled receptors. The amino-terminal region of secretin interacts with the seventh transmembrane segment of its receptor with structural specificity and with a charge-charge interaction helping to drive functional activation.

Discovery of a Positive Allosteric Modulator of Cholecystokinin Action at CCK1R in Normal and Elevated Cholesterol.

Drugs useful in prevention/treatment of obesity could improve health. Cholecystokinin (CCK) is a key regulator of appetite, working through the type 1 CCK receptor (CCK1R); however, full agonists have not stimulated more weight loss than dieting. We proposed an alternate strategy to target this receptor, while reducing likelihood of side effects and/or toxicity. Positive allosteric modulators (PAMs) with minimal intrinsic agonist activity would enhance CCK action, while maintaining spatial and temporal characteristics of physiologic signaling. This could correct abnormal stimulus-activity coupling observed in a high-cholesterol environment observed in obesity. We utilized high-throughput screening to identify a molecule with this pharmacological profile and studied its basis of action. Compound 1 was a weak partial agonist, with PAM activity to enhance CCK action at CCK1R, but not CCK2R, maintained in both normal and high cholesterol. Compound 1 (10 µM) did not exhibit agonist activity or stimulate internalization of CCK1R. It enhanced CCK activity by slowing the off-rate of bound hormone, increasing its binding affinity. Computational docking of Compound 1 to CCK1R yielded plausible poses. A radioiodinatable photolabile analogue retained Compound 1 pharmacology and covalently labeled CCK1R Thr211, consistent with one proposed pose. Our study identifies a novel, selective, CCK1R PAM that binds to the receptor to enhance action of CCK-8 and CCK-58 in both normal and disease-mimicking high-cholesterol environments. This facilitates the development of compounds that target the physiologic spatial and temporal engagement of CCK1R by CCK that underpins its critical role in metabolic regulation.

Structure of an AMPK complex in an inactive, ATP-bound state.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.

Roles of Cholecystokinin in the Nutritional Continuum. Physiology and Potential Therapeutics.

Cholecystokinin is a gastrointestinal peptide hormone with important roles in metabolic physiology and the maintenance of normal nutritional status, as well as potential roles in the prevention and management of obesity, currently one of the dominant causes of direct or indirect morbidity and mortality. In this review, we discuss the roles of this hormone and its receptors in maintaining nutritional homeostasis, with a particular focus on appetite control. Targeting this action led to the development of full agonists of the type 1 cholecystokinin receptor that have so far failed in clinical trials for obesity. The possible reasons for clinical failure are discussed, along with alternative pharmacologic strategies to target this receptor for prevention and management of obesity, including development of biased agonists and allosteric modulators. Cellular cholesterol is a natural modulator of the type 1 cholecystokinin receptor, with elevated levels disrupting normal stimulus-activity coupling. The molecular basis for this is discussed, along with strategies to overcome this challenge with a corrective positive allosteric modulator. There remains substantial scope for development of drugs to target the type 1 cholecystokinin receptor with these new pharmacologic strategies and such drugs may provide new approaches for treatment of obesity.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity.

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from "unwinding" of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.

Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

Discovery of small molecule positive allosteric modulators of the secretin receptor.

The secretin receptor (SCTR) is a prototypic Class B1 G protein-coupled receptor (GPCR) that represents a key target for the development of therapeutics for the treatment of cardiovascular, gastrointestinal, and metabolic disorders. However, no non-peptidic molecules targeting this receptor have yet been disclosed. Using a high-throughput screening campaign directed at SCTR to identify small molecule modulators, we have identified three structurally related scaffolds positively modulating SCTRs. Here we outline a comprehensive study comprising a structure-activity series based on commercially available analogs of the three hit scaffold sets A (2-sulfonyl pyrimidines), B (2-mercapto pyrimidines) and C (2-amino pyrimidines), which revealed determinants of activity, cooperativity and specificity. Structural optimization of original hits resulted in analog B2, which substantially enhances signaling of truncated secretin peptides and prolongs residence time of labeled secretin up to 13-fold in a dose-dependent manner. Furthermore, we found that investigated compounds display structural similarity to positive allosteric modulators (PAMs) active at the glucagon-like peptide-1 receptor (GLP-1R), and we were able to confirm cross-recognition of that receptor by a subset of analogs. Studies using SCTR and GLP-1R mutants revealed that scaffold A, but not B and C, likely acts via two distinct mechanisms, one of which constitutes covalent modification of Cys-347GLP-1R known from GLP-1R-selective modulators. The scaffolds identified in this study might not only serve as novel pharmacologic tools to decipher SCTR- or GLP-1R-specific signaling pathways, but also as structural leads to elucidate allosteric binding sites facilitating the future development of orally available therapeutic approaches targeting these receptors.

