Discovery process and pharmacological characterization of a novel dual orexin 1 and orexin 2 receptor antagonist useful for treatment of sleep disorders

The hypothalamic peptides orexin-A and orexin-B are potent agonists of two G-protein coupled receptors, namely the OX(1) and the OX(2) receptor. These receptors are widely distributed, though differentially, in the rat brain. In particular, the OX(1) receptor is highly expressed throughout the hypothalamus, whilst the OX(2) receptor is mainly located in the ventral posterior nucleus. A large body of compelling evidence, both pre-clinical and clinical, suggests that the orexin system is profoundly implicated in sleep disorders. In particular, modulation of the orexin receptors activation by appropriate antagonists was proven to be an efficacious strategy for the treatment of insomnia in man. A novel, drug-like bis-amido piperidine derivative was identified as potent dual OX(1) and OX(2) receptor antagonists, highly effective in a pre-clinical model of sleep.     

Hypersensitization of the Orexin 1 receptor by the CB1 receptor: evidence for cross-talk blocked by the specific CB1 antagonist,SR141716

In the present study, we observed evidence of cross-talk between the cannabinoid receptor CB1 and the orexin 1 receptor (OX1R) using a heterologous system. When the two receptors are co-expressed, we observed a major CB1-dependent enhancement of the orexin A potency to activate the mitogen-activated protein kinase pathway; dose-responses curves indicated a 100-fold increase in the potency of orexin-mediated mitogen-activated protein kinase activation. This effect required a functional CB1 receptor as evidenced by the blockade of the orexin response by the specific CB1 antagonist, N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716), but also by pertussis toxin, suggesting that this potentiation is Gi-mediated. In contrast to OX1R, the potency of direct activation of CB1 was not affected by co-expression with OX1R. In addition, electron microscopy experiments revealed that CB1 and OX1R are closely apposed at the plasma membrane level; they are close enough to form hetero-oligomers. Altogether, for the first time our data provide evidence that CB1 is able to potentiate an orexigenic receptor. Considering the antiobesity effect of SR141716, these results open new avenues to understand the mechanism by which the molecule may prevent weight gain through functional interaction between CB1 and other receptors involved in the control of appetite.     

Dynein light chain Tctex-type 1 modulates orexin signaling through its interaction with orexin 1 receptor

Orexins (OX-A, OX-B) are neuropeptides involved in the regulation of the sleep-wake cycle, feeding and reward, via activation of orexin receptors 1 and 2 (OX1R, OX2R). The loss of orexin peptides or functional OX2R has been shown to cause the sleep disorder, narcolepsy. Since the regulation of orexin receptors remains largely undefined, we searched for novel protein partners of the intracellular tail of orexin receptors. Using a yeast two-hybrid screening strategy in combination with co-immunoprecipitation experiments, we found interactions between OX1R and the dynein light chains Tctex-type 1 and 3 (Dynlt1, Dynlt3). These interactions were mapped to the C-terminal region of the dynein light chains and to specific residues within the last 10 amino acids of OX1R. Hence, we hypothesized that dynein light chains could regulate orexin signaling. In HEK293 cells expressing OX1R, stimulation with OX-A produced a less sustained extracellular signal-regulated kinases 1/2 (ERK1/2) activation when Dynlt1 was co-expressed, while it was prolonged under reduced Dynlt1 expression. The amount of OX1R located at the plasma membrane as well as the kinetics and extent of OX-A-induced internalization of OX1R (disappearance from membrane) were not altered by Dynlt1. However, Dynlt1 reduced the localization of OX1R in early endosomes following initial internalization. Taken together, these data suggest that Dynlt1 modulates orexin signaling by regulating OX1R, namely its intracellular localization following ligand-induced internalization.     

Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent coordinated alterations of receptor localization and function

Following inducible expression in HEK293 cells, the human orexin-1 receptor was targeted to the cell surface but became internalized following exposure to the peptide agonist orexin A. By contrast, constitutive expression of the human cannabinoid CB1 receptor resulted in a predominantly punctate, intracellular distribution pattern consistent with spontaneous, agonist-independent internalization. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors displaying the spontaneous internalization phenotype. Single cell fluorescence resonance energy transfer imaging indicated the two receptors were present as heterodimers/oligomers in intracellular vesicles. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor resulted in re-localization of the receptor to the cell surface. Although SR-141716A has no significant affinity for the orexin-1 receptor, in cells co-expressing the CB1 receptor, the orexin-1 receptor was also re-localized to the cell surface by treatment with SR-141716A. Treatment of cells co-expressing the orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in re-localization of both receptors to the cell surface. Treatment with SR-141716A resulted in decreased potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. Treatment with SB-674042 also reduced the potency of a CB1 receptor agonist to phosphorylate ERK1/2 only when the two receptors were co-expressed. These studies introduce an entirely novel pharmacological paradigm, whereby ligands modulate the function of receptors for which they have no significant inherent affinity by acting as regulators of receptor heterodimers.     

