RESUMO
Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Microscopia Crioeletrônica , Proteína 1 Modificadora da Atividade de Receptores , Humanos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/química , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/química , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/química , Animais , Ratos , Modelos Moleculares , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/química , Conformação ProteicaRESUMO
Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.
Assuntos
Doenças Metabólicas , Neuropeptídeos , Humanos , Receptores da Calcitonina/metabolismo , Proteínas Modificadoras da Atividade de Receptores , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Receptores de Peptídeos/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , ObesidadeRESUMO
Amylin receptors (AMYRs), heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins, are promising obesity targets. A hallmark of AMYR activation by Amy is the formation of a 'bypass' secondary structural motif (residues S19-P25). This study explored potential tuning of peptide selectivity through modification to residues 19-22, resulting in a selective AMYR agonist, San385, as well as nonselective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, and San45 bound to AMY3R or CTR. San45, via its conjugated lipid at position 21, was anchored at the edge of the receptor bundle, enabling a stable, alternative binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. Targeted lipid modification may provide a single intervention strategy for design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas , Receptores da Calcitonina , Humanos , Receptores da Calcitonina/agonistas , Receptores da Calcitonina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Obesidade , LipídeosRESUMO
The parathyroid hormone (PTH) 1 receptor (PTH1R) is a G protein-coupled receptor (GPCR) that regulates skeletal development and calcium homeostasis. Here, we describe cryo-EM structures of the PTH1R in complex with fragments of the two hormones, PTH and PTH-related protein, the drug abaloparatide, as well as the engineered tool compounds, long-acting PTH (LA-PTH) and the truncated peptide, M-PTH(1-14). We found that the critical N terminus of each agonist engages the transmembrane bundle in a topologically similar fashion, reflecting similarities in measures of Gαs activation. The full-length peptides induce subtly different extracellular domain (ECD) orientations relative to the transmembrane domain. In the structure bound to M-PTH, the ECD is unresolved, demonstrating that the ECD is highly dynamic when unconstrained by a peptide. High resolutions enabled identification of water molecules near peptide and G protein binding sites. Our results illuminate the action of orthosteric agonists of the PTH1R.
Assuntos
Hormônio Paratireóideo , Receptor Tipo 1 de Hormônio Paratireóideo , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Hormônio Paratireóideo/química , Hormônio Paratireóideo/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. Class B1 GPCRs constitute a subfamily of 15 receptors that characteristically contain large extracellular domains (ECDs) and respond to long polypeptide hormones. Class B1 GPCRs are critical regulators of homeostasis, and, as such, many are important drug targets. While most transmembrane proteins, including GPCRs, are recalcitrant to crystallization, recent advances in cryo-electron microscopy (cryo-EM) have facilitated a rapid expansion of the structural understanding of membrane proteins. As a testament to this success, structures for all the class B1 receptors bound to G proteins have been determined by cryo-EM in the past 5 years. Further advances in cryo-EM have uncovered dynamics of these receptors, ligands, and signaling partners. Here, we examine the recent structural underpinnings of the class B1 GPCRs with an emphasis on structure-function relationships.
Assuntos
Hormônios Peptídicos , Receptores Acoplados a Proteínas G , Humanos , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Hormônios Peptídicos/metabolismo , LigantesRESUMO
The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a comprehensive understanding of peptide binding and selectivity among different subfamily receptors is lacking. Here, we determine structures of active, Gs-coupled, VIP-VPAC1R, PACAP27-VPAC1R, and PACAP27-PAC1R complexes. Cryo-EM structural analyses and molecular dynamics simulations (MDSs) reveal fewer stable interactions between VPAC1R and VIP than for PACAP27, more extensive dynamics of VIP interaction with extracellular loop 3, and receptor-dependent differences in interactions of conserved N-terminal peptide residues with the receptor core. MD of VIP modelled into PAC1R predicts more transient VIP-PAC1R interactions in the receptor core, compared to VIP-VPAC1R, which may underlie the selectivity of VIP for VPAC1R over PAC1R. Collectively, our work improves molecular understanding of peptide engagement with the PAC1R and VPAC1R that may benefit the development of novel selective agonists.
Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Peptídeo Intestinal Vasoativo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Ligação Proteica , Simulação de Dinâmica MolecularRESUMO
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.
Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Peptídeo Intestinal Vasoativo , Sequência de Aminoácidos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Transdução de Sinais , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
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.