Evidence that specific interactions play a role in the cholesterol sensitivity of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the ß2-adrenergic receptor (ß2AR) and the cholecystokinin (CCK) receptor subfamily. The ß2AR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous ß2AR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCK1R shows functional sensitivity to membrane cholesterol while CCK2R does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with ß2AR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-Tm) shows that mutation of a conserved CRAC sequence on TM7 of the ß2AR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases.

Development of a Testing Funnel for Identification of Small-Molecule Modulators Targeting Secretin Receptors.

The secretin receptor (SCTR), a prototypical class B G protein-coupled receptor (GPCR), exerts its effects mainly by activating Gαs proteins upon binding of its endogenous peptide ligand secretin. SCTRs can be found in a variety of tissues and organs across species, including the pancreas, stomach, liver, heart, lung, colon, kidney, and brain. Beyond that, modulation of SCTR-mediated signaling has therapeutic potential for the treatment of multiple diseases, such as heart failure, obesity, and diabetes. However, no ligands other than secretin and its peptide analogs have been described to regulate SCTRs, probably due to inherent challenges in family B GPCR drug discovery. Here we report creation of a testing funnel that allowed targeted detection of SCTR small-molecule activators. Pursuing the strategy to identify positive allosteric modulators (PAMs), we established a unique primary screening assay employing a mixture of three orthosteric stimulators that was compared in a screening campaign testing 12,000 small-molecule compounds. Beyond that, we developed a comprehensive set of secondary assays, such as a radiolabel-free target engagement assay and a NanoBiT (NanoLuc Binary Technology)-based approach to detect ß-arrestin-2 recruitment, all feasible in a high-throughput environment as well as capable of profiling ligands and hits regarding their effect on binding and receptor function. This combination of methods enabled the discovery of five promising scaffolds, four of which have been validated and further characterized with respect to their allosteric activities. We propose that our results may serve as starting points for developing the first in vivo active small molecules targeting SCTRs.

Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists.

Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.

Structure and dynamics of the active Gs-coupled human secretin receptor.

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.

Structure and Dynamics of Adrenomedullin Receptors AM1 and AM2 Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal lymphatic and blood vascular development and angiogenesis. They play a pivotal role in embryo implantation and fertility and can provide protection against hypoxic and oxidative stress. CGRP and AM receptors are heterodimers of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as RAMP2 or RAMP3 (AM1R and AM2R, respectively). However, the mechanistic basis for RAMP modulation of CLR phenotype is unclear. In this study, we report the cryo-EM structure of the AM1R in complex with AM and Gs at a global resolution of 3.0 Å, and structures of the AM2R in complex with either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3 Å, respectively. The structures reveal distinctions in the primary orientation of the extracellular domains (ECDs) relative to the receptor core and distinct positioning of extracellular loop 3 (ECL3) that are receptor-dependent. Analysis of dynamic data present in the cryo-EM micrographs revealed additional distinctions in the extent of mobility of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting the TM helix and the ECD supports a role for this segment in controlling receptor phenotype. Moreover, a subset of the motions of the ECD appeared coordinated with motions of the G protein relative to the receptor core, suggesting that receptor ECD dynamics could influence G protein interactions. This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.

Rational development of a high-affinity secretin receptor antagonist.

