International Union of Basic and Clinical Pharmacology CXIV: Orexin Receptor Function, Nomenclature and Pharmacology.

The orexin system consists of the peptide transmitters orexin-A and -B and the G protein-coupled orexin receptors OX1 and OX2 Orexin receptors are capable of coupling to all four families of heterotrimeric G proteins, and there are also other complex features of the orexin receptor signaling. The system was discovered 25 years ago and was immediately identified as a central regulator of sleep and wakefulness; this is exemplified by the symptomatology of the disorder narcolepsy with cataplexy, in which orexinergic neurons degenerate. Subsequent translation of these findings into drug discovery and development has resulted to date in three clinically used orexin receptor antagonists to treat insomnia. In addition to sleep and wakefulness, the orexin system appears to be a central player at least in addiction and reward, and has a role in depression, anxiety and pain gating. Additional antagonists and agonists are in development to treat, for instance, insomnia, narcolepsy with or without cataplexy and other disorders with excessive daytime sleepiness, depression with insomnia, anxiety, schizophrenia, as well as eating and substance use disorders. The orexin system has thus proved an important regulator of numerous neural functions and a valuable drug target. Orexin prepro-peptide and orexin receptors are also expressed outside the central nervous system, but their potential physiological roles there remain unknown. SIGNIFICANCE STATEMENT: The orexin system was discovered 25 years ago and immediately emerged as an essential sleep-wakefulness regulator. This discovery has tremendously increased the understanding of these processes and has thus far resulted in the market approval of three orexin receptor antagonists, which promote more physiological aspects of sleep than previous hypnotics. Further, orexin receptor agonists and antagonists with different pharmacodynamic properties are in development since research has revealed additional potential therapeutic indications. Orexin receptor signaling is complex and may represent novel features.

Characterization of a putative orexin receptor in Ciona intestinalis sheds light on the evolution of the orexin/hypocretin system in chordates.

Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.

Azulene as a biphenyl mimetic in orexin/hypocretin receptor agonists.

Azulene is a rare ring structure in drugs, and we investigated whether it could be used as a biphenyl mimetic in known orexin receptor agonist Nag 26, which is binding to both orexin receptors OX1 and OX2 with preference towards OX2. The most potent azulene-based compound was identified as an OX1 orexin receptor agonist (pEC50 = 5.79 ± 0.07, maximum response = 81 ± 8% (s.e.m. of five independent experiments) of the maximum response to orexin-A in Ca2+ elevation assay). However, the azulene ring and the biphenyl scaffold are not identical in their spatial shape and electron distribution, and their derivatives may adopt different binding modes in the binding site.

Orexin receptors in GtoPdb v.2021.3.

Orexin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Orexin receptors [42]) are activated by the endogenous polypeptides orexin-A and orexin-B (also known as hypocretin-1 and -2; 33 and 28 aa) derived from a common precursor, preproorexin or orexin precursor, by proteolytic cleavage and some typical peptide modifications [109]. Currently the only orexin receptor ligands in clinical use are suvorexant and lemborexant, which are used as hypnotics. Orexin receptor crystal structures have been solved [134, 133, 54, 117, 46].

Cellular Signaling Mechanisms of Hypocretin/Orexin.

Orexin receptors (OXRs) are promiscuous G-protein-coupled receptors that signal via several G-proteins and, putatively, via other proteins. On which basis the signal pathways are selected and orchestrated is largely unknown. We also have an insufficient understanding of the kind of signaling that is important for specific types of cellular responses. OXRs are able to form complexes with several other G-protein-coupled receptors in vitro, and one possibility is that the complexing partners regulate the use of certain signal transducers. In the central nervous system neurons, the main acute downstream responses of OXR activation are the inhibition of K+ channels and the activation of the Na+/Ca2+ exchanger and non-selective cation channels of unknown identity. The exact nature of the intracellular signal chain between the OXRs and these downstream targets is yet to be elucidated, but the Gq-phospholipase C (PLC) protein kinase C pathway - which is a significant signaling pathway for OXRs in recombinant cells - may be one of the players in neurons. The Gq-PLC pathway may also, under certain circumstances, take the route to diacylglycerol lipase, which leads to the production of the potent endocannabinoid (eCB), 2-arachidonoyl glycerol, and thereby connects orexins with eCB signaling. In addition, OXRs have been studied in the context of neurodegeneration and cancer cell death. Overall, OXR signaling is complex, and it can change depending on the cell type and environment.

