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1
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Claff T, Mahardhika AB, Vaaßen VJ, Schlegel J, Vielmuth C, Weiße RH, Sträter N, Müller CE. Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A 2A Receptor. ACS Pharmacol Transl Sci 2024; 7:1415-1425. [PMID: 38751633 PMCID: PMC11091970 DOI: 10.1021/acsptsci.4c00051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2024] [Revised: 02/29/2024] [Accepted: 03/01/2024] [Indexed: 05/18/2024]
Abstract
The adenosine A2A receptor (A2AAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A2AAR agonist 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-p-hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A2AAR construct A2A-PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p-hydroxy group of LUF5834 with T883.36 was observed, an amino acid that was mutated to alanine in the most frequently used A2AAR crystallization constructs thus preventing the discovery of its interactions in most of the previous A2AAR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N-ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S913.39K mutation, although with an affinity lower than that at the native receptor. This could explain the compound's partial agonistic activity: while full A2AAR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs.
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Affiliation(s)
- Tobias Claff
- PharmaCenter
Bonn & Pharmaceutical Institute, Department of Pharmaceutical
& Medicinal Chemistry, University of
Bonn, Bonn 53113, Germany
| | - Andhika B. Mahardhika
- PharmaCenter
Bonn & Pharmaceutical Institute, Department of Pharmaceutical
& Medicinal Chemistry, University of
Bonn, Bonn 53113, Germany
- Research
Training Group 2873, University of Bonn, Bonn 53121, Germany
| | - Victoria J. Vaaßen
- PharmaCenter
Bonn & Pharmaceutical Institute, Department of Pharmaceutical
& Medicinal Chemistry, University of
Bonn, Bonn 53113, Germany
| | - Jonathan
G. Schlegel
- PharmaCenter
Bonn & Pharmaceutical Institute, Department of Pharmaceutical
& Medicinal Chemistry, University of
Bonn, Bonn 53113, Germany
| | - Christin Vielmuth
- PharmaCenter
Bonn & Pharmaceutical Institute, Department of Pharmaceutical
& Medicinal Chemistry, University of
Bonn, Bonn 53113, Germany
| | - Renato H. Weiße
- Institute
of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Leipzig 04103, Germany
| | - Norbert Sträter
- Institute
of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Leipzig 04103, Germany
| | - Christa E. Müller
- PharmaCenter
Bonn & Pharmaceutical Institute, Department of Pharmaceutical
& Medicinal Chemistry, University of
Bonn, Bonn 53113, Germany
- Research
Training Group 2873, University of Bonn, Bonn 53121, Germany
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2
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Nucleoside transporters and immunosuppressive adenosine signaling in the tumor microenvironment: Potential therapeutic opportunities. Pharmacol Ther 2022; 240:108300. [PMID: 36283452 DOI: 10.1016/j.pharmthera.2022.108300] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2022] [Revised: 10/17/2022] [Accepted: 10/19/2022] [Indexed: 11/30/2022]
Abstract
Adenosine compartmentalization has a profound impact on immune cell function by regulating adenosine localization and, therefore, extracellular signaling capabilities, which suppresses immune cell function in the tumor microenvironment. Nucleoside transporters, responsible for the translocation and cellular compartmentalization of hydrophilic adenosine, represent an understudied yet crucial component of adenosine disposition in the tumor microenvironment. In this review article, we will summarize what is known regarding nucleoside transporter's function within the purinome in relation to currently devised points of intervention (i.e., ectonucleotidases, adenosine receptors) for cancer immunotherapy, alterations in nucleoside transporter expression reported in cancer, and potential avenues for targeting of nucleoside transporters for the desired modulation of adenosine compartmentalization and action. Further, we put forward that nucleoside transporters are an unexplored therapeutic opportunity, and modulation of nucleoside transport processes could attenuate the pathogenic buildup of immunosuppressive adenosine in solid tumors, particularly those enriched with nucleoside transport proteins.
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3
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Raynaud P, Gauthier C, Jugnarain V, Jean-Alphonse F, Reiter E, Bruneau G, Crépieux P. Intracellular VHHs to monitor and modulate GPCR signaling. Front Endocrinol (Lausanne) 2022; 13:1048601. [PMID: 36465650 PMCID: PMC9708903 DOI: 10.3389/fendo.2022.1048601] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/19/2022] [Accepted: 10/24/2022] [Indexed: 11/17/2022] Open
Abstract
Single-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reflects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientific community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010's. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or β-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the field of GPCR signaling and trafficking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery.
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Affiliation(s)
- Pauline Raynaud
- Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Institut Français du Cheval et de l’Equitation (IFCE), Université de Tours, Nouzilly, France
| | - Camille Gauthier
- Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Institut Français du Cheval et de l’Equitation (IFCE), Université de Tours, Nouzilly, France
| | - Vinesh Jugnarain
- Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Institut Français du Cheval et de l’Equitation (IFCE), Université de Tours, Nouzilly, France
| | - Frédéric Jean-Alphonse
- Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Institut Français du Cheval et de l’Equitation (IFCE), Université de Tours, Nouzilly, France
- Inria, Inria Saclay-Ile-de-France, Palaiseau, France
| | - Eric Reiter
- Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Institut Français du Cheval et de l’Equitation (IFCE), Université de Tours, Nouzilly, France
- Inria, Inria Saclay-Ile-de-France, Palaiseau, France
| | - Gilles Bruneau
- Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Institut Français du Cheval et de l’Equitation (IFCE), Université de Tours, Nouzilly, France
| | - Pascale Crépieux
- Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Institut Français du Cheval et de l’Equitation (IFCE), Université de Tours, Nouzilly, France
- Inria, Inria Saclay-Ile-de-France, Palaiseau, France
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4
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Yeast-based directed-evolution for high-throughput structural stabilization of G protein-coupled receptors (GPCRs). Sci Rep 2022; 12:8657. [PMID: 35606532 PMCID: PMC9126886 DOI: 10.1038/s41598-022-12731-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Accepted: 05/10/2022] [Indexed: 11/08/2022] Open
Abstract
The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we present a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The yeast detergent-resistant cell wall presents a unique opportunity for compartmentalization, to physically link the receptor's phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method reveals a surprising amenability of the GPCR scaffold to stabilization, and suggests an intriguing possibility of amending the stability properties of GPCR by varying the structural status of the C-terminus.
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5
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Conrad M, Söldner CA, Miao Y, Sticht H. Agonist Binding and G Protein Coupling in Histamine H 2 Receptor: A Molecular Dynamics Study. Int J Mol Sci 2020; 21:ijms21186693. [PMID: 32932742 PMCID: PMC7554837 DOI: 10.3390/ijms21186693] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Revised: 09/04/2020] [Accepted: 09/08/2020] [Indexed: 02/06/2023] Open
Abstract
The histamine H2 receptor (H2R) plays an important role in the regulation of gastric acid secretion. Therefore, it is a main drug target for the treatment of gastroesophageal reflux or peptic ulcer disease. However, there is as of yet no 3D-structural information available hampering a mechanistic understanding of H2R. Therefore, we created a model of the histamine-H2R-Gs complex based on the structure of the ternary complex of the β2-adrenoceptor and investigated the conformational stability of this active GPCR conformation. Since the physiologically relevant motions with respect to ligand binding and conformational changes of GPCRs can only partly be assessed on the timescale of conventional MD (cMD) simulations, we also applied metadynamics and Gaussian accelerated molecular dynamics (GaMD) simulations. A multiple walker metadynamics simulation in combination with cMD was applied for the determination of the histamine binding mode. The preferential binding pose detected is in good agreement with previous data from site directed mutagenesis and provides a basis for rational ligand design. Inspection of the H2R-Gs interface reveals a network of polar interactions that may contribute to H2R coupling selectivity. The cMD and GaMD simulations demonstrate that the active conformation is retained on a μs-timescale in the ternary histamine-H2R-Gs complex and in a truncated complex that contains only Gs helix α5 instead of the entire G protein. In contrast, histamine alone is unable to stabilize the active conformation, which is in line with previous studies of other GPCRs.
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Affiliation(s)
- Marcus Conrad
- Bioinformatik, Institut für Biochemie, Emil-Fischer-Centrum, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Fahrstraße 17, 91054 Erlangen, Germany; (M.C.); (C.A.S.)
| | - Christian A. Söldner
- Bioinformatik, Institut für Biochemie, Emil-Fischer-Centrum, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Fahrstraße 17, 91054 Erlangen, Germany; (M.C.); (C.A.S.)
| | - Yinglong Miao
- Department of Computational Biology and Molecular Biosciences, University of Kansas, Lawrence, KS 66047, USA;
| | - Heinrich Sticht
- Bioinformatik, Institut für Biochemie, Emil-Fischer-Centrum, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Fahrstraße 17, 91054 Erlangen, Germany; (M.C.); (C.A.S.)
- Correspondence:
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6
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Lau CL, Beller JP, Boys JA, Zhao Y, Phillips J, Cosner M, Conaway MR, Petroni G, Charles EJ, Mehaffey JH, Mannem HC, Kron IL, Krupnick AS, Linden J. Adenosine A2A receptor agonist (regadenoson) in human lung transplantation. J Heart Lung Transplant 2020; 39:563-570. [PMID: 32503727 DOI: 10.1016/j.healun.2020.02.003] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2019] [Revised: 12/20/2019] [Accepted: 02/06/2020] [Indexed: 10/25/2022] Open
Abstract
BACKGROUND Currently, there are no clinically approved treatments for ischemia-reperfusion injury after lung transplantation. Pre-clinical animal models have demonstrated a promising efficacy of adenosine 2A receptor (A2AR) agonists as a treatment option for reducing ischemia-reperfusion injury. The purpose of this human study, is to conduct a Phase I clinical trial for evaluating the safety of continuous infusion of an A2AR agonist in lung transplant recipients. METHODS An adaptive, two-stage continual reassessment trial was designed to evaluate the safety of regadenoson (A2AR agonist) in the setting of lung transplantation. Continuous infusion of regadenoson was administered to lung transplant recipients that was started at the time of skin incision. Adverse events and dose-limiting toxicities, as pre-determined by a study team and assessed by a clinical team and an independent safety monitor, were the primary end-points for safety in this trial. RESULTS Between January 2018 and March 2019, 14 recipients were enrolled in the trial. Of these, 10 received the maximum infused dose of 1.44 µg/kg/min for 12 hours. No dose-limiting toxicities were observed. The steady-state plasma regadenoson levels sampled before the reperfusion of the first lung were 0.98 ± 0.46 ng/ml. There were no mortalities within 30 days. CONCLUSIONS Regadenoson, an A2AR agonist, can be safely infused in the setting of lung transplantation with no dose-limiting toxicities or drug-related mortality. Although not powered for the evaluation of secondary end-points, the results of this trial and the outcome of pre-clinical studies warrant further investigation with a Phase II randomized controlled trial.
