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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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate ...these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, IDO1, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
G protein-coupled receptors (GPCRs) continue to be important discovery targets for the treatment of type 2 diabetes mellitus (T2DM). Many GPCRs are directly involved in the development of insulin ...resistance and
-cell dysfunction, and in the etiology of inflammation that can lead to obesity-induced T2DM. This review summarizes the current literature describing a number of well-validated GPCR targets, but also outlines several new and promising targets for drug discovery. We highlight the importance of understanding the role of these receptors in the disease pathology, and their basic pharmacology, which will pave the way to the development of novel pharmacological probes that will enable these targets to fulfill their promise for the treatment of these metabolic disorders.
Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS where they control a variety of neuronal functions. Due to their roles in a number of CNS processes, mAChRs have long been ...a target of the drug discovery industry; however, the only mAChR ligands approved for use in the clinic are non-selective antagonists for the treatment of Parkinson's disease. This article briefly reviews recent progress made in mAChR drug discovery for Alzheimer's disease (AD), schizophrenia and Parkinson's disease, with particular emphasis on novel target validation, as well as highlighting novel indications such as drug addiction.
Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. ...Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.
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•2.1-, 2.1-, and 2.5-Å GLP-1R:Gs structures bound to GLP-1 and non-peptide agonists•Critical water networks stabilize peptide and small-molecule agonist binding•PF 06882961, but not CHU-128, closely mimics the GLP-1 signaling profile•Insight into how select non-peptide agonists mimic receptor activation by GLP-1
Zhang et al. show that the non-peptide GLP-1R agonist PF 06882961, but not CHU-128, was able to closely mimic the in vitro pharmacological fingerprint of GLP-1. These pharmacological activities could be rationalized from high-resolution structures that provide a novel template for the design of new agonists targeting the GLP-1R.
Chronic inflammation is a component of numerous diseases including autoimmune, metabolic, neurodegenerative, and cancer. The discovery and characterization of specialized pro-resolving mediators ...(SPMs) critical to the resolution of inflammation, and their cognate G protein-coupled receptors (GPCRs) has led to a significant increase in the understanding of this physiological process. Approximately 20 ligands, including lipoxins, resolvins, maresins, and protectins, and 6 receptors (FPR2/ALX, GPR32, GPR18, chemerin1, BLT1, and GPR37) have been identified highlighting the complex and multilayered nature of resolution. Therapeutic efforts in targeting these receptors have proved challenging, with very few ligands apparently progressing through to preclinical or clinical development. To date, some knowledge gaps remain in the understanding of how the activation of these receptors, and their downstream signaling, results in efficient resolution via apoptosis, phagocytosis, and efferocytosis of polymorphonuclear leukocytes (mainly neutrophils) and macrophages. SPMs bind and activate multiple receptors (ligand poly-pharmacology), while most receptors are activated by multiple ligands (receptor pleiotropy). In addition, allosteric binding sites have been identified signifying the capacity of more than one ligand to bind simultaneously. These fundamental characteristics of SPM receptors enable alternative targeting strategies to be considered, including biased signaling and allosteric modulation. This review describes those ligands and receptors involved in the resolution of inflammation, and highlights the most recent clinical trial results. Furthermore, we describe alternative mechanisms by which these SPM receptors could be targeted, paving the way for the identification of new therapeutics, perhaps with greater efficacy and fidelity.