Orexin-A measurement in narcolepsy: A stability study and a comparison of LC-MS/MS and immunoassays.

BACKGROUND: Orexin-A and -B are neuropeptides involved in sleep-wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus. Cerebrospinal fluid (CSF) orexin-A measurement is used in the diagnosis of narcolepsy type 1. Currently available immunoassays may lack specificity for accurate orexin quantification. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for CSF orexin-A and B. METHODS: We used CSF samples from narcolepsy type 1 (n = 22) and type 2 (n = 6) and non-narcoleptic controls (n = 44). Stable isotope-labeled orexin-A and -B internal standards were added to samples before solid-phase extraction and quantification by LC-MS/MS. The samples were also assayed by commercial radioimmunoassay (RIA, n = 42) and enzymatic immunoassay (EIA, n = 72) kits. Stability of orexins in CSF was studied for 12 months. RESULTS: Our assay has a good sensitivity (10 pmol/L = 35 pg/mL) and a wide linear range (35-3500 pg/mL). Added orexin-A and -B were stable in CSF for 12 and 3 months, respectively, when frozen. The median orexin-A concentration in CSF from narcolepsy type 1 patients was <35 pg/mL (range < 35-131 pg/mL), which was lower than that in CSF from control individuals (98 pg/mL, range < 35-424 pg/mL). Orexin-A concentrations determined using our LC-MS/MS assay were five times lower than those measured with a commercial RIA. Orexin-B concentrations were undetectable. CONCLUSIONS: Orexin-A concentrations measured by our LC-MS/MS assay were lower in narcolepsy type 1 patients as compared to controls. RIA yielded on average higher concentrations than LC-MS/MS.

Allosteric interactions via the orthosteric ligand binding sites in a constitutive G-protein-coupled receptor homodimer.

I interpret some recent data to indicate that co-operative effects take place between the (identical) orthosteric binding sites in a G-protein-coupled receptor dimer. In the current study, the reasonability of this concept was tested by creating a mathematical model. The model is composed of a symmetrical constitutive receptor dimer in which the protomers are able to affect each other allosterically, and it includes binding, receptor activation and signal amplification steps. The model was utilized for analyses of previous data as well as simulations of predicted behaviour. The model demonstrates the behaviour stated in the hypotheses, i.e. even an apparently neutral receptor ligand can allosterically affect agonist binding or receptor activation by binding to the normal orthosteric ligand binding site. Therewith the speculated allosteric action originating from the orthosteric binding site of the dimeric receptor is a realistic possibility. The results of the simulations and curve fitting constitute a reasonable starting point for further studies, and the model can be utilized to design meaningful experiments to investigate these questions.

Prolyl oligopeptidase inhibition by KYP-2407 increases alpha-synuclein fibril degradation in neuron-like cells.

Growing evidence emphasizes insufficient clearance of pathological alpha-synuclein (αSYN) aggregates in the progression of Parkinson's disease (PD). Consequently, cellular degradation pathways represent a potential therapeutic target. Prolyl oligopeptidase (PREP) is highly expressed in the brain and has been suggested to increase αSYN aggregation and negatively regulate the autophagy pathway. Inhibition of PREP with a small molecule inhibitor, KYP-2407, stimulates autophagy and reduces the oligomeric species of αSYN aggregates in PD mouse models. However, whether PREP inhibition has any effects on intracellular αSYN fibrils has not been studied before. In this study, the effect of KYP2407 on αSYN preformed fibrils (PFFs) was tested in SH-SY5Y cells and human astrocytes. Immunostaining analysis revealed that both cell types accumulated αSYN PFFs intracellularly but KYP-2047 decreased intracellular αSYN deposits only in SH-SY5Y cells, as astrocytes did not show any PREP activity. Western blot analysis confirmed the reduction of high molecular weight αSYN species in SH-SY5Y cell lysates, and secretion of αSYN from SH-SY5Y cells also decreased in the presence of KYP-2407. Accumulation of αSYN inside the SH-SY5Y cells resulted in an increase of the auto-lysosomal proteins p62 and LC3BII, as well as calpain 1 and 2, which have been shown to be associated with PD pathology. Notably, treatment with KYP-2407 significantly reduced p62 and LC3BII levels, indicating an increased autophagic flux, and calpain 1 and 2 levels returned to normal in the presence of KYP-2407. Our findings indicate that PREP inhibition can potentially be used as therapy to reduce the insoluble intracellular αSYN aggregates.