Intramolecular fluorescence resonance energy transfer (FRET) sensors of the orexin OX1 and OX2 receptors identify slow kinetics of agonist activation

Intramolecular fluorescence resonance energy transfer (FRET) sensors able to detect changes in distance or orientation between the 3rd intracellular loop and C-terminal tail of the human orexin OX(1) and OX(2) G protein-coupled receptors following binding of agonist ligands were produced and expressed stably. These were directed to the plasma membrane and, despite the substantial sequence alterations introduced, in each case were able to elevate [Ca(2+)](i), promote phosphorylation of the ERK1/2 MAP kinases and become internalized effectively upon addition of the native orexin peptides. Detailed characterization of the OX(1) sensor demonstrated that it was activated with rank order of potency orexin A > orexin B > orexin A 16-33, that it bound antagonist ligands with affinity similar to the wild-type receptor, and that mutation of a single residue, D203A, greatly reduced the binding and function of orexin A but not antagonist ligands. Addition of orexin A to individual cells expressing an OX(1) sensor resulted in a time- and concentration-dependent reduction in FRET signal consistent with mass-action and potency/affinity estimates for the peptide. Compared with the response kinetics of a muscarinic M(3) acetylcholine receptor sensor upon addition of agonist, response of the OX(1) and OX(2) sensors to orexin A was slow, consistent with a multistep binding and activation process. Such sensors provide means to assess the kinetics of receptor activation and how this may be altered by mutation and sequence variation of the receptors.     

The SK-N-MC cell line expresses an orexin binding site different from recombinant orexin 1-type receptor.

Orexin A and B (also known as hypocretins), two recently discovered neuropeptides, play an important role in food intake, sleep/wake cycle and neuroendocrine functions. Orexins are endogenous ligands of two G-protein-coupled receptors, termed OX1 and OX2. This work presents the first short orexin A and B analogues, orexin A 23-33 and orexin B 18-28, with high affinity (119 +/- 49 and 49 +/- 23 nm) for OX1 receptors expressed on SK-N-MC cells and indicates the importance of the C-terminal part of the orexin peptides for this ligand-receptor interaction. However, these C-terminal fragments of orexin did not displace the 125I-labelled orexin B from the recombinant orexin 1 receptor stably expressed in Chinese hamster ovary cells. To examine the role of the shortened orexin A 23-33 in feeding, its effects in mimicking or antagonizing the effects of orexin A were studied in rats after administration via the lateral hypothalamus. In contrast with orexin A, which potently induced feeding up to 4 h after administration, orexin A 23-33 neither induced feeding nor inhibited orexin A-induced feeding. Modafinil (Vigil), which was shown earlier to activate orexin neurons, displayed binding neither to the orexin receptor expressed on SK-N-MC cells nor to the recombinant orexin 1 receptor, which indicates that modafinil displays its antinarcoleptic action via another yet unknown mechanism. PCR and subsequent sequencing revealed expression of the full-length orexin 1 receptor mRNA in SK-N-MC and NT-2 cells. Interestingly, sequencing of several cDNA clones derived from RNA of both SK-N-MC and NT-2 cells differed from the published nucleotide sequence at position 1375. Amino acid prediction of this A -->G change results in an isoleucine --> valine substitution at the protein level, which may provide evidence for an editing process.     

N-Glycine-sulfonamides as potent dual orexin 1/orexin 2 receptor antagonists

A series of dual OX(1)R/OX(2)R orexin antagonists was prepared based on a N-glycine-sulfonamide core. SAR studies of a screening hit led to compounds with low nanomolar affinity for both receptors and good oral bioavailability. One of these compounds, 47, has demonstrated in vivo activity in rats following oral administration.     

Cannabinoid receptors CB1 and CB2 form functional heteromers in brain

Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.     

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

We wanted to analyze the basis for the distinction between OX(1) and OX(2) orexin receptors by the known agonists, orexin-A, orexin-B and Ala(11), D-Leu(15)-orexin-B, of which the latter two show some selectivity for OX(2). For this, chimaeric OX(1)/OX(2) and OX(2)/OX(1) orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca(2+) elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor's ligand profile.     