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Affiliation(s)
- Christine L Lau
- Department of Surgery, University of Maryland, Baltimore, Maryland.
| | - Jared P Beller
- Department of Surgery, University of Virginia, Charlottesville, Virginia
| | - Joshua A Boys
- Division of Cardiovascular and Thoracic Surgery, Department of Surgery, University of California, San Diego, California
| | - Yunge Zhao
- Department of Surgery, University of Maryland, Baltimore, Maryland
| | - Jennifer Phillips
- Department of Surgery, University of Virginia, Charlottesville, Virginia
| | - Michael Cosner
- Department of Surgery, University of Virginia, Charlottesville, Virginia
| | - Mark R Conaway
- Public Health Sciences, University of Virginia School of Medicine, Charlottesville, Virginia
| | - Gina Petroni
- Public Health Sciences, University of Virginia School of Medicine, Charlottesville, Virginia
| | - Eric J Charles
- Department of Surgery, University of Virginia, Charlottesville, Virginia
| | - J H Mehaffey
- Department of Surgery, University of Virginia, Charlottesville, Virginia
| | - Hannah C Mannem
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Virginia, Charlottesville, Virginia
| | - Irving L Kron
- Department of Surgery, University of Virginia, Charlottesville, Virginia; Department of Surgery, University of Arizona Health Sciences, Tucson, Arizona
| | | | - Joel Linden
- Division of Developmental Immunology, La Jolla Institute for Immunology and Department of Pharmacology, University of California, San Diego, California
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7
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Reconstruction of apo A2A receptor activation pathways reveal ligand-competent intermediates and state-dependent cholesterol hotspots. Sci Rep 2019; 9:14199. [PMID: 31578448 PMCID: PMC6775061 DOI: 10.1038/s41598-019-50752-6] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Accepted: 08/27/2019] [Indexed: 02/07/2023] Open
Abstract
G-protein coupled receptors (GPCRs) play a pivotal role in transmitting signals at the cellular level. Structural insights can be exploited to support GPCR structure-based drug discovery endeavours. Despite advances in GPCR crystallography, active state structures are scarce. Molecular dynamics (MD) simulations have been used to explore the conformational landscape of GPCRs. Efforts have been made to retrieve active state conformations starting from inactive structures, however to date this has not been possible without using an energy bias. Here, we reconstruct the activation pathways of the apo adenosine receptor (A2A), starting from an inactive conformation, by applying adaptive sampling MD combined with a goal-oriented scoring function. The reconstructed pathways reconcile well with experiments and help deepen our understanding of A2A regulatory mechanisms. Exploration of the apo conformational landscape of A2A reveals the existence of ligand-competent states, active intermediates and state-dependent cholesterol hotspots of relevance for drug discovery. To the best of our knowledge this is the first time an activation process has been elucidated for a GPCR starting from an inactive structure only, using a non-biased MD approach, opening avenues for the study of ligand binding to elusive yet pharmacologically relevant GPCR states.
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8
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Mahmod Al-Qattan MN, Mordi MN. Molecular Basis of Modulating Adenosine Receptors Activities. Curr Pharm Des 2019; 25:817-831. [DOI: 10.2174/1381612825666190304122624] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2019] [Accepted: 02/26/2019] [Indexed: 01/04/2023]
Abstract
Modulating cellular processes through extracellular chemical stimuli is medicinally an attractive approach to control disease conditions. GPCRs are the most important group of transmembranal receptors that produce different patterns of activations using intracellular mediators (such as G-proteins and Beta-arrestins). Adenosine receptors (ARs) belong to GPCR class and are divided into A1AR, A2AAR, A2BAR and A3AR. ARs control different physiological activities thus considered valuable target to control neural, heart, inflammatory and other metabolic disorders. Targeting ARs using small molecules essentially works by binding orthosteric and/or allosteric sites of the receptors. Although targeting orthosteric site is considered typical to modulate receptor activity, allosteric sites provide better subtype selectivity, saturable modulation of activity and variable activation patterns. Each receptor exists in dynamical equilibrium between conformational ensembles. The equilibrium is affected by receptor interaction with other molecules. Changing the population of conformational ensembles of the receptor is the method by which orthosteric, allosteric and other cellular components control receptor signaling. Herein, the interactions of ARs with orthosteric, allosteric ligands as well as intracellular mediators are described. A quinary interaction model for the receptor is proposed and energy wells for major conformational ensembles are retrieved.
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Affiliation(s)
| | - Mohd Nizam Mordi
- Centre For Drug Research, Universiti Sains Malaysia, 11800 Gelugor, Penang, Malaysia
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9
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Lee S, Nivedha AK, Tate CG, Vaidehi N. Dynamic Role of the G Protein in Stabilizing the Active State of the Adenosine A 2A Receptor. Structure 2019; 27:703-712.e3. [PMID: 30713025 PMCID: PMC6531377 DOI: 10.1016/j.str.2018.12.007] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2018] [Revised: 07/28/2018] [Accepted: 12/09/2018] [Indexed: 11/20/2022]
Abstract
Agonist binding in the extracellular region of the G protein-coupled adenosine A2A receptor increases its affinity to the G proteins in the intracellular region, and vice versa. The structural basis for this effect is not evident from the crystal structures of A2AR in various conformational states since it stems from the receptor dynamics. Using atomistic molecular dynamics simulations on four different conformational states of the adenosine A2A receptor, we observed that the agonists show decreased ligand mobility, lower entropy of the extracellular loops in the active-intermediate state compared with the inactive state. In contrast, the entropy of the intracellular region increases to prime the receptor for coupling the G protein. Coupling of the G protein to A2AR shrinks the agonist binding site, making tighter receptor agonist contacts with an increase in the strength of allosteric communication compared with the active-intermediate state. These insights provide a strong basis for structure-based ligand design studies.
GPCR conformation dynamics reveals the forward and backward allosteric mechanism Agonist binding increases the entropy in the intracellular region of the GPCR G protein binding shrinks the receptor-ligand contacts in the extracellular region Increased allostery between G protein and agonist in the GPCR-G protein complex
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Affiliation(s)
- Sangbae Lee
- Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Anita K Nivedha
- Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Christopher G Tate
- MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK
| | - Nagarajan Vaidehi
- Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA.
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10
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Carlin JL, Jain S, Duroux R, Suresh RR, Xiao C, Auchampach JA, Jacobson KA, Gavrilova O, Reitman ML. Activation of adenosine A 2A or A 2B receptors causes hypothermia in mice. Neuropharmacology 2018; 139:268-278. [PMID: 29548686 PMCID: PMC6067974 DOI: 10.1016/j.neuropharm.2018.02.035] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Revised: 01/26/2018] [Accepted: 02/28/2018] [Indexed: 12/25/2022]
Abstract
Extracellular adenosine is a danger/injury signal that initiates protective physiology, such as hypothermia. Adenosine has been shown to trigger hypothermia via agonism at A1 and A3 adenosine receptors (A1AR, A3AR). Here, we find that adenosine continues to elicit hypothermia in mice null for A1AR and A3AR and investigated the effect of agonism at A2AAR or A2BAR. The poorly brain penetrant A2AAR agonists CGS-21680 and PSB-0777 caused hypothermia, which was not seen in mice lacking A2AAR. MRS7352, a likely non-brain penetrant A2AAR antagonist, inhibited PSB-0777 hypothermia. While vasodilation is probably a contributory mechanism, A2AAR agonism also caused hypometabolism, indicating that vasodilation is not the sole mechanism. The A2BAR agonist BAY60-6583 elicited hypothermia, which was lost in mice null for A2BAR. Low intracerebroventricular doses of BAY60-6583 also caused hypothermia, indicating a brain site of action, with neuronal activation in the preoptic area and paraventricular nucleus of the hypothalamus. Thus, agonism at any one of the canonical adenosine receptors, A1AR, A2AAR, A2BAR, or A3AR, can cause hypothermia. This four-fold redundancy in adenosine-mediated initiation of hypothermia may reflect the centrality of hypothermia as a protective response.
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Affiliation(s)
- Jesse Lea Carlin
- Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA
| | - Shalini Jain
- Mouse Metabolism Core, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA
| | - Romain Duroux
- Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA
| | - R Rama Suresh
- Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA
| | - Cuiying Xiao
- Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA
| | - John A Auchampach
- Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
| | - Kenneth A Jacobson
- Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA
| | - Oksana Gavrilova
- Mouse Metabolism Core, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA.
| | - Marc L Reitman
- Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA.
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11
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Cuzzolin A, Deganutti G, Salmaso V, Sturlese M, Moro S. AquaMMapS: An Alternative Tool to Monitor the Role of Water Molecules During Protein-Ligand Association. ChemMedChem 2018; 13:522-531. [DOI: 10.1002/cmdc.201700564] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2017] [Revised: 11/21/2017] [Indexed: 12/11/2022]
Affiliation(s)
- Alberto Cuzzolin
- Molecular Modeling Section, MMS, Department of Pharmaceutical and Pharmacological Sciences; University of Padova; via Marzolo 5 35131 Padova Italy
| | - Giuseppe Deganutti
- Molecular Modeling Section, MMS, Department of Pharmaceutical and Pharmacological Sciences; University of Padova; via Marzolo 5 35131 Padova Italy
| | - Veronica Salmaso
- Molecular Modeling Section, MMS, Department of Pharmaceutical and Pharmacological Sciences; University of Padova; via Marzolo 5 35131 Padova Italy
| | - Mattia Sturlese
- Molecular Modeling Section, MMS, Department of Pharmaceutical and Pharmacological Sciences; University of Padova; via Marzolo 5 35131 Padova Italy
| | - Stefano Moro
- Molecular Modeling Section, MMS, Department of Pharmaceutical and Pharmacological Sciences; University of Padova; via Marzolo 5 35131 Padova Italy
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12
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Carpenter B, Lebon G. Human Adenosine A 2A Receptor: Molecular Mechanism of Ligand Binding and Activation. Front Pharmacol 2017; 8:898. [PMID: 29311917 PMCID: PMC5736361 DOI: 10.3389/fphar.2017.00898] [Citation(s) in RCA: 57] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Accepted: 11/24/2017] [Indexed: 11/29/2022] Open
Abstract
Adenosine receptors (ARs) comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs). ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR), making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes.