No evidence of autoimmunity to human OX1 or OX2 orexin receptors in Pandemrix-vaccinated narcoleptic children.

Narcolepsy type 1, likely an immune-mediated disease, is characterized by excessive daytime sleepiness and cataplexy. The disease is strongly associated with human leukocyte antigen (HLA) DQB1∗06:02. A significant increase in the incidence of childhood and adolescent narcolepsy was observed after a vaccination campaign with AS03-adjuvanted Pandemrix influenza vaccine in Nordic and several other countries in 2010 and 2011. Previously, it has been suggested that a surface-exposed region of influenza A nucleoprotein, a structural component of the Pandemrix vaccine, shares amino acid residues with the first extracellular domain of the human OX2 orexin/hypocretin receptor eliciting the development of autoantibodies. Here, we analyzed, whether H1N1pdm09 infection or Pandemrix vaccination contributed to the development of autoantibodies to the orexin precursor protein or the OX1 or OX2 receptors. The analysis was based on the presence or absence of autoantibody responses against analyzed proteins. Entire OX1 and OX2 receptors or just their extracellular N-termini were transiently expressed in HuH7 cells to determine specific antibody responses in human sera. Based on our immunofluorescence analysis, none of the 56 Pandemrix-vaccinated narcoleptic patients, 28 patients who suffered from a laboratory-confirmed H1N1pdm09 infection or 19 Pandemrix-vaccinated controls showed specific autoantibody responses to prepro-orexin, orexin receptors or the isolated extracellular N-termini of orexin receptors. We also did not find any evidence for cell-mediated immunity against the N-terminal epitopes of OX2. Our findings do not support the hypothesis that the surface-exposed region of the influenza nucleoprotein A would elicit the development of an immune response against orexin receptors.

CRK2 Enhances Salt Tolerance by Regulating Callose Deposition in Connection with PLDα1.

High salinity is an increasingly prevalent source of stress to which plants must adapt. The receptor-like protein kinases, including members of the Cys-rich receptor-like kinase (CRK) subfamily, are a highly expanded family of transmembrane proteins in plants that are largely responsible for communication between cells and the extracellular environment. Various CRKs have been implicated in biotic and abiotic stress responses; however, their functions on a cellular level remain largely uncharacterized. Here we have shown that CRK2 enhances salt tolerance at the germination stage in Arabidopsis (Arabidopsis thaliana) and also modulates root length. We established that functional CRK2 is required for salt-induced callose deposition. In doing so, we revealed a role for callose deposition in response to increased salinity and demonstrated its importance for salt tolerance during germination. Using fluorescently tagged proteins, we observed specific changes in the subcellular localization of CRK2 in response to various stress treatments. Many of CRK2's cellular functions were dependent on phospholipase D activity, as were the subcellular localization changes. Thus, we propose that CRK2 acts downstream of phospholipase D during salt stress, promoting callose deposition and regulating plasmodesmal permeability, and that CRK2 adopts specific stress-dependent subcellular localization patterns that allow it to carry out its functions.

Structure-Activity Relationships of 1-Benzoylazulenes at the OX1 and OX2 Orexin Receptors.

