RESUMEN
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.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/química , Péptidos/farmacología , Ponzoñas/farmacología , Animales , Células CHO , Calcio/metabolismo , Línea Celular , Cricetulus , AMP Cíclico/metabolismo , Exenatida , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxintomodulina/química , Oxintomodulina/metabolismo , Péptidos/química , Ratas , Transducción de Señal , Ponzoñas/químicaRESUMEN
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.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Animales , Sitios de Unión/fisiología , Microscopía por Crioelectrón/métodos , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/química , Humanos , Péptidos/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.
Asunto(s)
Colesterol , Proteínas de Unión al GTP , Unión Proteica , Receptor de Colecistoquinina A , Receptor de Colecistoquinina B , Animales , Humanos , Células CHO , Colesterol/metabolismo , Cricetulus , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Células HEK293 , Ligandos , Mutación , Conformación Proteica , Receptor de Colecistoquinina A/metabolismo , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina B/metabolismo , Receptor de Colecistoquinina B/genéticaRESUMEN
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.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Isoquinolinas/farmacología , Fenilalanina/análogos & derivados , Piridinas/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Receptor del Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Isoquinolinas/química , Cinética , Modelos Moleculares , Fenilalanina/química , Fenilalanina/farmacología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Piridinas/química , Homología Estructural de ProteínaRESUMEN
Wnt/ß-catenin signaling is activated when extracellular Wnt ligands bind Frizzled (FZD) receptors at the cell membrane. Wnts bind FZD cysteine-rich domains (CRDs) with high affinity through a palmitoylated N-terminal "thumb" and a disulfide-stabilized C-terminal "index finger," yet how these binding events trigger receptor activation and intracellular signaling remains unclear. Here we report the crystal structure of the Frizzled-4 (FZD4) CRD in complex with palmitoleic acid, which reveals a CRD tetramer consisting of two cross-braced CRD dimers. Each dimer is stabilized by interactions of one hydrophobic palmitoleic acid tail with two CRD palmitoleoyl-binding grooves oriented end to end, suggesting that the Wnt palmitoleoyl group stimulates CRD-CRD interaction. Using bioluminescence resonance energy transfer (BRET) in live cells, we show that WNT5A stimulates dimerization of membrane-anchored FZD4 CRDs and oligomerization of full-length FZD4, which requires the integrity of CRD palmitoleoyl-binding residues. These results suggest that FZD receptors may form signalosomes in response to Wnt binding through the CRDs and that the Wnt palmitoleoyl group is important in promoting these interactions. These results complement our understanding of lipoprotein receptor-related proteins 5 and 6 (LRP5/6), Dishevelled, and Axin signalosome assembly and provide a more complete model for Wnt signalosome assembly both intracellularly and at the membrane.
Asunto(s)
Cisteína/química , Ácidos Grasos Monoinsaturados/química , Receptores Frizzled/química , Proteína Wnt-5a/metabolismo , Cristalografía por Rayos X , Cisteína/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Dimerización , Conformación Proteica , Receptores Acoplados a Proteínas G/químicaRESUMEN
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.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/metabolismo , Colecistoquinina/metabolismo , Colesterol/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Receptores de Colecistoquinina/ultraestructura , Transducción de SeñalRESUMEN
Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the Gs-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteína Similar al Receptor de Calcitonina/ultraestructura , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Proteína 1 Modificadora de la Actividad de Receptores/ultraestructura , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/ultraestructura , Sitios de Unión , Péptido Relacionado con Gen de Calcitonina/química , Proteína Similar al Receptor de Calcitonina/química , Proteína Similar al Receptor de Calcitonina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Humanos , Simulación de Dinámica Molecular , Dominios Proteicos , Estabilidad Proteica , Proteína 1 Modificadora de la Actividad de Receptores/química , Proteína 1 Modificadora de la Actividad de Receptores/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/química , Proteínas ras/química , Proteínas ras/metabolismoRESUMEN
The class B glucagon-like peptide-1 (GLP-1) G protein-coupled receptor is a major target for the treatment of type 2 diabetes and obesity. Endogenous and mimetic GLP-1 peptides exhibit biased agonism-a difference in functional selectivity-that may provide improved therapeutic outcomes. Here we describe the structure of the human GLP-1 receptor in complex with the G protein-biased peptide exendin-P5 and a Gαs heterotrimer, determined at a global resolution of 3.3 Å. At the extracellular surface, the organization of extracellular loop 3 and proximal transmembrane segments differs between our exendin-P5-bound structure and previous GLP-1-bound GLP-1 receptor structure. At the intracellular face, there was a six-degree difference in the angle of the Gαs-α5 helix engagement between structures, which was propagated across the G protein heterotrimer. In addition, the structures differed in the rate and extent of conformational reorganization of the Gαs protein. Our structure provides insights into the molecular basis of biased agonism.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/ultraestructura , Sitios de Unión , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/química , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.
Asunto(s)
Receptores Frizzled/química , Sitios de Unión , Cristalografía por Rayos X , Cisteína/metabolismo , Proteínas Dishevelled/metabolismo , Receptores Frizzled/genética , Humanos , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Vía de Señalización WntRESUMEN
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.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Desarrollo de Medicamentos , Receptores Acoplados a Proteínas G/agonistas , Sitio Alostérico , Humanos , Ligandos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαsßγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gß subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Unión al GTP Heterotriméricas/ultraestructura , Receptores de Calcitonina/clasificación , Receptores de Calcitonina/ultraestructura , Sitios de Unión , Membrana Celular/metabolismo , Secuencia Conservada , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Modelos Moleculares , Conformación Proteica , Receptores de Calcitonina/agonistas , Receptores de Calcitonina/metabolismoRESUMEN
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.