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Affiliation(s)
- Byron Carpenter
- Warwick Integrative Synthetic Biology Centre, School of Life Sciences, University of Warwick, Coventry, United Kingdom
| | - Guillaume Lebon
- Institut de Génomique Fonctionnelle, Neuroscience Department, UMR CNRS 5203, INSERM U1191, Université de Montpellier, Montpellier, France
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Du L, Gao ZG, Paoletta S, Wan TC, Gizewski ET, Barbour S, van Veldhoven JPD, IJzerman AP, Jacobson KA, Auchampach JA. Species differences and mechanism of action of A 3 adenosine receptor allosteric modulators. Purinergic Signal 2017; 14:59-71. [PMID: 29170977 DOI: 10.1007/s11302-017-9592-1] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2017] [Accepted: 11/01/2017] [Indexed: 02/01/2023] Open
Abstract
Activity of the A3 adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A3AR species homologs used in preclinical drug development. In guanosine 5'-[γ-[35S]thio]triphosphate ([35S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A3ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A3ARs but provided only weak activity at mouse A3ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A3AR agonist 2-chloro-N 6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N 6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methylcarboxamide ([125I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A3AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [125I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A3AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.
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Affiliation(s)
- Lili Du
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA
| | - Zhan-Guo Gao
- Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 200892-0810, USA
| | - Silvia Paoletta
- Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 200892-0810, USA
| | - Tina C Wan
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA
| | - Elizabeth T Gizewski
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA
| | - Samantha Barbour
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA
| | - Jacobus P D van Veldhoven
- Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, PO Box 9502, 2300 RA, Leiden, The Netherlands
| | - Adriaan P IJzerman
- Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, PO Box 9502, 2300 RA, Leiden, The Netherlands
| | - Kenneth A Jacobson
- Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 200892-0810, USA
| | - John A Auchampach
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA.
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Carpenter B, Tate CG. Active state structures of G protein-coupled receptors highlight the similarities and differences in the G protein and arrestin coupling interfaces. Curr Opin Struct Biol 2017; 45:124-132. [DOI: 10.1016/j.sbi.2017.04.010] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2016] [Revised: 04/17/2017] [Accepted: 04/20/2017] [Indexed: 10/19/2022]
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Abstract
Ligand-induced activation of G protein-coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist-bound structures of hormone-activated GPCRs and have provided insight into the dynamic character of receptors. Nanobodies have also been used to stabilize transient GPCR transmembrane signaling complexes, yielding the first structural insights into GPCR signal transduction across the cellular membrane. Beyond their in vitro uses, nanobodies have served as conformational biosensors in living systems and have provided novel ways to modulate GPCR function. Here, we highlight several examples of how nanobodies have enabled the study of GPCR function and give insights into potential future uses of these important tools.
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Affiliation(s)
- Aashish Manglik
- Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305; ,
| | - Brian K Kobilka
- Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305; ,
| | - Jan Steyaert
- Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium;
- VIB Structural Biology Research Center, Vrije Universiteit Brussel, 1050 Brussels, Belgium
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McNeely PM, Naranjo AN, Forsten-Williams K, Robinson AS. A 2AR Binding Kinetics in the Ligand Depletion Regime. SLAS DISCOVERY 2016; 22:166-175. [PMID: 27577981 DOI: 10.1177/1087057116667256] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Ligand binding plays a fundamental role in stimulating the downstream signaling of membrane receptors. Here, ligand-binding kinetics of the full-length human adenosine A2A receptor (A2AR) reconstituted in detergent micelles were measured using a fluorescently labeled ligand via fluorescence anisotropy. Importantly, to optimize the signal-to-noise ratio, these experiments were conducted in the ligand depletion regime. In the ligand depletion regime, the assumptions used to determine analytical solutions for one-site binding models for either one or two ligands in competition are no longer valid. We therefore implemented a numerical solution approach to analyze kinetic binding data as experimental conditions approach the ligand depletion regime. By comparing the results from the numerical and the analytical solutions, we highlight the ligand-receptor ratios at which the analytical solution begins to lose predictive accuracy. Using the numerical solution approach, we determined the kinetic rate constants of the fluorescent ligand, FITC-APEC, and those for three unlabeled ligands using competitive association experiments. The association and dissociation rate constants of the unlabeled ligands determined from the competitive association experiments were then independently validated using competitive dissociation data. Based on this study, a numerical solution is recommended to determine kinetic ligand-binding parameters for experiments conducted in the ligand-depletion regime.
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Affiliation(s)
- Patrick M McNeely
- 1 Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA
| | - Andrea N Naranjo
- 1 Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA
| | | | - Anne Skaja Robinson
- 1 Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA.,2 Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA, USA
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Structure of the adenosine A(2A) receptor bound to an engineered G protein. Nature 2016; 536:104-7. [PMID: 27462812 PMCID: PMC4979997 DOI: 10.1038/nature18966] [Citation(s) in RCA: 330] [Impact Index Per Article: 36.7] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2016] [Accepted: 06/23/2016] [Indexed: 12/17/2022]
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Chavez-Valdez R, Ahlawat R, Wills-Karp M, Gauda EB. Mechanisms of modulation of cytokine release by human cord blood monocytes exposed to high concentrations of caffeine. Pediatr Res 2016; 80:101-9. [PMID: 26982450 PMCID: PMC4929021 DOI: 10.1038/pr.2016.50] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/01/2015] [Accepted: 12/23/2015] [Indexed: 01/12/2023]
Abstract
BACKGROUND Serum caffeine concentrations >20 μg/ml (100 μmol/l) in infants treated for apnea of prematurity increases TNF-α and decreases IL-10, changes that perhaps are linked to comorbidities. We hypothesize that this proinflammatory cytokine profile may be linked to differential binding of caffeine to adenosine receptor subtypes (AR), inhibition of phosphodiesterases (PDEs), and modulation of toll-like receptors (TLR). METHODS Lipopolysaccharide-activated cord blood monocytes (CBM) from 19 infants were exposed to caffeine (0-200 μmol/l) with or without previous exposure to A1R, A3R, or PDE IV antagonists to determine changes in dose-response curves. Cytokines levels (enzyme-linked immunosorbent assay (ELISA)), intracellular cyclic adenosine monophosphate (cAMP) accumulation (enzyme immunoassay (EIA)), and TLR gene expression (real time qRT PCR) were measured. RESULTS Caffeine at ≤100 μmol/l decreased TNF-α levels (~25%, P = 0.01) and cAMP. All caffeine concentrations decreased IL-10 levels (17-35%, P < 0.01). A1R, A3R, and PDE blockades decreased TNF-α (31, 21, and 88%, P ≤ 0.01), but not IL-10. Caffeine further decreased TNF-α following A3R and PDE blockades. Caffeine concentrations directly correlated to TLR4 gene expression (r = 0.84; P < 0.001). CONCLUSION Neither A3R, nor PDE blockades are involved in caffeine's modulation of cytokine release by CBM at any concentration. Besides A1R blockade, caffeine's upregulation of TLR4 may promote inflammation at high concentrations.
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Affiliation(s)
- Raul Chavez-Valdez
- Department of Pediatrics, Division of Neonatology. Johns Hopkins Hospital. Johns Hopkins University - School of Medicine. Baltimore, Maryland 21287, United States,CORRESPONDING AUTHOR: Dr. Raul Chavez-Valdez, Department of Pediatrics - Division of Neonatology, Johns Hopkins Hospital, 600 N. Wolfe Street, CMSC 6-104, Baltimore, MD 21287-3200, United States. Telephone: +1 410-955 5259; Fax: +1 410 614 8388;
| | - Rajni Ahlawat
- Department of Pediatrics, Division of Pediatric Gastroenterology. North Shore LIJ Health System. Lake Success, NY 11042, United States
| | - Marsha Wills-Karp
- Department of Environmental Health Sciences. Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205
| | - Estelle B. Gauda
- Department of Pediatrics, Division of Neonatology. Johns Hopkins Hospital. Johns Hopkins University - School of Medicine. Baltimore, Maryland 21287, United States
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De Filippo E, Namasivayam V, Zappe L, El-Tayeb A, Schiedel AC, Müller CE. Role of extracellular cysteine residues in the adenosine A2A receptor. Purinergic Signal 2016; 12:313-29. [PMID: 26969588 DOI: 10.1007/s11302-016-9506-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2016] [Accepted: 02/29/2016] [Indexed: 12/13/2022] Open
Abstract
The G protein-coupled A2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71(2.69)-Cys159(45.43); II, Cys74(3.22)-Cys146(45.30), and III, Cys77(3.25)-Cys166(45.50)). However, the A2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists' efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A2A and A2BARs.
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Affiliation(s)
- Elisabetta De Filippo
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Vigneshwaran Namasivayam
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Lukas Zappe
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Ali El-Tayeb
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Anke C Schiedel
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Christa E Müller
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany.
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20
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Alnouri MW, Jepards S, Casari A, Schiedel AC, Hinz S, Müller CE. Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors. Purinergic Signal 2015; 11:389-407. [PMID: 26126429 PMCID: PMC4529847 DOI: 10.1007/s11302-015-9460-9] [Citation(s) in RCA: 98] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2014] [Accepted: 06/17/2015] [Indexed: 12/14/2022] Open
Abstract
Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.