Hypocretin/orexin type 1 receptor in brain: role in cardiovascular control and the neuroendocrine response to immobilization stress

Hypocretin/orexin acts pharmacologically in the hypothalamus to stimulate stress hormone secretion at least in part by an action in the hypothalamic paraventricular nucleus, where the peptide's receptors have been localized. In addition, orexin acts in the brain to increase sympathetic tone and, therefore, mean arterial pressure and heart rate. We provide evidence for the role of endogenously produced hypocretin/orexin in the physiological response to immobilization stress and identify the receptor subtype responsible for this action of the peptide. Antagonism of the orexin type 1 receptor (OX(1)R) in the brain prevented the ACTH-stimulating effect of centrally administered hypocretin/orexin. Furthermore, pretreatment of animals with the OX(1)R antagonist blocked the ACTH response to immobilization/restraint stress. The OX(1)R antagonist did not, however, block the pharmacological or physiological release of prolactin in these two models. Antagonism of the OX(1)R also blocked the central action of orexin to elevate mean arterial pressures and heart rates in conscious rats. These data suggest receptor subtype-selective responses to hypocretin/orexin and provide further evidence for the importance of endogenously produced peptide in the physiological control of stress hormone secretion.     

A novel phosphorylation site on orexin receptor 1 regulating orexinA-induced GRK2-biased signaling

Drug discovery efforts targeting G protein–coupled receptors (GPCRs) have succeeded in developing multiple medications for treating various human diseases including cancer, metabolic disorders, and inflammatory disorders. These medications are broadly classified as either agonists or antagonists that respectively promote or inhibit receptor activation by endogenous stimuli. However, there has been a growing appreciation that GPCR biased signaling between G protein- and β-arrestin-dependent signaling in particular is a promising method for improving drug efficacy and therapy. Orexin receptor 1 (OX1R), a member of the GPCRs, is an important drug target in the central nervous system. In this study, we identified a novel regulatory phosphorylation site (Ser-262) on OX1R that abolished its capability to interact with GRK2, but did not affect its interaction with G proteins, GRK5, or β-arrestin1/2 activation, indicating that Ser-262 is a key amino acid for OX1R internalization that contributes to induction of GRK2-dependent biased signaling via orexin A. Our findings could potentially lead to the development of new drug targets for the prevention and treatment of insomnia, narcolepsy, and substance abuse, with fewer side effects than existing therapies.     

Selective orexin receptor antagonists

The orexin, or hypocretin, neuropeptides (orexin-A and orexin-B) are produced on neurons in the hypothalamus which project to key areas of the brain that control sleep–wake states, modulation of food intake, panic, anxiety, emotion, reward and addictive behaviors. These neuropeptides exert their effects on a pair of G-protein coupled receptors termed the orexin-1 (OX1) and orexin-2 (OX2) receptors. Emerging biology suggests the involvement of these receptors in psychiatric disorders as they are thought to play a key role in the regulation of multiple systems. This review is intended to highlight key selective OX1 or OX2 small-molecule antagonists.     

Adrenal expression of orexin receptor subtypes is differentially regulated in experimental streptozotocin induced type-1 diabetes

Orexins (hypocretins) are involved in the regulation of energy homeostasis and sleeping behavior. Orexins were also implicated in the regulation of neuroendocrine and autonomic functions. Recent data show the expression of orexin receptors within the hypothalamic-pituitary-adrenal (HPA) axis and suggest specific actions of orexins at the pituitary and adrenal glands. To further evaluate the role of orexin in the HPA axis, we investigated the mRNA expression of prepro-orexin (PPO) and orexin receptors within the HPA axis of streptozotocin-injected (STZ) rats showing type-1 like diabetes. PPO, as well as OX(1) and OX(2) receptor levels were analyzed by quantitative real-time PCR (qPCR). STZ rats were characterized by decreased body weight, plasma insulin, and leptin levels and by increased plasma glucose. Hypothalamic PPO mRNA levels were significantly reduced in STZ compared to non-diabetic control rats. No differences were found in the mRNA levels of hypothalamic or pituitary OX(1) and OX(2) receptors between control and STZ rats. In adrenals, OX(1) receptor mRNA levels were significantly elevated in STZ rats while OX(2) receptors were significantly reduced. Our results imply distinct functions of adrenal orexin receptor subtypes during type-1 like diabetes.     

Novel substituted 4-phenyl-[1,3]dioxanes: potent and selective orexin receptor 2 (OX(2)R) antagonists

Orexins, also termed hypocretins, consist of two neuropeptide agonists (orexin A and B) interacting with two known G-protein coupled receptors (OX(1)R and OX(2)R). In addition to other biological functions, the orexin-2 receptor is thought to be an important modulator of sleep and wakefulness. Herein we describe a series of novel, selective OX(2)R antagonists consisting of substituted 4-phenyl-[1,3]dioxanes. One such antagonist is compound 9, 1-(2,4-dibromo-phenyl)-3-((4S,5S)-2,2-dimethyl-4-phenyl-[1,3]dioxan-5-yl)-urea, which is bound by the OX(2)R with a pK(i) of 8.3, has a pK(b) of 7.9, and is 600-fold selective for the OX(2)R over the OX(1)R.     