Schizophrenia remains a sizable socio‐economic burden that continues to be treated with therapeutics based on 70‐year old science. All currently approved therapeutics primarily target the dopamine D2 ...receptor to achieve their efficacy. Whilst dopaminergic dysregulation is a key feature in this disorder, the targeting of dopaminergic machinery has yielded limited efficacy and an appreciable side effect burden. Over the recent decades, numerous drugs that engage non‐dopaminergic G protein‐coupled receptors (GPCRs) have yielded a promise of efficacy without the deleterious side effect profile, yet none have successfully completed clinical studies and progressed to the market. More recently, there has been increased attention around non‐dopaminergic GPCR‐targeting drugs, which demonstrated efficacy in some schizophrenia symptom domains. This provides renewed hope that effective schizophrenia treatment may lie outside of the dopaminergic space. Despite the potential for muscarinic receptor‐ (and other well‐characterised GPCR families) targeting drugs to treat schizophrenia, they are often plagued with complications such as lack of receptor subtype selectivity and peripheral on‐target side effects. Orphan GPCR studies have opened a new avenue of exploration with many demonstrating schizophrenia‐relevant mechanisms and a favourable expression profile, thus offering potential for novel drug development. This review discusses centrally expressed orphan GPCRs: GPR3, GPR6, GPR12, GPR52, GPR85, GPR88 and GPR139 and their relationship to schizophrenia. We review their expression, signalling mechanisms and cellular function, in conjunction with small molecule development and structural insights. We seek to provide a snapshot of the growing evidence and development potential of new classes of schizophrenia therapeutics.
LINKED ARTICLES
This article is part of a themed issue Therapeutic Targeting of G Protein‐Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc
Orexins are neuropeptides that activate the rhodopsin-like G proteincoupled receptors OX1R and OX2R. The orexin system plays an important role in the regulation of the sleep-wake cycle and the ...regulation of feeding and emotions. The nonselective orexin receptor antagonist suvorexant has been the first drug on the market targeting the orexin system and is prescribed for the treatment of insomnia. Subtype-selective OX1R antagonists are valuable tools to further investigate the functions and physiological role of the OX1R in vivo and promising lead compounds for the treatment of drug addiction, anxiety, pain or obesity. Starting from the OX1R and OX2R crystal structures bound to suvorexant, we exploited a single amino acid difference in the orthosteric binding site by using molecular docking and structure-based drug design to optimize ligand interactions with the OX1R while introducing repulsive interactions with the OX2R. A newly established enantiospecific synthesis provided ligands showing up to 75-fold selectivity for the OX1R over the OX2R subtype. The structure of a new OX1R antagonist with subnanomolar affinity (JH112) was determined by crystallography in complex with the OX1R and corresponded closely to the dockingpredicted geometry. JH112 exhibits high selectivity over a panel of different GPCRs, is able to cross the blood–brain barrier and acts as slowly diffusing and insurmountable antagonist for Gq protein activation and in particular β-arrestin-2 recruitment at OX1R. This study demonstrates the potential of structure-based drug design to develop more subtype-selective GPCR ligands with potentially reduced side effects and provides an attractive probe molecule and lead compound.
Despite 50+ years of drug discovery, current antipsychotics have limited efficacy against negative and cognitive symptoms of schizophrenia, and are ineffective-with the exception of clozapine-against ...any symptom domain for patients who are treatment resistant. Novel therapeutics with diverse non-dopamine D
receptor targets have been explored extensively in clinical trials, yet often fail due to a lack of efficacy despite showing promise in preclinical development. This lack of translation between preclinical and clinical efficacy suggests a systematic failure in current methods that determine efficacy in preclinical rodent models. In this review, we critically evaluate rodent models and behavioural tests used to determine preclinical efficacy, and look to clinical research to provide a roadmap for developing improved translational measures. We highlight the dependence of preclinical models and tests on dopamine-centric theories of dysfunction and how this has contributed towards a self-reinforcing loop away from clinically meaningful predictions of efficacy. We review recent clinical findings of distinct dopamine-mediated dysfunction of corticostriatal circuits in patients with treatment-resistant vs. non-treatment-resistant schizophrenia and suggest criteria for establishing rodent models to reflect such differences, with a focus on objective, translational measures. Finally, we review current schizophrenia drug discovery and propose a framework where preclinical models are validated against objective, clinically informed measures and preclinical tests of efficacy map onto those used clinically.
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