The secretin receptor is a prototypic class B GPCR with substantial and broad pharmacologic importance. The aim of this project was to develop a high affinity selective antagonist as a new and important pharmacologic tool and to aid stabilization of this receptor in an inactive conformation for ultimate structural characterization. Amino-terminal truncation of the natural 27-residue ligand reduced biological activity, but also markedly reduced binding affinity. This was rationally and experimentally overcome with lactam stabilization of helical structure and with replacement of residues with natural and unnatural amino acids. A key new step in this effort was the replacement of peptide residue Leu22 with L-cyclohexylalanine (Cha) to enhance potential hydrophobic interactions with receptor residues Leu31, Val34, and Phe92 that were predicted from molecular modeling. Alanine-replacement mutagenesis of these residues markedly affected ligand binding and biological activity. The optimal antagonist ligand, (Y10,c[E16,K20],I17,Cha22,R25)sec(6-27), exhibited high binding affinity (4 nM), similar to natural secretin, and exhibited no demonstrable biological activity to stimulate cAMP accumulation, intracellular calcium mobilization, or ß-arrestin-2 translocation. It acts as an orthosteric competitive antagonist, predicted to bind within the peptide-binding groove in the receptor extracellular domain. The analogous peptide that was one residue longer, retaining Thr5, exhibited partial agonist activity, while further truncation of even a single residue (Phe6) reduced binding affinity. This sec(6-27)-based peptide will be an important new tool for pharmacological and structural studies.

Secretin effects on gastric functions, hormones and symptoms in functional dyspepsia and health: randomized crossover trial.

Abnormal gastric accommodation (GA) and gastric emptying contribute to pathophysiology in functional dyspepsia (FD). Secretin is a key regulator of GA in animal studies. Our aim was to study the effects of secretin on gastric motility, satiation, postprandial symptoms, and key hormones. We performed two double-blind, randomized, saline-controlled crossover trials in 10 healthy volunteers and 10 patients with FD by Rome IV criteria. We used measured GA (by validated SPECT method) after a 111In radiolabeled Ensure 300-mL meal and quantified gastric emptying for 30 min by scintigraphy. Satiation was measured by volume to fullness (VTF) and maximum tolerated volume (MTV) on an Ensure nutrient drink test and postprandial symptoms 30 min post-MTV. Fasting and postprandial GLP-1, GIP, and HPP were measured. The ages and sex distribution of healthy controls and patients with FD were similar. Compared with placebo, secretin delayed gastric emptying at 30 min in both health [-11% (-16, -4), P = 0.004]; and FD [-8% (-9, 0), P = 0.03]. Satiation (VTF and MTV), GA, and plasma levels of GLP-1, GIP, and HPP did not differ between treatment arms in health or FD. On ANCOVA analysis (adjusting for age and sex), secretin did not consistently increase postprandial symptoms in health or FD. Secretin delayed gastric emptying in both health and FD without significantly altering GA, VTF, or MTV or selected hormones. Thus, secretin receptor activation may provide a novel therapeutic mechanism for patients with FD and rapid gastric emptying.NEW & NOTEWORTHY The naturally occurring hormone secretin retards gastric emptying of solids without deleteriously affecting gastric accommodation, satiation, other upper gastrointestinal hormones, or postprandial symptoms. Given these findings, a subset of patients with rapid gastric emptying (e.g., the estimated 20% of patients with functional dyspepsia) could be candidates for treatments that stimulate a secretin receptor such as sacubitril, which inhibits neprilysin, an enzyme that degrades secretin.

Activation of the GLP-1 receptor by a non-peptidic agonist.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.

Structural Basis for Allosteric Modulation of Class B G Protein-Coupled Receptors.

Recent advances in our understanding of the structure and function of class B G protein-coupled receptors (GPCRs) provide multiple opportunities for targeted development of allosteric modulators. Given the pleiotropic signaling patterns emanating from these receptors in response to a variety of natural agonist ligands, modulators have the potential to sculpt the responses to meet distinct needs of different groups of patients. In this review, we provide insights into how this family of GPCRs differs from the rest of the superfamily, how orthosteric agonists bind and activate these receptors, the potential for allosteric modulators to interact with various regions of these targets, and the allosteric influence of endogenous proteins on the pharmacology of these receptors, all of which are important considerations when developing new therapies.