Assuntos
Receptores Acoplados a Proteínas G , Secretina , Sequência de Aminoácidos , Mutagênese , Peptídeos/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais , Secretina/química , Secretina/genética , Secretina/metabolismo , Relação Estrutura-AtividadeRESUMO
G protein-coupled receptors (GPCRs) are the largest class of membrane proteins and in recent years there has been a growing appreciation of the importance in understanding temporal aspects of GPCR behaviour, including the kinetics of ligand binding and downstream receptor mediated signalling. Class B1 GPCRs are activated by peptide agonists and are validated therapeutic targets for numerous diseases. However, the kinetics of ligand binding and how this is linked to downstream activation of signalling cascades is not routinely assessed in development of peptide agonists for this receptor class. The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical class B1 GPCR and a validated target for treatment of global health burdens, including type 2 diabetes and obesity. In this study we examined the kinetics of different steps in GLP-1R activation and subsequent cAMP production mediated by a series of GLP-1R peptide agonists, including the ligand-receptor interaction, ligand-receptor-mediated G protein engagement and conformational change and cAMP production. Our results revealed GLP-1R peptide agonist dissociation kinetics (Koff), but not association kinetics (Kon), were positively correlated with the onset of receptor-G protein coupling/conformational change, onset of cAMP production and duration of cAMP signalling. Thus, this study advances the understanding of molecular events that couple GLP-1R ligand binding to intracellular signaling, with the findings likely to have implications for mechanistic understanding of agonist action at other related class B1 GPCRs.
Assuntos
Diabetes Mellitus Tipo 2 , Receptor do Peptídeo Semelhante ao Glucagon 1 , Diabetes Mellitus Tipo 2/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Cinética , Ligantes , Peptídeos/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.
Assuntos
Agonistas dos Receptores da Amilina/química , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/química , Animais , Microscopia Crioeletrônica , Humanos , Fenótipo , Conformação Proteica , Multimerização Proteica , SalmãoRESUMO
Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.
Assuntos
Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Exenatida , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Peptídeos/química , Domínios ProteicosRESUMO
Class B1 G protein-coupled receptors (GPCRs) are important targets for many diseases, including cancer, diabetes, and heart disease. All the approved drugs for this receptor family are peptides that mimic the endogenous activating hormones. An understanding of how agonists bind and activate class B1 GPCRs is fundamental for the development of therapeutic small molecules. We combined supervised molecular dynamics (SuMD) and classic molecular dynamics (cMD) simulations to study the binding of the calcitonin gene-related peptide (CGRP) to the CGRP receptor (CGRPR). We also evaluated the association and dissociation of the antagonist telcagepant from the extracellular domain (ECD) of CGRPR and the water network perturbation upon binding. This study, which represents the first example of dynamic docking of a class B1 GPCR peptide, delivers insights on several aspects of ligand binding to CGRPR, expanding understanding of the role of the ECD and the receptor-activity modifying protein 1 (RAMP1) on agonist selectivity.
RESUMO
Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/química , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Microscopia Crioeletrônica/métodos , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Domínios Proteicos , Elementos Estruturais de ProteínasRESUMO
The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.
Assuntos
Analgesia , Receptor A1 de Adenosina/metabolismo , Adenosina/química , Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Analgesia/métodos , Animais , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/química , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Hiperalgesia/tratamento farmacológico , Lipídeos , Masculino , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor A1 de Adenosina/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) are part of the incretin system that regulates glucose homeostasis. A series of GIPR residues putatively important for ligand binding and receptor activation were mutated and pharmacologically evaluated using GIPR selective agonists in cAMP accumulation, ERK1/2 phosphorylation (pERK1/2) and ß-arrestin 2 recruitment assays. The impact of mutation on ligand efficacy was determined by operational modelling of experimental data for each mutant, with results mapped onto the full-length, active-state GIPR structure. Two interaction networks, comprising transmembrane helix (TM) 7, TM1 and TM2, and extracellular loop (ECL) 2, TM5 and ECL3 were revealed, respectively. Both networks were critical for Gαs-mediated cAMP accumulation and the recruitment of ß-arrestin 2, however, cAMP response was more sensitive to alanine substitution, with most mutated residues displaying reduced signaling. Unlike the other two assays, activation of ERK1/2 was largely independent of the network involving ECL2, TM5 and ECL3, indicating that pERK1/2 is at least partially distinct from Gαs or ß-arrestin pathways and this network is also crucial for potential biased agonism at GIPR. Collectively, our work advances understanding of the structure-function relationship of GIPR and provides a framework for the design and/or interpretation of GIP analogues with unique signaling profiles.
