RÉSUMÉ
Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.
Sujet(s)
Peptides , Récepteur de la mélanocortine de type 4 , Humains , Peptides/pharmacologie , Ligands , Conception de médicament , Récepteur de la mélanocortine de type 3 , Récepteurs à la mélanocortineRÉSUMÉ
The hydroxycarboxylic acid receptor 2 (HCA2) agonist niacin has been used as treatment for dyslipidemia for several decades albeit with skin flushing as a common side-effect in treated individuals. Extensive efforts have been made to identify HCA2 targeting lipid lowering agents with fewer adverse effects, despite little being known about the molecular basis of HCA2 mediated signalling. Here, we report the cryo-electron microscopy structure of the HCA2-Gi signalling complex with the potent agonist MK-6892, along with crystal structures of HCA2 in inactive state. These structures, together with comprehensive pharmacological analysis, reveal the ligand binding mode and activation and signalling mechanisms of HCA2. This study elucidates the structural determinants essential for HCA2 mediated signalling and provides insights into ligand discovery for HCA2 and related receptors.
Sujet(s)
Acide nicotinique , Humains , Acide nicotinique/pharmacologie , Ligands , Cryomicroscopie électronique , Transduction du signal , Récepteurs couplés aux protéines G/métabolismeRÉSUMÉ
GPR20 is a class-A orphan G protein-coupled receptor (GPCR) and a potential therapeutic target for gastrointestinal stromal tumors (GIST) owing to its differentially high expression. An antibody-drug conjugate (ADC) containing a GPR20-binding antibody (Ab046) was recently developed in clinical trials for GIST treatment. GPR20 constitutively activates Gi proteins in the absence of any known ligand, but it remains obscure how this high basal activity is achieved. Here we report three cryo-EM structures of human GPR20 complexes including Gi-coupled GPR20 in the absence or presence of the Fab fragment of Ab046 and Gi-free GPR20. Remarkably, the structures demonstrate a uniquely folded N-terminal helix capping onto the transmembrane domain and our mutagenesis study suggests a key role of this cap region in stimulating the basal activity of GPR20. We also uncover the molecular interactions between GPR20 and Ab046, which may enable the design of tool antibodies with enhanced affinity or new functionality for GPR20. Furthermore, we report the orthosteric pocket occupied by an unassigned density which might be essential for exploring opportunities for deorphanization.
RÉSUMÉ
Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson's disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds.
Sujet(s)
Nucléosides , Récepteur A2A à l'adénosine , Antagonistes des récepteurs A2 à l'adénosine/composition chimique , Antagonistes des récepteurs A2 à l'adénosine/pharmacologie , Cristallographie aux rayons X , Conformation moléculaire , Récepteur A2A à l'adénosine/composition chimique , ThiophènesRÉSUMÉ
The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AAR) has been the subject of much research in recent years. In this paper, we report the design, synthesis and pharmacological analysis of quinazoline derivatives as A2AAR antagonists with high ligand efficiency. This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM-241385. More precisely, we identified a series of 2-aminoquinazoline as promising A2AAR antagonists. Among them, one compound showed a high affinity towards A2AAR (21a, Ki = 20 nM). We crystallized this ligand in complex with A2AAR, confirming one of our predicted docking poses and opening up possibilities for further optimization to derive selective ligands for specific adenosine receptor subtypes.
Sujet(s)
Antagonistes des récepteurs A2 à l'adénosine , Antagonistes des récepteurs purinergiques P1 , Antagonistes des récepteurs A2 à l'adénosine/composition chimique , Antagonistes des récepteurs A2 à l'adénosine/pharmacologie , Ligands , Simulation de docking moléculaire , Antagonistes des récepteurs purinergiques P1/pharmacologie , Quinazolines/pharmacologie , Récepteur A2A à l'adénosine/composition chimique , Relation structure-activitéRÉSUMÉ
Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT2R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT2R. We repurposed EMA401, an AT2R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT2R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT2R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or ß-arrestin recruitment. The architecture and interactions of EMA401 in AT2R differ drastically from complexes of AT2R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2-tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.