We previously demonstrated the potential of di- or trisubstituted azulenes as ligands (potentiators, weak agonists, and antagonists) of the orexin receptors. In this study we investigated 27 1-benzoylazulene derivatives, uncovering seven potentiators of the orexin response on OX1 and two weak dual orexin receptor agonists. For potentiators, replacement of the azulene scaffold by indole retained the activity of four out of six compounds. The structure-activity relationships for agonism and potentiation can be summarized into a bicyclic aromatic ring system substituted with two hydrogen-bond acceptors (1-position, benzoyl; 6-position, carboxyl/ester) within 7-8 Šof each other; a third acceptor at the 3-position is also well tolerated. The same pharmacophoric signature is found in the preferred conformations of the orexin receptor agonist Nag26 from molecular dynamics simulations. Subtle changes switch the activity between weak agonism and potentiation, suggesting overlapping binding sites.

Pharmacological characterization of the orexin/hypocretin receptor agonist Nag 26.

One promising series of small-molecule orexin receptor agonists has been described, but the molecular pharmacological properties, i.e. ability and potency to activate the different orexin receptor-regulated signal pathways have not been reported for any of these ligands. We have thus here assessed these properties for the most potent ligand of the series, 4'-methoxy-N,N-dimethyl-3'-[N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl sulfamoyl]-(1,1'-biphenyl)-3-carboxamide (Nag 26). Chinese hamster ovary-K1 cells expressing human orexin receptor subtypes OX1 and OX2 were used. Ca2+ elevation and cell viability and death were assessed by fluorescent methods, the extracellular signal-regulated kinase pathway by a luminescent Elk-1 reporter assay, and phospholipase C and adenylyl cyclase activities by radioactive methods. The data suggest that for the Gq-dependent responses, Ca2+, phospholipase C and Elk-1, Nag 26 is a full agonist for both receptors, though of much lower potency. However, saturation was not always reached for OX1, partially due to Nag 26's low solubility and partially because the response decreased at high concentrations. The latter occurs in the same range as some reduction of cell viability, which is independent of orexin receptors. Based on the EC50, Nag 26 was OX2-selective by 20-200 fold in different assays, with some indication of biased agonism (as compared to orexin-A). Nag 26 is a potent orexin receptor agonist with a largely similar pharmacological profile as orexin-A. However, its weaker potency (low-mid micromolar) and low water solubility as well as the non-specific effect in the mid-micromolar range may limit its usefulness under physiological conditions.

Azulene-based compounds for targeting orexin receptors.

A library of 70 000 synthetically accessible azulene-based compounds was virtually screened at the OX2 receptor. Based on the results, a series of azulene derivatives was synthesized and the binding to and activation of both orexin receptor subtypes were assessed. Two most promising binders were determined to have inhibition constants in the 3-9 µM range and two other compounds showed weak OX2 receptor agonism. Furthermore, three compounds exhibited a concentration-dependent potentiation of the response to orexin-A at the OX1 but not the OX2 receptors. Altogether this data opens new approaches for further development of antagonists, agonists, and potentiators of orexin response based on the azulene scaffold.

Endocannabinoid Signaling in Embryonic Neuronal Motility and Cell-Cell Contact - Role of mGluR5 and TRPC3 Channels.

Cell-cell communication plays a central role in the guidance of migrating neuronal precursor cells during the development of the cerebral cortex. Endocannabinoids (eCBs) have previously been shown to be one of the central factors regulating neuronal migration. In this study the effects of eCBs on different parameters, expected to affect embryonic cortical neuronal motility have been analyzed in neurosphere-derived neuroblasts using time-lapse microscopy. Increased endogenous production of the endocannabinoid 2-arachidonyl glycerol (2-AG) causes bursts of neuroblast motility. The neuroblasts move longer distances and show a low frequency of turning, and the number of neuron-neuron contacts are reduced. Similar changes occur interfering with the function of the metabotropic glutamate receptor 5 (mGluR5) or its transducer canonical transient receptor potential channel 3 (TRPC3) or the neuregulin receptor ErbB4. Blocking of 2-AG production reverses these effects. The data suggest that eCB-regulated neuronal motility is controlled by mGluR5/TRPC3 activity possibly via NRG/ErbB4 signaling.

Stapled truncated orexin peptides as orexin receptor agonists.