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MESH Headings
- Adenosine A1 Receptor Agonists/metabolism
- Adenosine A1 Receptor Agonists/pharmacology
- Adenosine A1 Receptor Antagonists/metabolism
- Adenosine A1 Receptor Antagonists/pharmacology
- Adenosine A2 Receptor Agonists/metabolism
- Adenosine A2 Receptor Agonists/pharmacology
- Adenosine A2 Receptor Antagonists/metabolism
- Adenosine A2 Receptor Antagonists/pharmacology
- Adenosine A3 Receptor Agonists/metabolism
- Adenosine A3 Receptor Agonists/pharmacology
- Adenosine A3 Receptor Antagonists/metabolism
- Adenosine A3 Receptor Antagonists/pharmacology
- Animals
- Arrestin/metabolism
- Binding, Competitive/drug effects
- CHO Cells
- Cell Membrane/drug effects
- Cell Membrane/metabolism
- Cricetinae
- Cricetulus
- Cyclic AMP/metabolism
- DNA, Complementary/drug effects
- DNA, Complementary/genetics
- Humans
- Mice
- Rats
- Receptor, Adenosine A2A/drug effects
- Receptor, Adenosine A2A/genetics
- Receptor, Adenosine A2A/metabolism
- Receptor, Adenosine A2B/drug effects
- Receptor, Adenosine A2B/genetics
- Receptor, Adenosine A2B/metabolism
- Receptors, Purinergic P1/drug effects
- Receptors, Purinergic P1/genetics
- Receptors, Purinergic P1/metabolism
- Species Specificity
- Structure-Activity Relationship
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Affiliation(s)
- Mohamad Wessam Alnouri
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Stephan Jepards
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Alessandro Casari
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Anke C. Schiedel
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Sonja Hinz
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Christa E. Müller
- Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
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The infarct-sparing effect of IB-MECA against myocardial ischemia/reperfusion injury in mice is mediated by sequential activation of adenosine A3 and A 2A receptors. Basic Res Cardiol 2015; 110:16. [PMID: 25711314 DOI: 10.1007/s00395-015-0473-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/09/2014] [Revised: 01/26/2015] [Accepted: 02/18/2015] [Indexed: 01/30/2023]
Abstract
Conflicting results exist regarding the role of A3 adenosine receptors (A3ARs) in mediating cardioprotection during reperfusion following myocardial infarction. We hypothesized that the effects of the A3AR agonist IB-MECA to produce cardioprotection might involve activation of other adenosine receptor subtypes. C57Bl/6 (B6), A3AR KO, A2AAR KO, and A2AAR KO/WT bone marrow chimeric mice were assigned to 12 groups undergoing either hemodynamic studies or 45 min of LAD occlusion and 60 min of reperfusion. IB-MECA (100 μg/kg) or vehicle was administered by iv bolus 5 min before reperfusion. Radioligand binding assays showed that IB-MECA has high affinity for the mouse A3AR (K i = 0.17 ± 0.05 nM), but also can bind with lower affinity to the A1AR (9.0 ± 2.4 nM) or the A2AAR (56.5 ± 10.2 nM). IB-MECA caused bi-phasic hemodynamic changes, which were completely absent in A3AR KO mice and were modified by A2AAR blockade or deletion. IB-MECA stimulated histamine release, increased heart rate, and significantly reduced IF size in B6 mice from 61.5 ± 1.4 to 48.6 ± 2.4% of risk region (RR; 21% reduction, p < 0.05) but not in A3AR KO mice. Compared to B6, A3AR KO mice had significantly reduced IF size (p < 0.05). In B6/B6 bone marrow chimeras, IB-MECA caused a 47% reduction of IF size (from 47.3 ± 3.9 to 24.7 ± 4.5, p < 0.05). However, no significant cardioprotective effect of IB-MECA was observed in A2AARKO/B6 mice, which lacked A2AARs only on their bone marrow-derived cells. Activation of A3ARs induces a bi-phasic hemodynamic response, which is partially mediated by activation of A2AARs. The cardioprotective effect of IB-MECA is due to the initial activation of A3AR followed by activation of A2AARs in bone marrow-derived cells.
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Yuan G, Gedeon NG, Jankins TC, Jones GB. Novel approaches for targeting the adenosine A2Areceptor. Expert Opin Drug Discov 2014; 10:63-80. [DOI: 10.1517/17460441.2015.971006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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Dix AV, Moss SM, Phan K, Hoppe T, Paoletta S, Kozma E, Gao ZG, Durell SR, Jacobson KA, Appella DH. Programmable nanoscaffolds that control ligand display to a G-protein-coupled receptor in membranes to allow dissection of multivalent effects. J Am Chem Soc 2014; 136:12296-303. [PMID: 25116377 PMCID: PMC4156868 DOI: 10.1021/ja504288s] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
![]()
A programmable
ligand display system can be used to dissect the
multivalent effects of ligand binding to a membrane receptor. An antagonist
of the A2A adenosine receptor, a G-protein-coupled receptor
that is a drug target for neurodegenerative conditions, was displayed
in 35 different multivalent configurations, and binding to A2A was determined. A theoretical model based on statistical mechanics
was developed to interpret the binding data, suggesting the importance
of receptor dimers. Using this model, extended multivalent arrangements
of ligands were constructed with progressive improvements in binding
to A2A. The results highlight the ability to use a highly
controllable multivalent approach to determine optimal ligand valency
and spacing that can be subsequently optimized for binding to a membrane
receptor. Models explaining the multivalent binding data are also
presented.
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Affiliation(s)
- Andrew V Dix
- Laboratory of Bioorganic Chemistry, NIDDK, ‡Laboratory of Biochemistry and Genetics, NIDDK, and §Laboratory of Cell Biology, CCR, NCI, National Institutes of Health , Bethesda, Maryland 20892, United States
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de Lera Ruiz M, Lim YH, Zheng J. Adenosine A2A Receptor as a Drug Discovery Target. J Med Chem 2013; 57:3623-50. [DOI: 10.1021/jm4011669] [Citation(s) in RCA: 204] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Affiliation(s)
- Manuel de Lera Ruiz
- Department
of Chemical Research, Merck Research Laboratories, 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States
| | - Yeon-Hee Lim
- Department
of Chemical Research, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - Junying Zheng
- Department
of Chemical Research, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
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Li Y, Figler RA, Kolling G, Bracken TC, Rieger J, Stevenson RW, Linden J, Guerrant RL, Warren CA. Adenosine A2A receptor activation reduces recurrence and mortality from Clostridium difficile infection in mice following vancomycin treatment. BMC Infect Dis 2012; 12:342. [PMID: 23217055 PMCID: PMC3523970 DOI: 10.1186/1471-2334-12-342] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2012] [Accepted: 12/03/2012] [Indexed: 11/10/2022] Open
Abstract
Background Activation of the A2A adenosine receptor (A2AAR) decreases production of inflammatory cytokines, prevents C. difficile toxin A-induced enteritis and, in combination with antibiotics, increases survival from sepsis in mice. We investigated whether A2AAR activation improves and A2AAR deletion worsens outcomes in a murine model of C. difficile (strain VPI10463) infection (CDI). Methods C57BL/6 mice were pretreated with an antibiotic cocktail prior to infection and then treated with vancomycin with or without an A2AAR agonist. A2AAR-/- and littermate wild-type (WT) mice were similarly infected, and IFNγ and TNFα were measured at peak of and recovery from infection. Results Infected, untreated mice rapidly lost weight, developed diarrhea, and had mortality rates of 50-60%. Infected mice treated with vancomycin had less weight loss and diarrhea during antibiotic treatment but mortality increased to near 100% after discontinuation of antibiotics. Infected mice treated with both vancomycin and an A2AAR agonist, either ATL370 or ATL1222, had minimal weight loss and better long-term survival than mice treated with vancomycin alone. A2AAR KO mice were more susceptible than WT mice to death from CDI. Increases in cecal IFNγ and blood TNFα were pronounced in the absence of A2AARs. Conclusion In a murine model of CDI, vancomycin treatment resulted in reduced weight loss and diarrhea during acute infection, but high recurrence and late-onset death, with overall mortality being worse than untreated infected controls. The administration of vancomycin plus an A2AAR agonist reduced inflammation and improved survival rates, suggesting a possible benefit of A2AAR agonists in the management of CDI to prevent recurrent disease.
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Affiliation(s)
- Yuesheng Li
- Division of Infectious Diseases and International Health, Carter Harrison Bldg, Charlottesville, VA 22908, USA
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Lebon G, Warne T, Tate CG. Agonist-bound structures of G protein-coupled receptors. Curr Opin Struct Biol 2012; 22:482-90. [PMID: 22480933 DOI: 10.1016/j.sbi.2012.03.007] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2012] [Revised: 03/12/2012] [Accepted: 03/14/2012] [Indexed: 01/14/2023]
Abstract
G protein-coupled receptors (GPCRs) play a major role in intercellular communication by binding small diffusible ligands (agonists) at the extracellular surface. Agonist-binding induces a conformational change in the receptor, which results in the binding and activation of heterotrimeric G proteins within the cell. Ten agonist-bound structures of non-rhodopsin GPCRs published last year defined for the first time the molecular details of receptor activated states and how inverse agonists, partial agonists and full agonists bind to produce different effects on the receptor. In addition, the structure of the β(2)-adrenoceptor coupled to a heterotrimeric G protein showed how the opening of a cleft in the cytoplasmic face of the receptor as a consequence of agonist binding results in G protein coupling and activation of the G protein.