Asunto(s)
Receptores Acoplados a Proteínas G , Secretina , Secuencia de Aminoácidos , Mutagénesis , Péptidos/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de la Hormona Gastrointestinal , Secretina/química , Secretina/genética , Secretina/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Péptido Intestinal Vasoactivo , Secuencia de Aminoácidos , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Transducción de Señal , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-Lrp5/6 signaling complex.
Asunto(s)
Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Receptores Frizzled/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Animales , Sitios de Unión , Células COS , Cristalografía por Rayos X , Dimerización , Proteínas del Ojo/genética , Receptores Frizzled/química , Células HEK293 , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Tetraspaninas/metabolismo , Factor de Crecimiento Transformador beta/química , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Dispepsia/tratamiento farmacológico , Vaciamiento Gástrico/efectos de los fármacos , Secretina/farmacología , Adulto , Estudios Cruzados , Sacarosa en la Dieta , Método Doble Ciego , Femenino , Alimentos Formulados , Humanos , Masculino , Comidas , Persona de Mediana Edad , Periodo Posprandial , SaciedadRESUMEN
Allosteric modulation of receptors provides mechanistic safety while effectively achieving biologic endpoints otherwise difficult or impossible to obtain by other means. The theoretical case has been made for the development of a positive allosteric modulator (PAM) of the type 1 cholecystokinin receptor (CCK1R) having minimal intrinsic agonist activity to enhance meal-induced satiety for the treatment of obesity, while reducing the risk of side effects and/or toxicity. Unfortunately, such a drug does not currently exist. In this work, we have identified a PAM agonist of the CCK1R, SR146131, and determined its putative binding mode and receptor activation mechanism by combining molecular modeling, chimeric CCK1R/CCK2R constructs, and site-directed mutagenesis. We probed the structure-activity relationship of analogs of SR146131 for impact on agonism versus cooperativity of the analogs. This identified structural features that might be responsible for binding affinity and potency while retaining PAM activity. SR146131 and several of its analogs were docked into the receptor structure, which had the natural endogenous peptide agonist, cholecystokinin, already in the bound state (by docking), providing a refined structural model of the intact CCK1R holoreceptor. Both SR146131 and its analogs exhibited unique probe-dependent cooperativity with orthosteric peptide agonists and were simultaneously accommodated in this model, consistent with the derived structure-activity relationships. This provides improved understanding of the molecular basis for CCK1R-directed drug development.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Colecistoquinina/metabolismo , Receptores de Colecistoquinina/agonistas , Receptores de Colecistoquinina/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células CHO , Línea Celular , Cricetulus , Indoles/farmacología , Mutagénesis Sitio-Dirigida/métodos , Péptidos/metabolismo , Relación Estructura-Actividad , Tiazoles/farmacologíaRESUMEN
Class B G protein-coupled receptors (GPCRs) respond to paracrine or endocrine peptide hormones involved in control of bone homeostasis, glucose regulation, satiety, and gastro-intestinal function, as well as pain transmission. These receptors are targets for existing drugs that treat osteoporosis, hypercalcaemia, Paget's disease, type II diabetes, and obesity and are being actively pursued as targets for numerous other diseases. Exploitation of class B receptors has been limited by difficulties with small molecule drug discovery and development and an under appreciation of factors governing optimal therapeutic efficacy. Recently, there has been increasing awareness of novel attributes of GPCR function that offer new opportunity for drug development. These include the presence of allosteric binding sites on the receptor that can be exploited as drug binding pockets and the ability of individual drugs to enrich subpopulations of receptor conformations to selectively control signaling, a phenomenon termed biased agonism. In this review, current knowledge of biased signaling and small molecule allostery within class B GPCRs is discussed, highlighting areas that have progressed significantly over the past decade, in addition to those that remain largely unexplored with respect to these phenomena.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Regulación Alostérica , Dimerización , Receptores Acoplados a Proteínas G/agonistas , Relación Estructura-ActividadRESUMEN
The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain-binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders.
Asunto(s)
Péptido 1 Similar al Glucagón , Receptores Acoplados a Proteínas G , Animales , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
One of the hallmarks of Alzheimer's disease is the formation of extracellular amyloid plaques that consist mainly of abnormally aggregated forms of amyloid ß (Aß) peptides. These peptides are generated by γ-secretase-catalyzed cleavage of a dimeric membrane-bound C-terminal fragment (C99) of the amyloid precursor protein. Although C99 homodimerization has been linked to Aß production and changes in the aggregation-determining Aß42/Aß40 ratio, the motif through which C99 dimerizes has remained controversial. Here, we have used two independent assays to gain insight into C99 homodimerization in the context of the membrane of live cells: bioluminescence resonance energy transfer and Tango membrane protein-protein interaction assays, which were further confirmed by traditional pull-down assays. Our results indicate a four-amino acid region within the C99 transmembrane helix (43TVIV46) as well as its local secondary structure as critical determinants for homodimerization. These four amino acids are also a hot spot of familial Alzheimer's disease-linked mutations that both decrease C99 homodimerization and γ-secretase cleavage and alter the initial cleavage site to increase the Aß42/40 ratio.