Sujet(s)
Antagonistes du récepteur de type 2 de l'angiotensine-II , Composés benzhydryliques , Tumeurs du cerveau , Repositionnement des médicaments , Glioblastome , Isoquinoléines , Récepteur de type 2 à l'angiotensine-II , Analgésiques/pharmacologie , Angiotensine-II/composition chimique , Angiotensine-II/pharmacologie , Antagonistes du récepteur de type 2 de l'angiotensine-II/usage thérapeutique , Apoptose , Composés benzhydryliques/composition chimique , Composés benzhydryliques/pharmacologie , Composés benzhydryliques/usage thérapeutique , Tumeurs du cerveau/traitement médicamenteux , Glioblastome/traitement médicamenteux , Humains , Isoquinoléines/composition chimique , Isoquinoléines/pharmacologie , Isoquinoléines/usage thérapeutique , Structure en hélice alpha , Récepteur de type 2 à l'angiotensine-II/composition chimique , Récepteur de type 2 à l'angiotensine-II/métabolisme , Charge tumorale/effets des médicaments et des substances chimiquesRÉSUMÉ
The technique of cryogenic-electron microscopy (cryo-EM) has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species. This report describes the structural characterization of a fully active human apelin receptor (APJR) complexed with heterotrimeric G protein observed in both 2:1 and 1:1 stoichiometric ratios. We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation. Protein preparations, in the presence of the endogenous peptide ligand ELA or a synthetic small molecule, both demonstrate these mixed stoichiometric states. Structural differences in G protein engagement between dimeric and monomeric APJR suggest a role for the stoichiometry of G protein-coupled receptor- (GPCR-)G protein coupling on downstream signaling and receptor pharmacology. Furthermore, a small, hydrophobic dimer interface provides a starting framework for additional class A GPCR dimerization studies. Together, these findings uncover a mechanism of versatile regulation through oligomerization by which GPCRs can modulate their signaling.
Sujet(s)
Protéines G , Récepteurs couplés aux protéines G , Récepteur de l'apeline/composition chimique , Récepteur de l'apeline/métabolisme , Protéines de transport/métabolisme , Protéines G/métabolisme , Humains , Récepteurs couplés aux protéines G/composition chimique , Transduction du signalRÉSUMÉ
γ-Aminobutyric acid (GABA) transporter 1 (GAT1)1 regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy2. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine3, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition4. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.
Sujet(s)
Transporteurs de GABA , Inhibiteurs de la capture du GABA , Acide gamma-amino-butyrique , Anticonvulsivants/composition chimique , Anticonvulsivants/pharmacologie , Cryomicroscopie électronique , Transporteurs de GABA/composition chimique , Transporteurs de GABA/métabolisme , Transporteurs de GABA/ultrastructure , Inhibiteurs de la capture du GABA/composition chimique , Inhibiteurs de la capture du GABA/pharmacologie , Humains , Agents neuromédiateurs/métabolisme , Conformation des protéines/effets des médicaments et des substances chimiques , Tiagabine/composition chimique , Tiagabine/métabolisme , Tiagabine/pharmacologie , Acide gamma-amino-butyrique/métabolismeRÉSUMÉ
The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.
Sujet(s)
Hypoglycémiants/composition chimique , Récepteurs aux leucotriènes B4/ultrastructure , Animaux , Sites de fixation/génétique , Cristallographie aux rayons X , Diabète de type 2 , Cellules HEK293 , Humains , Hypoglycémiants/pharmacologie , Hypoglycémiants/usage thérapeutique , Ligands , Simulation de docking moléculaire , Mutagenèse dirigée , Récepteurs aux leucotriènes B4/agonistes , Récepteurs aux leucotriènes B4/antagonistes et inhibiteurs , Récepteurs aux leucotriènes B4/génétique , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Protéines recombinantes/ultrastructure , Cellules Sf9 , Spodoptera , Relation structure-activitéRÉSUMÉ
Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A2A receptor (A2AAR) at 2.0â Å resolution is determined and compared with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
RÉSUMÉ
We report the development of the orthogonal amber-suppressor pair Archaeoglobus fulgidus seryl-tRNA (Af-tRNASer)/Methanosarcina mazei seryl-tRNA synthetase (MmSerRS) in Escherichia coli. Furthermore, the crystal structure of MmSerRS was solved at 1.45 Å resolution, which should enable structure-guided engineering of its active site to genetically encode small, polar noncanonical amino acids (ncAAs).
Sujet(s)
Acides aminés/métabolisme , Escherichia coli/métabolisme , ARN de transfert/métabolisme , Serine-tRNA ligase/métabolisme , Acides aminés/génétique , Archaeoglobus fulgidus/enzymologie , Methanosarcina/enzymologie , Ingénierie des protéines , ARN de transfert/composition chimique , Serine-tRNA ligase/composition chimiqueRÉSUMÉ
Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis1. GABAB belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits2. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABAB. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.