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Affiliation(s)
- Guillaume Lebon
- Institut de Génomique Fonctionnelle, UMR 5203 CNRS - U 661 INSERM - Univ. Montpellier I & II, 141, rue de la cardonille, 34094 Montpellier Cedex 05, France
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Du L, Gao ZG, Nithipatikom K, Ijzerman AP, Veldhoven JPDV, Jacobson KA, Gross GJ, Auchampach JA. Protection from myocardial ischemia/reperfusion injury by a positive allosteric modulator of the A₃ adenosine receptor. J Pharmacol Exp Ther 2012; 340:210-7. [PMID: 22011434 PMCID: PMC3251031 DOI: 10.1124/jpet.111.187559] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2011] [Accepted: 10/18/2011] [Indexed: 01/10/2023] Open
Abstract
Adenosine is increased in ischemic tissues where it serves a protective role by activating adenosine receptors (ARs), including the A₃ AR subtype. We investigated the effect of N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarboxamide (LUF6096), a positive allosteric modulator of the A₃ AR, on infarct size in a barbital-anesthetized dog model of myocardial ischemia/reperfusion injury. Dogs were subjected to 60 min of coronary artery occlusion and 3 h of reperfusion. Infarct size was assessed by macrohistochemical staining. Three experimental groups were included in the study. Groups I and II received two doses of vehicle or LUF6096 (0.5 mg/kg i.v. bolus), one administered before ischemia and the other immediately before reperfusion. Group III received a single dose of LUF6096 (1 mg/kg i.v. bolus) immediately before reperfusion. In preliminary in vitro studies, LUF6096 was found to exert potent enhancing activity (EC₅₀ 114.3 ± 15.9 nM) with the canine A₃ AR in a guanosine 5'-[γ-[³⁵S]thio]triphosphate binding assay. LUF6096 increased the maximal efficacy of the partial A₃ AR agonist 2-chloro-N⁶-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide and the native agonist adenosine more than 2-fold while producing a slight decrease in potency. In the dog studies, administration of LUF6096 had no effect on any hemodynamic parameter measured. Pretreatment with LUF6096 before coronary occlusion and during reperfusion in group II dogs produced a marked reduction in infarct size (∼50% reduction) compared with group I vehicle-treated dogs. An equivalent reduction in infarct size was observed when LUF6096 was administered immediately before reperfusion in group III dogs. This is the first study to demonstrate efficacy of an A₃ AR allosteric enhancer in an in vivo model of infarction.
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Affiliation(s)
- Lili Du
- Department of Pharmacology and the Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
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van der Hoeven D, Wan TC, Gizewski ET, Kreckler LM, Maas JE, Van Orman J, Ravid K, Auchampach JA. A role for the low-affinity A2B adenosine receptor in regulating superoxide generation by murine neutrophils. J Pharmacol Exp Ther 2011; 338:1004-12. [PMID: 21693629 PMCID: PMC3164346 DOI: 10.1124/jpet.111.181792] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2011] [Accepted: 06/16/2011] [Indexed: 12/27/2022] Open
Abstract
The formation of adenosine dampens inflammation by inhibiting most cells of the immune system. Among its actions on neutrophils, adenosine suppresses superoxide generation and regulates chemotactic activity. To date, most evidence implicates the G(s) protein-coupled A(2A) adenosine receptor (AR) as the primary AR subtype responsible for mediating the actions of adenosine on neutrophils by stimulating cAMP production. Given that the A(2B)AR is now known to be expressed in neutrophils and that it is a G(s) protein-coupled receptor, we examined in this study whether it signals to suppress neutrophil activities by using 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY 60-6583), a new agonist for the human A(2B)AR that was confirmed in preliminary studies to be a potent and highly selective agonist for the murine A(2B)AR. We found that treating mouse neutrophils with low concentrations (10(-9) and 10(-8) M) of BAY 60-6583 inhibited formylated-methionine-leucine-phenylalanine (fMLP)-stimulated superoxide production by either naive neutrophils, tumor necrosis factor-α-primed neutrophils, or neutrophils isolated from mice treated systemically with lipopolysaccharide. This inhibitory action of BAY 60-6583 was confirmed to involve the A(2B)AR in experiments using neutrophils obtained from A(2B)AR gene knockout mice. It is noteworthy that BAY 60-6583 increased fMLP-stimulated superoxide production at higher concentrations (>1 μM), which was attributed to an AR-independent effect. In a standard Boyden chamber migration assay, BAY 60-6583 alone did not stimulate neutrophil chemotaxis or influence chemotaxis in response to fMLP. These results indicate that the A(2B)AR signals to suppress oxidase activity by murine neutrophils, supporting the idea that this low-affinity receptor for adenosine participates along with the A(2A)AR in regulating the proinflammatory actions of neutrophils.
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Affiliation(s)
- Dharini van der Hoeven
- Department of Pharmacology and the Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA
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Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AGW, Tate CG. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 2011; 474:521-5. [PMID: 21593763 PMCID: PMC3146096 DOI: 10.1038/nature10136] [Citation(s) in RCA: 688] [Impact Index Per Article: 49.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2011] [Accepted: 04/21/2011] [Indexed: 12/18/2022]
Abstract
Adenosine receptors and β-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine or noradrenaline, respectively. GPCRs have similar structures consisting of seven transmembrane helices that contain well-conserved sequence motifs, indicating that they are probably activated by a common mechanism. Recent structures of β-adrenoceptors highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, indicating that these residues have an important role in agonist-induced activation of receptors. Here we present two crystal structures of the thermostabilized human adenosine A(2A) receptor (A(2A)R-GL31) bound to its endogenous agonist adenosine and the synthetic agonist NECA. The structures represent an intermediate conformation between the inactive and active states, because they share all the features of GPCRs that are thought to be in a fully activated state, except that the cytoplasmic end of transmembrane helix 6 partially occludes the G-protein-binding site. The adenine substituent of the agonists binds in a similar fashion to the chemically related region of the inverse agonist ZM241385 (ref. 8). Both agonists contain a ribose group, not found in ZM241385, which extends deep into the ligand-binding pocket where it makes polar interactions with conserved residues in H7 (Ser 277(7.42) and His 278(7.43); superscripts refer to Ballesteros-Weinstein numbering) and non-polar interactions with residues in H3. In contrast, the inverse agonist ZM241385 does not interact with any of these residues and comparison with the agonist-bound structures indicates that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A(2A)R with the agonist-bound structures of β-adrenoceptors indicates that the contraction of the ligand-binding pocket caused by the inward motion of helices 3, 5 and 7 may be a common feature in the activation of all GPCRs.
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Affiliation(s)
- Guillaume Lebon
- MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK
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Lebon G, Bennett K, Jazayeri A, Tate CG. Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor. J Mol Biol 2011; 409:298-310. [PMID: 21501622 PMCID: PMC3145977 DOI: 10.1016/j.jmb.2011.03.075] [Citation(s) in RCA: 90] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2011] [Revised: 03/31/2011] [Accepted: 03/31/2011] [Indexed: 11/02/2022]
Abstract
The adenosine A(2A) receptor (A(2A)R) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A(2A)R was determined recently in an antagonist-bound conformation, which was facilitated by the T4 lysozyme fusion in cytoplasmic loop 3 and the considerable stabilisation conferred on the receptor by the bound inverse agonist ZM241385. Unfortunately, the natural agonist adenosine does not sufficiently stabilise the receptor for the formation of diffraction-quality crystals. As a first step towards determining the structure of A(2A)R bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist [(3)H]5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A(2A)R-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A(2A)R-GL26 is stabilised in an agonist-bound conformation because antagonists bind with up to 320-fold decreased affinity. None of the thermostabilising mutations are in the ZM241385 binding pocket, suggesting that the mutations affect ligand binding by altering the conformation of the receptor rather than through direct interactions with ligands. A(2A)R-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation.
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Bigler Wang D, Sherman NE, Shannon JD, Leonhardt SA, Mayeenuddin LH, Yeager M, McIntire WE. Binding of β4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry. Biochemistry 2010; 50:207-20. [PMID: 21128647 DOI: 10.1021/bi101227y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Characterization of G protein βγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native βγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and βγ labeled with heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched βγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing β and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three β isoforms, β(1), β(2), and β(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. β(1) and γ(5) were most abundant in the enriched βγ fraction, and this βγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more β(4) and γ(5) compared to the enriched βγ fraction; also, more β(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched βγ fraction. These results suggest that preferences for particular βγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.
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Affiliation(s)
- Dora Bigler Wang
- Department of Pharmacology, University of Virginia Health System, Charlottesville, 22908, United States
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Sf9 cells: a versatile model system to investigate the pharmacological properties of G protein-coupled receptors. Pharmacol Ther 2010; 128:387-418. [PMID: 20705094 DOI: 10.1016/j.pharmthera.2010.07.005] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2010] [Accepted: 07/12/2010] [Indexed: 11/23/2022]
Abstract
The Sf9 cell/baculovirus expression system is widely used for high-level protein expression, often with the purpose of purification. However, proteins may also be functionally expressed in the defined Sf9 cell environment. According to the literature, the pharmacology of G-protein-coupled receptors (GPCRs) functionally reconstituted in Sf9 cells is similar to the receptor properties in mammalian cells. Sf9 cells express both recombinant GPCRs and G-proteins at much higher levels than mammalian cells. Sf9 cells can be grown in suspension culture, providing an inexpensive way of obtaining large protein amounts. Co-infection with various baculoviruses allows free combination of GPCRs with different G-proteins. The absence of constitutively active receptors in Sf9 cells provides an excellent signal-to background ratio in functional assays, allowing the detection of agonist-independent receptor activity and of small ligand-induced signals including partial agonistic and inverse agonistic effects. Insect cell Gα(i)-like proteins mostly do not couple productively to mammalian GPCRs. Thus, unlike in mammalian cells, Sf9 cells do not require pertussis toxin treatment to obtain a Gα(i)-free environment. Co-expression of GPCRs with Gα(i1), Gα(i2), Gα(i3) or Gα(o) in Sf9 cells allows the generation of a selectivity profile for these Gα(i/o)-isoforms. Additionally, GPCR-G-protein combinations can be compared with defined 1:1 stoichiometry by expressing GPCR-Gα fusion proteins. Sf9 cells can also be employed for ligand screening in medicinal chemistry programs, using radioligand binding assays or functional assays, like the steady-state GTPase- or [(35)S]GTPγS binding assay. This review shows that Sf9 cells are a versatile model system to investigate the pharmacological properties of GPCRs.
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Schwindinger WF, Mihalcik LJM, Giger KE, Betz KS, Stauffer AM, Linden J, Herve D, Robishaw JD. Adenosine A2A receptor signaling and golf assembly show a specific requirement for the gamma7 subtype in the striatum. J Biol Chem 2010; 285:29787-96. [PMID: 20639202 DOI: 10.1074/jbc.m110.142620] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The adenosine A(2A) receptor (A(2A)R) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α(olf) subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ(7) subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ(7) protein controls the hierarchial assembly of a specific G-protein α(olf)β(2)γ(7) heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A(2A)R activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ(7) subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A(2A)R G-protein α(olf)β(2)γ(7) interface as a possible therapeutic target for Parkinson disease.