The peptides orexin-A and -B, the endogenous agonists of the orexin receptors, have similar 19-amino-acid C-termini which retain full maximum response as truncated peptides with only marginally reduced potency, while further N-terminal truncations successively reduce the activity. The peptides have been suggested to bind in an α-helical conformation, and truncation beyond a certain critical length is likely to disrupt the overall helical structure. In this study, we set out to stabilize the α-helical conformation of orexin-A15-33 via peptide stapling at four different sites. At a suggested hinge region, we varied the length of the cross-linker as well as replaced the staple with two α-aminoisobutyric acid residues. Modifications close to the peptide C-terminus, which is crucial for activity, were not allowed. However, central and N-terminal modifications yielded bioactive peptides, albeit with decreased potencies. This provides evidence that the orexin receptors can accommodate and be activated by α-helical peptides. The decrease in potency is likely linked to a stabilization of suboptimal peptide conformation or blocking of peptide backbone-receptor interactions at the hinge region by the helical stabilization or the modified amino acids.

Orexin Receptor Multimerization versus Functional Interactions: Neuropharmacological Implications for Opioid and Cannabinoid Signalling and Pharmacogenetics.

Orexins/hypocretins are neuropeptides formed by proteolytic cleavage of a precursor peptide, which are produced by neurons found in the lateral hypothalamus. The G protein-coupled receptors (GPCRs) for these ligands, the OX1 and OX2 orexin receptors, are more widely expressed throughout the central nervous system. The orexin/hypocretin system has been implicated in many pathways, and its dysregulation is under investigation in a number of diseases. Disorders in which orexinergic mechanisms are being investigated include narcolepsy, idiopathic sleep disorders, cluster headache and migraine. Human narcolepsy has been associated with orexin deficiency; however, it has only rarely been attributed to mutations in the gene encoding the precursor peptide. While gene variations within the canine OX2 gene hcrtr2 have been directly linked with narcolepsy, the majority of human orexin receptor variants are weakly associated with diseases (the idiopathic sleep disorders, cluster headache and polydipsia-hyponatremia in schizophrenia) or are of potential pharmacogenetic significance. Evidence for functional interactions and/or heterodimerization between wild-type and variant orexin receptors and opioid and cannabinoid receptors is discussed in the context of its relevance to depression and epilepsy.

Orexin receptor agonist Yan 7874 is a weak agonist of orexin/hypocretin receptors and shows orexin receptor-independent cytotoxicity.

Two promising lead structures of small molecular orexin receptor agonist have been reported, but without detailed analyses of the pharmacological properties. One of them, 1-(3,4-dichlorophenyl)-2-[2-imino-3-(4-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl]ethan-1-ol (Yan 7874), is commercially available, and we set out to analyze its properties. As test system we utilized human OX1 and OX2 orexin receptor-expressing Chinese hamster ovary (CHO) K1 cells as well as control CHO-K1 and neuro-2a neuroblastoma cells. Gq-coupling was assessed by measurement of intracellular Ca2+ and phospholipase C activity, and the coupling to Gi and Gs by adenylyl cyclase inhibition and stimulation, respectively. At concentrations above 1 µM, strong Ca2+ and low phospholipase C responses to Yan 7874 were observed in both OX1- and OX2-expressing cells. However, a major fraction of the response was not mediated by orexin receptors, as determined utilizing the non-selective orexin receptor antagonist N-biphenyl-2-yl-1-{[(1-methyl-1H-benzimidazol-2-yl)sulfanyl]acetyl}-L-prolinamide (TCS 1102) as well as control CHO-K1 cells. Yan 7874 did not produce any specific adenylyl cyclase response. Some experiments suggested an effect on cell viability by Yan 7874, and we thus analyzed this. Within a few hours of exposure, Yan 7874 markedly changed cell morphology (shrunken, rich in vacuoles), reduced growth, promoted cell detachment, and induced necrotic cell death. The effect was equal in cells expressing orexin receptors or not. Thus, Yan 7874 is a weak partial agonist of orexin receptors. It also displays strong off-target effects in the same concentration range, culminating in necrotic cell demise. This makes Yan 7874 unsuitable as orexin receptor agonist.