Sujet(s)
Cryomicroscopie électronique , Récepteurs GABA-B/composition chimique , Récepteurs GABA-B/ultrastructure , Régulation allostérique/effets des médicaments et des substances chimiques , Apoprotéines/composition chimique , Apoprotéines/métabolisme , Apoprotéines/ultrastructure , Sites de fixation/effets des médicaments et des substances chimiques , Agonistes du recepteur GABA-B/composition chimique , Agonistes du recepteur GABA-B/métabolisme , Agonistes du recepteur GABA-B/pharmacologie , Humains , Modèles moléculaires , Domaines protéiques/effets des médicaments et des substances chimiques , Multimérisation de protéines/effets des médicaments et des substances chimiques , Récepteurs GABA-B/métabolisme , Transduction du signal , Relation structure-activitéRÉSUMÉ
The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte-stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)-independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein-coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.
Sujet(s)
Calcium/composition chimique , Récepteur de la mélanocortine de type 4/composition chimique , Récepteurs couplés aux protéines G/composition chimique , Cristallographie aux rayons X , AMP cyclique/composition chimique , Humains , Ligands , Hormones mélanotropes/composition chimique , Hormones mélanotropes/pharmacologie , Mutation , Canaux potassiques rectifiants entrants/composition chimique , Liaison aux protéines , Multimérisation de protéines , Structure secondaire des protéines , Récepteur de la mélanocortine de type 4/antagonistes et inhibiteurs , Récepteur de la mélanocortine de type 4/génétique , Récepteurs couplés aux protéines G/antagonistes et inhibiteurs , Récepteurs couplés aux protéines G/génétique , Transduction du signalRÉSUMÉ
Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family.
Sujet(s)
Récepteur du peptide-1 similaire au glucagon/composition chimique , Séquence d'acides aminés , Apoprotéines/composition chimique , Disulfures/métabolisme , Récepteur du peptide-1 similaire au glucagon/ultrastructure , Humains , Ligands , Simulation de dynamique moléculaire , Conformation des protéines , Stabilité protéique , Récepteurs au glucagon/composition chimiqueRÉSUMÉ
GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington's disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52.
Sujet(s)
Récepteurs couplés aux protéines G/composition chimique , Récepteurs couplés aux protéines G/métabolisme , Régulation allostérique , Site allostérique , Motifs d'acides aminés , Séquence d'acides aminés , Apoprotéines/agonistes , Apoprotéines/composition chimique , Apoprotéines/métabolisme , Sites de fixation , Cryomicroscopie électronique , Cristallographie aux rayons X , Sous-unités alpha Gs des protéines G/composition chimique , Sous-unités alpha Gs des protéines G/métabolisme , Sous-unités alpha Gs des protéines G/ultrastructure , Humains , Ligands , Modèles moléculaires , Récepteurs couplés aux protéines G/agonistes , Récepteurs couplés aux protéines G/ultrastructureRÉSUMÉ
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding, and understanding this diversity is key for designing selective or biased drugs for them. Here, we determine two crystal structures of the α2A adrenergic receptor (α2AAR) in complex with a partial agonist and an antagonist. Key non-conserved residues from the ligand-binding pocket (Phe7.39 and Tyr6.55) to G-protein coupling region (Ile34.51 and Lys34.56) are discovered to play a key role in the interplay between partial agonism and biased signaling of α2AAR, which provides insights into the diversity of ligand binding and G-protein coupling preference of adrenergic receptors and lays the foundation for the discovery of next-generation drugs targeting these receptors.
Sujet(s)
Récepteurs alpha-2 adrénergiques/métabolisme , Animaux , Sites de fixation/physiologie , Cellules CHO , Lignée cellulaire , Cricetulus , Protéines G/métabolisme , Cellules HEK293 , Humains , Ligands , Liaison aux protéines/physiologie , Transduction du signal/physiologieRÉSUMÉ
Adrenergic G-protein-coupled receptors (GPCRs) mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions. Extensive studies have been carried out to investigate the structure and function of ß adrenergic receptors (ßARs). However, the structure of α adrenergic receptors (αARs) remains to be determined. Here, we report the structure of the human α2C adrenergic receptor (α2CAR) with the non-selective antagonist, RS79948, at 2.8 Å. Our structure, mutations, modeling, and functional experiments indicate that a α2CAR-specific D206ECL2-R409ECL3-Y4056.58 network plays a role in determining α2 adrenergic subtype selectivity. Furthermore, our results show that a specific loosened helix at the top of TM4 in α2CAR is involved in receptor activation. Together, our structure of human α2CAR-RS79948 provides key insight into the mechanism underlying the α2 adrenergic receptor activation and subtype selectivity.