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Stewart GD, Sexton PM, Christopoulos A. Detection of novel functional selectivity at M3 muscarinic acetylcholine receptors using a Saccharomyces cerevisiae platform. ACS Chem Biol 2010; 5:365-75. [PMID: 20155933 DOI: 10.1021/cb900276p] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
"Functional selectivity", although new to many chemists and biologists only a few years ago, has now become a dominant theme in drug discovery. This concept posits that different ligands engender unique receptor conformations such that only a subset of signaling pathways linked to a given receptor are recruited. However, successful exploitation of the phenomenon to achieve pathway-based selectivity requires the ability to routinely detect it when assessing ligand behavior. We have utilized different strains of the yeast S. cerevisiae, each expressing a specific human Galpha/yeast Gpa1 protein chimera coupled to a MAP kinase-linked reporter gene readout, to investigate the signaling of the M(3) muscarinic receptor, a G protein-coupled receptor (GPCR) for which various antagonists are used clinically. Using this novel platform, we found that the "antagonists", atropine, N-methylscopolamine, and pirenzepine, were inverse agonists for Gpa1/Galpha(q) but low efficacy agonists for Gpa1/Galpha(12.) Subsequent studies with atropine performed in mammalian 3T3 cells validated these findings by demonstrating inverse agonism for G(q/11)-mediated calcium mobilization but positive agonism for G(12)-mediated membrane ruffling. This is the first study to utilize a yeast platform to discover pathway-biased functional selectivity in a GPCR. In addition to the likely applicability of this approach for identifying biased signaling by novel chemical entities, our findings also suggest that currently marketed medications may exhibit hitherto unappreciated functional selectivity.
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Affiliation(s)
- Gregory D. Stewart
- Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash University, Parkville, Victoria, Australia 3052
| | - Patrick M. Sexton
- Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash University, Parkville, Victoria, Australia 3052
| | - Arthur Christopoulos
- Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash University, Parkville, Victoria, Australia 3052
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Devine SM, Gregg A, Figler H, McIntosh K, Urmaliya V, Linden J, Pouton CW, White PJ, Bottle SE, Scammells PJ. Synthesis and evaluation of new N6-substituted adenosine-5'-N-methylcarboxamides as A3 adenosine receptor agonists. Bioorg Med Chem 2010; 18:3078-87. [PMID: 20385496 DOI: 10.1016/j.bmc.2010.03.047] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2010] [Revised: 03/16/2010] [Accepted: 03/17/2010] [Indexed: 11/25/2022]
Abstract
A number of N(6)-substituted adenosine-5'-N-methylcarboxamides were synthesised and their pharmacology, in terms of their receptor affinity, selectivity and cardioprotective effects, were explored. The first series of compounds, 4a-4f and 5a-5f, showed modest receptor affinity for the A(3)AR with K(i) values in the low to mid muM range. However, the incorporation of a 4-(2-aminoethyl)-2,6-di-tert-butylphenol group in the N(6)-position (in compounds 4g and 5g) significantly improved the affinity with K(i) values of 30 and 9 nM, respectively. Improvements in affinity, as well as selectivity were seen when a functionalized linker was introduced. The N(6)-phenyl series, compounds 7a-7d, demonstrated low to mid nanomolar receptor affinities (K(i)=2.3-45.0 nM), with 7b displaying 109-fold selectivity for the A(3)AR (vs A(1)). The N(6)-benzyl series 9a-9c also proved to be potent and selective A(3)AR agonists and the longer chain length linker 13 was tolerated at the A(3)AR without abrogation of affinity or selectivity. Cardioprotection was demonstrated by a simulated ischaemia cell culture assay, whereby 7b, 7c, 9a, 9b and 9c all showed cardioprotective effects at 100 nM comparable or better than the benchmark A(3)AR agonist IB-MECA, but which were indistinguishable by statistical analysis. For example, compound 9c reduced cell death by 68.0+/-3.6%.
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Affiliation(s)
- Shane M Devine
- Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville VIC 3052, Australia
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Auchampach JA, Kreckler LM, Wan TC, Maas JE, van der Hoeven D, Gizewski E, Narayanan J, Maas GE. Characterization of the A2B adenosine receptor from mouse, rabbit, and dog. J Pharmacol Exp Ther 2009; 329:2-13. [PMID: 19141710 PMCID: PMC2670590 DOI: 10.1124/jpet.108.148270] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2008] [Accepted: 01/12/2009] [Indexed: 11/22/2022] Open
Abstract
We have cloned and pharmacologically characterized the A(2B) adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A(2B)AR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A(2B)AR cDNA was obtained by screening a rabbit brain cDNA library. It is noteworthy that an additional clone was isolated by library screening that was identical in sequence to the full-length rabbit A(2B)AR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A(2B)AR(103-111)). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A(2B)AR and seems to result from the use of an additional 5' donor site found in the rabbit and dog but not in the human, rat, or mouse sequences. [(3)H]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-(3)H]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine ([(3)H]MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A(2B)ARs. Competition binding studies performed with a panel of agonist (adenosine and 2-amino-3,5-dicyano-4-phenylpyridine analogs) and antagonist ligands identified similar potency orders for the A(2B)AR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A(2B)AR compared with A(2B)ARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A(2B)AR(103-111) variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A(2B)AR exist between species and that some species express nonfunctional variants of the A(2B)AR due to "leaky" splicing.
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Affiliation(s)
- John A Auchampach
- Department of Pharmacology and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
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McIntire WE. Structural determinants involved in the formation and activation of G protein betagamma dimers. Neurosignals 2009; 17:82-99. [PMID: 19212142 DOI: 10.1159/000186692] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2008] [Accepted: 05/13/2008] [Indexed: 01/08/2023] Open
Abstract
Heterotrimeric G proteins, composed of an alpha, beta and gamma subunit, represent one of the most important and dynamic families of signaling proteins. As a testament to the significance of G protein signaling, the hundreds of seven-transmembrane-spanning receptors that interact with G proteins are estimated to occupy 1-2% of the human genome. This broad diversity of receptors is echoed in the number of potential heterotrimer combinations that can arise from the 23 alpha subunit, 7 beta subunit and 12 gamma subunit isoforms that have been identified. The potential for such vast complexity implies that the receptor G protein interface is the site of much regulation. The historical model for the activation of a G protein holds that activated receptor catalyzes the exchange of GDP for GTP on the alpha subunit, inducing a conformational change that substantially lowers the affinity of alpha for betagamma. This decreased affinity enables dissociation of betagamma from alpha and receptor. The free form of betagamma is thought to activate effectors, until the hydrolysis of GTP by G alpha (aided by RGS proteins) allows the subunits to re-associate, effectively deactivating the G protein until another interaction with activated receptor.
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Affiliation(s)
- William E McIntire
- Department of Pharmacology, University of Virginia Health System, Charlottesville, VA 22908, USA.
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Abstract
The development of potent and selective agonists and antagonists of adenosine receptors (ARs) has been a target of medicinal chemistry research for several decades, and recently the US Food and Drug Administration has approved Lexiscan, an adenosine derivative substituted at the 2 position, for use as a pharmacologic stress agent in radionuclide myocardial perfusion imaging. Currently, some other adenosine A(2A) receptor (A(2A)AR) agonists and antagonists are undergoing preclinical testing and clinical trials. While agonists are potent antiinflammatory agents also showing hypotensive effects, antagonists are being developed for the treatment of Parkinson's disease.However, since there are still major problems in this field, including side effects, low brain penetration (for the targeting of CNS diseases), short half-life, or lack of in vivo effects, the design and development of new AR ligands is a hot research topic.This review presents an update on the medicinal chemistry of A(2A)AR agonists and antagonists, and stresses the strong need for more selective ligands at the human A(2A)AR subtype, in particular in the case of agonists.
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Affiliation(s)
- Gloria Cristalli
- Dipartimento di Scienze Chimiche, Università di Camerino, 62032 Camerino (MC), Italy.
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Rork TH, Wallace KL, Kennedy DP, Marshall MA, Lankford AR, Linden J. Adenosine A2A receptor activation reduces infarct size in the isolated, perfused mouse heart by inhibiting resident cardiac mast cell degranulation. Am J Physiol Heart Circ Physiol 2008; 295:H1825-33. [PMID: 18757481 DOI: 10.1152/ajpheart.495.2008] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A2A adenosine receptors (A2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A2AAR+/+ mice, A2AAR(-/-) mice, mast cell-deficient (Kit(W-sh/W-sh)) mice, and chimeric mice prepared by transplanting bone marrow from A2AAR(-/-) or A2AAR+/+ mice to radiation-ablated A2AAR+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 +/- 2% to 24 +/- 2% and 22 +/- 2% in ATL146e- and CGS-21680-treated hearts, respectively (P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A2AAR(-/-) hearts and in hearts from chimeric mice with A2AAR(-/-) bone marrow. Vehicle-treated Kit(W-sh/W-sh) mice had lower IS (11 +/- 3%) than WT mice, and ATL146e had no significant protective effect (16 +/- 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.
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Affiliation(s)
- Tyler H Rork
- Robert M. Berne Cardiovascular Research Center, University of Virginia Health System, Charlottesville, VA, USA
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Wan TC, Ge ZD, Tampo A, Mio Y, Bienengraeber MW, Tracey WR, Gross GJ, Kwok WM, Auchampach JA. The A3 adenosine receptor agonist CP-532,903 [N6-(2,5-dichlorobenzyl)-3'-aminoadenosine-5'-N-methylcarboxamide] protects against myocardial ischemia/reperfusion injury via the sarcolemmal ATP-sensitive potassium channel. J Pharmacol Exp Ther 2008; 324:234-43. [PMID: 17906066 PMCID: PMC2435594 DOI: 10.1124/jpet.107.127480] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
We examined the cardioprotective profile of the new A(3) adenosine receptor (AR) agonist CP-532,903 [N(6)-(2,5-dichlorobenzyl)-3'-aminoadenosine-5'-N-methylcarboxamide] in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using human embryonic kidney 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A(3)ARs (K(i) = 9.0 +/- 2.5 nM) and with high selectivity versus mouse A(1)AR (100-fold) and A(2A)ARs (1000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 microg/kg CP-532,903 reduced infarct size from 59.2 +/- 2.1% of the risk region in vehicle-treated mice to 42.5 +/- 2.3 and 39.0 +/- 2.9%, respectively. Likewise, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration dependently improved recovery of contractile function after 20 min of global ischemia and 45 min of reperfusion, including developed pressure and maximal rate of contraction/relaxation. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A(3)AR gene, A(3) knockout (KO) mice. In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by three brief ischemia/reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATP-sensitive potassium (K(ATP)) channel. Whole-cell patch-clamp recordings provided evidence that the A(3)AR is functionally coupled to the sarcolemmal K(ATP) channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A(3)AR that protects against ischemia/reperfusion injury by activating sarcolemmal K(ATP) channels.