Signaling transduction regulated by 5-hydroxytryptamine 1A receptor and orexin receptor 2 heterodimers

As G-protein-coupled receptors (GPCRs), 5-hydroxytryptamine 1A receptor (5-HT1AR) and orexin receptor 2 (OX2R) regulate the levels of the cellular downstream molecules. The heterodimers of different GPCRs play important roles in various of neurological diseases. Moreover, 5-HT1AR and OX2R are involved in the pathogenesis of neurological diseases such as depression with deficiency of hippocampus plasticity. However, the direct interaction of the two receptors remains elusive. In the present study, we firstly demonstrated the heterodimer formation of 5-HT1AR and OX2R. Exchange protein directly activated by cAMP (Epac) cAMP bioluminescence resonance energy transfer (BRET) biosensor analysis revealed that the expression levels of cellular cAMP significantly increased in HEK293T cells transfected with the two receptors compared with the 5-HT1AR group. Additionally, the cellular level of calcium was upregulated robustly in HEK293T cells co-transfected with 5-HT1AR and OX2R group after agonist treatment. Furthermore, western blotting data showed that 5-HT1AR and OX2R heterodimer decreased the levels of phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element-binding protein (CREB). These results not only unraveled the formation of 5-HT1AR and OX2R heterodimer but also suggested that the heterodimer affected the downstream signaling pathway, which will provide new insights into the function of the two receptors in the brain.     

Inverse agonism and neutral antagonism at cannabinoid CB1 receptors

There are at least two types of cannabinoid receptor, CB1 and CB2, both G protein coupled. CB1 receptors are expressed predominantly at nerve terminals and mediate inhibition of transmitter release whereas CB2 receptors are found mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous cannabinoid receptor agonists also exist and these "endocannabinoids" together with their receptors constitute the "endocannabinoid system". These discoveries were followed by the development of a number of CB1- and CB2-selective antagonists that in some CB1 or CB2 receptor-containing systems also produce "inverse cannabimimetic effects", effects opposite in direction from those produced by cannabinoid receptor agonists. This review focuses on the CB1-selective antagonists, SR141716A, AM251, AM281 and LY320135, and discusses possible mechanisms by which these ligands produce their inverse effects: (1) competitive surmountable antagonism at CB1 receptors of endogenously released endocannabinoids, (2) inverse agonism resulting from negative, possibly allosteric, modulation of the constitutive activity of CB1 receptors in which CB1 receptors are shifted from a constitutively active "on" state to one or more constitutively inactive "off" states and (3) CB1 receptor-independent mechanisms, for example antagonism of endogenously released adenosine at A1 receptors. Recently developed neutral competitive CB1 receptor antagonists, which are expected to produce inverse effects through antagonism of endogenously released endocannabinoids but not by modulating CB1 receptor constitutive activity, are also discussed. So too are possible clinical consequences of the production of inverse cannabimimetic effects, there being convincing evidence that released endocannabinoids can have "autoprotective" roles.     

The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptor-stimulated cannabinoid CB1 receptor activity

The cannabinoid CB1 receptor (CB1R) is a G protein-coupled receptor, which couples to the Gi/o family of heterotrimeric G proteins. The receptor displays both basal and agonist-induced signaling and internalization. Although basal activity of CB1Rs is attributed to constitutive (agonist-independent) receptor activity, studies in neurons suggested a role of postsynaptic endocannabinoid (eCB) release in the persistent activity of presynaptic CB1Rs. To elucidate the role of eCBs in basal CB1R activity, we have investigated the role of diacylglycerol lipase (DAGL) in this process in Chinese hamster ovary (CHO) cells, which are not targeted specifically with eCBs. Agonist-induced G protein activation was determined by detecting dissociation G protein subunits expressed in CHO cells with bioluminescence resonance energy transfer (BRET), after labeling the alpha and beta subunits with Renilla luciferase and enhanced yellow fluorescent protein (EYFP), respectively. Preincubation of the cells with tetrahydrolipstatin (THL), a known inhibitor of DAGLs, caused inhibition of the basal activity of CB1R. Moreover, preincubation of CHO and cultured hippocampal neurons with THL increased the number of CB1Rs on the cell membrane, which reflects its inhibitory action on CB1R internalization in non-simulated cells. In CHO cells co-expressing CB1R and angiotensin AT1 receptors, angiotensin II-induced Go protein activation that was blocked by both a CB1R antagonist and THL. These data indicate that cell-derived eCB mediators have a general role in the basal activity of CB1Rs in non-neural cells and neurons, and that this mechanism can be stimulated by AT1 receptor activation.     

Proline bis-amides as potent dual orexin receptor antagonists

A series of OX(2)R/OX(1)R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered. N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed peripherally in a pharmacodynamic model of orexin activity.     

Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.