Assuntos
Polipeptídeo Inibidor Gástrico/genética , Polipeptídeo Inibidor Gástrico/metabolismo , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Polipeptídeo Inibidor Gástrico/química , Polipeptídeo Inibidor Gástrico/farmacologia , Células HEK293 , Humanos , Mutação/fisiologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Receptores dos Hormônios Gastrointestinais/químicaRESUMO
The glucagon-like peptide-1 receptor (GLP-1R) regulates insulin secretion, carbohydrate metabolism, and appetite and is an important target for treatment of type 2 diabetes and obesity. Multiple GLP-1R agonists have entered into clinical trials, with some, such as semaglutide, progressing to approval. Others, including taspoglutide, failed due to the high incidence of side effects or insufficient efficacy. GLP-1R agonists have a broad spectrum of signaling profiles, but molecular understanding is limited by a lack of structural information on how different agonists engage with the GLP-1R. Here, we report cryoelectron microscopy (cryo-EM) structures and cryo-EM 3D variability analysis of semaglutide- and taspoglutide-bound GLP-1R-Gs protein complexes. These reveal similar peptide interactions to GLP-1 but different motions within the receptor and bound peptides, providing insights into the molecular determinants of GLP-1R peptide engagement.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Peptídeos Semelhantes ao Glucagon/química , Peptídeos Semelhantes ao Glucagon/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Adenosine A1 receptors (A1R) are a potential target for cardiac injury treatment due to their cardioprotective/antihypertrophic actions, but drug development has been hampered by on-target side effects such as bradycardia and altered renal hemodynamics. Biased agonism has emerged as an attractive mechanism for A1R-mediated cardioprotection that is haemodynamically safe. Here we investigate the pre-clinical pharmacology, efficacy and side-effect profile of the A1R agonist neladenoson, shown to be safe but ineffective in phase IIb trials for the treatment of heart failure. We compare this agent with the well-characterized, pan-adenosine receptor (AR) agonist NECA, capadenoson, and the A1R biased agonist VCP746, previously shown to be safe and cardioprotective in pre-clinical models of heart failure. We show that like VCP746, neladenoson is biased away from Ca2+ influx relative to NECA and the cAMP pathway at the A1R, a profile predictive of a lack of adenosine-like side effects. Additionally, neladenoson was also biased away from the MAPK pathway at the A1R. In contrast to VCP746, which displays more 'adenosine-like' signaling at the A2BR, neladenoson was a highly selective A1R agonist, with biased, weak agonism at the A2BR. Together these results show that unwanted hemodynamic effects of A1R agonists can be avoided by compounds biased away from Ca2+ influx relative to cAMP, relative to NECA. The failure of neladenoson to reach primary endpoints in clinical trials suggests that A1R-mediated cAMP inhibition may be a poor indicator of effectiveness in chronic heart failure. This study provides additional information that can aid future screening and/or design of improved AR agonists that are safe and efficacious in treating heart failure in patients.
RESUMO
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.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Animais , Sítios de Ligação/fisiologia , Microscopia Crioeletrônica/métodos , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Humanos , Peptídeos/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas G/química , Receptores dos Hormônios Gastrointestinais/química , Secretina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , Microscopia Crioeletrônica , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Insetos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores dos Hormônios Gastrointestinais/ultraestrutura , Secretina/metabolismoRESUMO
Metabolic diseases such as obesity, diabetes, and their comorbidities have converged as one of the most serious health concerns on a global scale. Selective glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists are one of the major therapeutics for type 2 diabetes and obesity. Polypharmacological approaches that enable modulation of multiple metabolic targets in a single drug have emerged as a potential avenue to improve therapeutic outcomes. Among numerous peptides under development are those targeting the GLP-1R and either the glucagon receptor (GCGR), glucose-dependent insulinotropic peptide receptor (GIPR) or all 3 receptors, as dual- or tri- peptide agonists. Despite many of them entering into clinical trials, current development has been based on only a limited understanding of the spectrum of potential pharmacological properties of these ligands beyond binding selectivity. In the present study, we examined the potential for agonists that target both GLP-1R and GCGR to exhibit biased agonism, comparing activity across proximal activation of Gs protein, cAMP accumulation, pERK1/2 and ß-arrestin recruitment. Three distinct dual agonists that have different relative cAMP production potency for GLP-1R versus GCGR, "peptide 15", MEDI0382 and SAR425899, and one triagonist of the GLP-1R, GCGR and GIPR were examined. We demonstrated that all novel peptides have distinct biased agonism profiles relative to either of the cognate agonists of the receptors, and to each other. This is an important feature of the pharmacology of this drug class that needs to be considered alongside selectivity, bioavailability and pharmacokinetics for rational optimization of new therapeutics.