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Affiliation(s)
- Tina C Wan
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
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Haskó G, Pacher P. A2A receptors in inflammation and injury: lessons learned from transgenic animals. J Leukoc Biol 2007; 83:447-55. [PMID: 18160539 DOI: 10.1189/jlb.0607359] [Citation(s) in RCA: 184] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Adenosine regulates the function of the innate and adaptive immune systems through targeting virtually every cell type that is involved in orchestrating an immune/inflammatory response. Of the four adenosine receptors (A(1), A(2A), A(2B), A(3)), A(2A) receptors have taken center stage as the primary anti-inflammatory effectors of extracellular adenosine. This broad, anti-inflammatory effect of A(2A) receptor activation is a result of the predominant expression of A(2A) receptors on monocytes/macrophages, dendritic cells, mast cells, neutrophils, endothelial cells, eosinophils, epithelial cells, as well as lymphocytes, NK cells, and NKT cells. A(2A) receptor activation inhibits early and late events occurring during an immune response, which include antigen presentation, costimulation, immune cell trafficking, immune cell proliferation, proinflammatory cytokine production, and cytotoxicity. In addition to limiting inflammation, A(2A) receptors participate in tissue remodeling and reparation. Consistent with their multifaceted, immunoregulatory action on immune cells, A(2A) receptors have been shown to impact the course of a wide spectrum of ischemic, autoimmune, infectious, and allergic diseases. Here, we review the regulatory roles of A(2A) receptors in immune/inflammatory diseases of various organs, including heart, lung, gut, liver, kidney, joints, and brain, as well as the role of A(2A) receptors in regulating multiple organ failure and sepsis.
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Affiliation(s)
- György Haskó
- UMDNJ-New Jersey Medical School, 185 South Orange Avenue, University Heights, Newark, NJ 07103, USA.
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42
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Chang CZ, Dumont AS, Simsek S, Titus BJ, Kwan AL, Kassell NF, Solenski NJ. THE ADENOSINE 2A RECEPTOR AGONIST ATL-146E ATTENUATES EXPERIMENTAL POSTHEMORRHAGIC VASOSPASM. Neurosurgery 2007; 60:1110-7; discussion 1117-8. [PMID: 17538386 DOI: 10.1227/01.neu.0000255467.22387.5c] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
OBJECTIVE Selective adenosine 2A receptor agonists, such as ATL-146e, are known to be potent anti-inflammatory agents devoid of systemic side effects and have been used clinically in a number of disease states. However, adenosine 2A receptor agonists have not been studied in the treatment of cerebral vasospasm after subarachnoid hemorrhage. The present study investigated the efficacy of ATL-146e in the prevention of leukocyte infiltration and attenuation of posthemorrhagic vasospasm. METHODS The rodent femoral artery model of vasospasm was used. Forty male Sprague-Dawley rats were randomly assigned to four different groups (vehicle, 1 ng/kg/min, 10 ng/kg/min, or 100 ng/kg/min ATL-146e administered via subcutaneous osmotic minipump). Vasospasm was evaluated at posthemorrhage Day 8 (period of peak constriction) by calculating the lumen cross-sectional area (expressed as percent change in luminal area: ratio of blood-exposed vessel to normal saline-exposed vessel) and radial wall thickness. Immunostaining with anti-CD45 monoclonal antibody to detect leukocytes was used to evaluate localized inflammation. RESULTS Significant vasospasm was noted in the vehicle-treated (blood-exposed) control group (78.5%, P < 0.001; expressed as a ratio of luminal area of the saline [no blood] control), but not in the ATL-146e-treated groups (lumen ratio to control: 105.0, 83.4, and 91.3% for the 1, 10, and 100 ng/kg/min groups, respectively). Additionally, infiltration of inflammatory cells was reduced significantly and radial wall thickness was decreased in the ATL-146e-treated groups compared with the vehicle-treated control group. CONCLUSION Selective activation of the adenosine 2A receptor with ATL-146e prevented posthemorrhagic vasospasm and reduced leukocyte infiltration in this experimental model. This agent is worthy of further investigation and lends credence to the hypothesis supporting a role for inflammation in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage.
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Affiliation(s)
- Chih-Zen Chang
- Department of Neurological Surgery, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA
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Ge ZD, Peart JN, Kreckler LM, Wan TC, Jacobson MA, Gross GJ, Auchampach JA. Cl-IB-MECA [2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide] reduces ischemia/reperfusion injury in mice by activating the A3 adenosine receptor. J Pharmacol Exp Ther 2006; 319:1200-10. [PMID: 16985166 DOI: 10.1124/jpet.106.111351] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
We used pharmacological agents and genetic methods to determine whether the potent A(3) adenosine receptor (AR) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A(3)AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 mug/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A(3)AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A(2A)AR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A(3)AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A(3)AR gene "knock-out" (A(3)KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A(3)KO mice in vivo and did not protect isolated perfused hearts obtained from A(3)KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A(3)AR.
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Affiliation(s)
- Zhi-Dong Ge
- Department of Pharmacology and Toxicology and the Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA
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Ravyn V, Bostwick JR. Functional coupling of the Galpha(olf) variant XLGalpha(olf) with the human adenosine A2A receptor. J Recept Signal Transduct Res 2006; 26:241-58. [PMID: 16818375 DOI: 10.1080/10799890600710592] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
A recently identified novel Galphaolf variant, XLGalphaolf, is shown to functionally couple to the human adenosine A2A receptor (A2AR). In Sf9 cells expressing A2AR, beta1, and gamma2, co-expression of XLGalphaolf increased NECA-induced [35S]GTPgammaS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A2AR ligands on these cells were evaluated by using [3H]ZM241385- and [35S]GTPgammaS- binding assays. The rank order of the equilibrium binding constants (Kd or Ki) of adenosine receptor ligands were [3H]ZM241385 approximately CGS15943 < MRS1220 < < CV1808 approximately NECA < CGS21680 approximately adenosine < IBMECA < HEMADO approximately CPA approximately CCPA. The rank order of EC50 values for agonists were CV1808 approximately NECA < adenosine approximately CGS26180 < IBMECA < HEMADO approximately CPA approximately CCPA. This pharmacology is consistent with the literature for A2AR and suggests that Sf9 cells co-expressing A2AR, beta1, gamma2, and XLGalphaolf could serve as a heterologous expression system for A2AR drug screening.
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Affiliation(s)
- Vipa Ravyn
- Lead Discovery, AstraZeneca Pharmaceuticals, Wilmington, Delaware 19850, USA
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Odashima M, Otaka M, Jin M, Horikawa Y, Matsuhashi T, Ohba R, Linden J, Watanabe S. A selective adenosine A2A receptor agonist, ATL-146e, prevents concanavalin A-induced acute liver injury in mice. Biochem Biophys Res Commun 2006; 347:949-54. [PMID: 16859640 DOI: 10.1016/j.bbrc.2006.06.185] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2006] [Accepted: 06/28/2006] [Indexed: 10/24/2022]
Abstract
BACKGROUND AND AIMS Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production. METHODS Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell. RESULTS Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production. CONCLUSION A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.
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Affiliation(s)
- Masaru Odashima
- Department of Gastroenterology, Akita University School of Medicine, Akita, Japan.
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Ernens I, Rouy D, Velot E, Devaux Y, Wagner DR. Adenosine inhibits matrix metalloproteinase-9 secretion by neutrophils: implication of A2a receptor and cAMP/PKA/Ca2+ pathway. Circ Res 2006; 99:590-7. [PMID: 16917093 DOI: 10.1161/01.res.0000241428.82502.d4] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Matrix metalloproteinases (MMPs), and in particular MMP-9 secreted by neutrophils, are capable of degrading the matrix components of the heart and are thought to be the driving force behind myocardial matrix remodeling after infarction. Adenosine, a naturally produced nucleoside, has been shown to have cardioprotective effects and to inhibit secretion of various cytokines. The aim of our study was to determine the effect of adenosine on the secretion of MMP-9 by neutrophils. Neutrophils were isolated from healthy volunteers through Ficoll and Dextran sedimentation. Neutrophils were activated by N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence or absence of adenosine or adenosine analogs. Zymography and enzyme linked immunosorbent assay were used to measure MMP-9 secretion. Adenosine (1 micromol/L) decreased the fMLP-induced MMP-9 secretion by 30+/-2% (n=8, P<0.001). The effect was dose-dependent and was not specific to fMLP because adenosine also inhibited MMP-9 secretion by LPS- or H(2)O(2)-stimulated neutrophils. The effect of adenosine was mimicked by the adenosine A2a receptor agonist CGS21680 and was inhibited by both the A2a antagonist SCH5826 and A2a RNA silencing. The A3 agonist IB-MECA moderately decreased fMLP-induced MMP-9 secretion. Agonists and antagonists of the other types of adenosine receptors had no significant effect. Adenosine increased intracellular cAMP concentration and accelerated the return to baseline of the intracytoplasmic calcium peak. The inhibition of MMP-9 secretion by adenosine, as well as the calcium effect, was prevented by the protein kinase A inhibitor H-89. In conclusion, we show here that adenosine inhibits MMP-9 secretion by neutrophils. Our results suggest that this effect implies the A2a receptor and is mediated through the cAMP/PKA/Ca(2+) pathway. Therefore, adenosine may represent a new approach to prevent matrix degradation and remodeling after myocardial injury.
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Affiliation(s)
- Isabelle Ernens
- Laboratory of Cardiovascular Research, CRP-Santé, Luxembourg
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Characterization of human and rodent native and recombinant adenosine A(2B) receptors by radioligand binding studies. Purinergic Signal 2006; 2:559-71. [PMID: 18404493 PMCID: PMC2096648 DOI: 10.1007/s11302-006-9012-4] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2006] [Accepted: 04/21/2006] [Indexed: 10/25/2022] Open
Abstract
Adenosine A(2B) receptors of native human and rodent cell lines were investigated using [(3)H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [(3)H]PSB-298 showed saturable and reversible binding. It exhibited a K(D) value of 60 +/- 1 nM and limited capacity (B(max) = 3.511 fmol per milligram protein) at recombinant human adenosine A(2B) receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5'-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A(2B) receptors. Adenosine A(2B) receptors were shown to be the major adenosine A(2) receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [(3)H]PSB-298 is a selective radioligand for adenosine A(2B) binding sites in this cell line.
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48
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Yan X, Koos BJ, Kruger L, Linden J, Murray TF. Characterization of [125I]ZM 241385 binding to adenosine A2A receptors in the pineal of sheep brain. Brain Res 2006; 1096:30-9. [PMID: 16764836 DOI: 10.1016/j.brainres.2006.04.072] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2006] [Revised: 04/04/2006] [Accepted: 04/10/2006] [Indexed: 11/29/2022]
Abstract
Adenosine is a ubiquitous neuromodulator and homeostatic regulator that exerts its physiologic actions through activation of A(1), A(2A), A(2B) and A(3) adenosine receptor subtypes. In the central nervous system, adenosine's action in neurons is manifested in its modulation of tonic inhibitory control. Adenosine released in the brain during hypoxia has critical depressant effects on breathing in fetal and newborn mammals, an action suggested to be mediated by A(2A) receptors in the posteromedial thalamus. In an effort to more accurately define the spatial distribution of adenosine A(2A) receptors in fetal sheep diencephalon, we have used a receptor autoradiographic technique utilizing an iodinated radioligand [(125)I]ZM 241385, which has greater sensitivity and resolution than the tritiated compound. The distribution of ligand binding sites in the fetal sheep diencephalon indicated that the highest levels of binding were in select thalamic nuclei, including those implicated in hypoxic depression of fetal breathing, and the pineal. Given the high density of labeled A(2A) receptors in the pineal, these sites were characterized more fully in homogenate radioligand binding assays. These data indicate that [(125)I]ZM 241385 binding sites display a pharmacological signature consistent with that of adenosine A(2A) receptors and are expressed at similar levels in fetal, lamb and adult ovine brain. The adenosine A(2A) receptor pharmacologic signature of the [(125)I]ZM 241385 binding site in pineal cell membranes generalized to the site characterized in membranes derived from other portions of the lamb thalamus, including the sector involved in hypoxic inhibition of fetal breathing. These results have important implications for the functional roles of adenosine A(2A) receptors in the thalamus and pineal of sheep brain.
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Affiliation(s)
- X Yan
- Department of Pharmacology, Creighton University School of Medicine, Omaha, NE 68178, USA
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Bhattacharya S, Youkey R, Ghartey K, Leonard M, Linden J, Tucker A. The allosteric enhancer PD81,723 increases chimaeric A1/A2A adenosine receptor coupling with Gs. Biochem J 2006; 396:139-46. [PMID: 16390330 PMCID: PMC1449996 DOI: 10.1042/bj20051422] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2005] [Revised: 11/22/2005] [Accepted: 01/03/2006] [Indexed: 11/17/2022]
Abstract
PD81,723 {(2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]methanone} is a selective allosteric enhancer of the G(i)-coupled A1 AR (adenosine receptor) that is without effect on G(s)-coupled A2A ARs. PD81,723 elicits a decrease in the dissociation kinetics of A1 AR agonist radioligands and an increase in functional agonist potency. In the present study, we sought to determine whether enhancer sensitivity is dependent on coupling domains or G-protein specificity of the A1 AR. Using six chimaeric A1/A2A ARs, we show that the allosteric effect of PD81,723 is maintained in a chimaera in which the predominant G-protein-coupling domain of the A1 receptor, the 3ICL (third intracellular loop), is replaced with A2A sequence. These chimaeric receptors are dually coupled with G(s) and G(i), and PD81,723 increases the potency of N6-cyclopentyladenosine to augment cAMP accumulation with or without pretreatment of cells with pertussis toxin. Thus PD81,723 has similar functional effects on chimaeric receptors with A1 transmembrane sequences that couple with G(i) or G(s). This is the first demonstration that an allosteric regulator can function in the context of a switch in G-protein-coupling specificity. There is no enhancement by PD81,723 of G(i)-coupled A2A chimaeric receptors with A1 sequence replacing A2A sequence in the 3ICL. The results suggest that the recognition site for PD81,723 is on the A1 receptor and that the enhancer acts to directly stabilize the receptor to a conformational state capable of coupling with G(i) or G(s).
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Key Words
- adenosine receptor
- allosteric enhancer
- canine
- g-protein
- n6-cyclopentyladenosine (cpa)
- pd81,723
- 3icl, third intracellular loop
- ada, adenosine deaminase
- ar, adenosine receptor
- cpa, n6-cyclopentyladenosine
- cpx, 1,3-dipropyl-8-cyclopentylxanthine
- gpcr, g-protein-coupled receptor
- gtp[s], guanosine 5′-[γ-thio]triphosphate
- hek-293 cell, human embryonic kidney 293 cell
- 125i-aba, 125i-n6-4-amino-3-iodo-benzyladenosine
- 125i-ape, 125i-2-[2-(4-amino-3-iodo-phenyl)ethylamino]adenosine
- neca, 5′-n-ethylcarboxamidoadenosine
- r–g complex, receptor–g-protein complex
- tm, transmembrane domain
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MESH Headings
- Adenosine/analogs & derivatives
- Adenosine/pharmacology
- Adrenergic alpha-Agonists/pharmacology
- Adrenergic alpha-Antagonists/pharmacology
- Allosteric Regulation/drug effects
- Animals
- Cell Line
- Cyclic AMP/metabolism
- Dogs
- GTP-Binding Protein alpha Subunits, Gi-Go/metabolism
- GTP-Binding Protein alpha Subunits, Gs/metabolism
- Humans
- Iodobenzenes/pharmacology
- Kidney/cytology
- Protein Conformation
- Protein Interaction Mapping
- Protein Structure, Tertiary
- Radioligand Assay
- Receptor, Adenosine A1/chemistry
- Receptor, Adenosine A1/drug effects
- Receptor, Adenosine A1/genetics
- Receptor, Adenosine A1/metabolism
- Receptor, Adenosine A2A/chemistry
- Receptor, Adenosine A2A/drug effects
- Receptor, Adenosine A2A/genetics
- Recombinant Fusion Proteins/chemistry
- Recombinant Fusion Proteins/drug effects
- Recombinant Fusion Proteins/metabolism
- Thiophenes/pharmacology
- Transfection
- Xanthines/pharmacology
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Affiliation(s)
- Samita Bhattacharya
- *Department of Internal Medicine, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
| | - Rebecca L. Youkey
- *Department of Internal Medicine, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
| | - Kobina Ghartey
- *Department of Internal Medicine, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
| | - Matthew Leonard
- *Department of Internal Medicine, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
| | - Joel Linden
- *Department of Internal Medicine, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
- †Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
- ‡Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
| | - Amy L. Tucker
- *Department of Internal Medicine, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
- †Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
- ‡Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A
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Kreckler LM, Wan TC, Ge ZD, Auchampach JA. Adenosine inhibits tumor necrosis factor-alpha release from mouse peritoneal macrophages via A2A and A2B but not the A3 adenosine receptor. J Pharmacol Exp Ther 2006; 317:172-80. [PMID: 16339914 DOI: 10.1124/jpet.105.096016] [Citation(s) in RCA: 165] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Adenosine is elaborated in injured tissues where it suppresses inflammatory responses of essentially all immune cells, including production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the A(2A) adenosine receptor (A(2A)AR). Previously, however, it has been shown that the A(3)AR agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA) potently inhibited TNF-alpha release from macrophages obtained from A(2A)AR "knockout" (A(2A)KO) mice, suggesting that the A(3)AR may also regulate cytokine expression. Here, we confirmed that the A(2A)AR is the primary AR subtype that suppresses TNF-alpha release from thioglycollate-elicited mouse peritoneal macrophages induced by both Toll-like receptor-dependent (TLR) and TLR-independent stimuli, but we determined that the A(2B)AR rather than the A(3)AR mediates the non-A(2A)AR actions of adenosine since 1) the ability of IB-MECA to inhibit TNF-alpha release was not altered in macrophages isolated from A(3)KO mice, and 2) the A(2B)AR antagonist 1,3-dipropyl-8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]xanthine (MRS 1754) blocked the ability of the nonselective AR agonist adenosine-5'-N-ethylcarboxamide (NECA) to inhibit TNF-alpha release from macrophages isolated from A(2A)KO mice. Although A(2B)ARs seem capable of inhibiting TNF-alpha release, the A(2A)AR plays a dominant suppressive role since MRS 1754 did not block the ability of NECA to inhibit TNF-alpha release from macrophages isolated from wild-type (WT) mice. Furthermore, the potency and efficacy of adenosine to inhibit TNF-alpha release from WT macrophages were not influenced by blocking A(2B)ARs with MRS 1754. The data indicate that adenosine suppresses TNF-alpha release from macrophages primarily via A(2A)ARs, although the A(2B)AR seems to play an underlying inhibitory role that may contribute to the anti-inflammatory actions of adenosine under select circumstances.
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MESH Headings
- Adenosine/physiology
- Adenosine A2 Receptor Agonists
- Adenosine A2 Receptor Antagonists
- Adenosine A3 Receptor Agonists
- Adenosine A3 Receptor Antagonists
- Animals
- In Vitro Techniques
- Macrophages, Peritoneal/drug effects
- Macrophages, Peritoneal/metabolism
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Receptor, Adenosine A2A/genetics
- Receptor, Adenosine A2A/metabolism
- Receptor, Adenosine A2B/genetics
- Receptor, Adenosine A2B/metabolism
- Receptor, Adenosine A3/genetics
- Receptor, Adenosine A3/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Tumor Necrosis Factor-alpha/antagonists & inhibitors
- Tumor Necrosis Factor-alpha/biosynthesis
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Affiliation(s)
- Laura M Kreckler
- Department of Pharmacology, Medical College of Wisconsin, Milwaukee, 53226, USA
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