Structure-based discovery of potent and selective melatonin receptor agonists.

Melatonin receptors MT1 and MT2 are involved in synchronizing circadian rhythms and are important targets for treating sleep and mood disorders, type-2 diabetes and cancer. Here, we performed large scale structure-based virtual screening for new ligand chemotypes using recently solved high-resolution 3D crystal structures of agonist-bound MT receptors. Experimental testing of 62 screening candidates yielded the discovery of 10 new agonist chemotypes with sub-micromolar potency at MT receptors, with compound 21 reaching EC50 of 0.36 nM. Six of these molecules displayed selectivity for MT2 over MT1. Moreover, two most potent agonists, including 21 and a close derivative of melatonin, 28, had dramatically reduced arrestin recruitment at MT2, while compound 37 was devoid of Gi signaling at MT1, implying biased signaling. This study validates the suitability of the agonist-bound orthosteric pocket in the MT receptor structures for the structure-based discovery of selective agonists.

Virtual discovery of melatonin receptor ligands to modulate circadian rhythms.

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT1 and MT2. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle1-4. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep5,6 and depression1-4,7-9. Despite their importance, few in vivo active MT1-selective ligands have been reported2,8,10-12, hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT1 inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT1-selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT1- but not in MT2-knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT1-selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.

Melatonin MT1 and MT2 Receptors Exhibit Distinct Effects in the Modulation of Body Temperature across the Light/Dark Cycle.

Melatonin (MLT) is a neurohormone that regulates many physiological functions including sleep, pain, thermoregulation, and circadian rhythms. MLT acts mainly through two G-protein-coupled receptors named MT1 and MT2, but also through an MLT type-3 receptor (MT3). However, the role of MLT receptor subtypes in thermoregulation is still unknown. We have thus investigated the effects of selective and non-selective MLT receptor agonists/antagonists on body temperature (Tb) in rats across the 12/12-h light-dark cycle. Rectal temperature was measured every 15 min from 4:00 a.m. to 9:30 a.m. and from 4:00 p.m. to 9:30 p.m., following subcutaneous injection of each compound at either 5:00 a.m. or 5:00 p.m. MLT (40 mg/kg) had no effect when injected at 5 a.m., whereas it decreased Tb during the light phase only when injected at 5:00 p.m. This effect was blocked by the selective MT2 receptor antagonist 4P-PDOT and the non-selective MT1/MT2 receptor antagonist, luzindole, but not by the α1/MT3 receptors antagonist prazosin. However, unlike MLT, neither the selective MT1 receptor partial agonist UCM871 (14 mg/kg) nor the selective MT2 partial agonist UCM924 (40 mg/kg) altered Tb during the light phase. In contrast, UCM871 injected at 5:00 p.m. increased Tb at the beginning of the dark phase, whereas UCM924 injected at 5:00 a.m. decreased Tb at the end of the dark phase. These effects were blocked by luzindole and 4P-PDOT, respectively. The MT3 receptor agonist GR135531 (10 mg/kg) did not affect Tb. These data suggest that the simultaneous activation of both MT1 and MT2 receptors is necessary to regulate Tb during the light phase, whereas in a complex but yet unknown manner, they regulate Tb differently during the dark phase. Overall, MT1 and MT2 receptors display complementary but also distinct roles in modulating circadian fluctuations of Tb.

Structural basis of ligand recognition at the human MT1 melatonin receptor.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Melatonin receptor activation provides cerebral protection after traumatic brain injury by mitigating oxidative stress and inflammation via the Nrf2 signaling pathway.

Traumatic brain injury (TBI) is a principal cause of death and disability worldwide. Melatonin, a hormone made by the pineal gland, is known to have anti-inflammatory and antioxidant properties. In this study, using a weight-drop model of TBI, we investigated the protective effects of ramelteon, a melatonin MT1/MT2 receptor agonist, and its underlying mechanisms of action. Administration of ramelteon (10 mg/kg) daily at 10:00 a.m. alleviated TBI-induced early brain damage on day 3 and long-term neurobehavioral deficits on day 28 in C57BL/6 mice. Ramelteon also increased the protein levels of interleukin (IL)-10, IL-4, superoxide dismutase (SOD), glutathione, and glutathione peroxidase and reduced the protein levels of IL-1ß, tumor necrosis factor, and malondialdehyde in brain tissue and serum on days 1, 3, and 7 post-TBI. Similarly, ramelteon attenuated microglial and astrocyte activation in the perilesional cortex on day 3. Furthermore, ramelteon decreased Keap 1 expression, promoted nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear accumulation, and increased levels of downstream proteins, including SOD-1, heme oxygenase-1, and NQO1 on day 3 post-TBI. However, in Nrf2 knockout mice with TBI, ramelteon did not decrease the lesion volume, neuronal degeneration, or myelin loss on day 3; nor did it mitigate depression-like behavior or most motor behavior deficits on day 28. Thus, timed ramelteon treatment appears to prevent inflammation and oxidative stress via the Nrf2-antioxidant response element pathway and might represent a potential chronotherapeutic strategy for treating TBI.

Antidepressant potential of Uncaria rhynchophylla and its active flavanol, catechin, targeting melatonin receptors.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicines (TCMs) are fascinating sources for natural drug candidates. Uncaria rhynchophylla (Gouteng) is a famous TCM used for alleviating central nervous system (CNS) disorders, while its antidepressant constituents are still disputed. AIM OF THE STUDY: The present study was designed to assess the antidepressant property of U. rhynchophylla and characterize the active constituents targeting melatonin receptors which are closely related to CNS diseases. MATERIALS AND METHODS: The total extract and each fraction of U. rhynchophylla were extensively assessed for their agonistic activity on melatonin receptors in vitro. The following bioassay-guided fractionation yielded the active constituents, whose activity was confirmed by dose-dependent bioassay and antagonistic experiment on HEK293 cells. Their antidepressant effects were evaluated on forced swimming test (FST), tail suspension test (TST) and open-field test (OFT) mice models in vivo. Their metabolic profiles in mice plasma were analyzed by LCMS-IT-TOF. RESULTS: The stems and hooks of U. rhynchophylla were revealed with agonistic activity on melatonin receptors (MT1 and MT2). Under the guidance of bioassay, two flavanols, catechin and epicatechin were obtained and showed obviously activity agitating MT1 (EC50 = 25.8 and 156.1 µM) and MT2 (EC50 = 47.3 and 208.8 µM) receptors. The agonistic activity of catechin on melatonin receptors can be antagonized by luzindole at the concentrations of 1.57-100 µM. Catechin could significantly reduce the immobility time in both FST and TST mice models at doses of 80 and 40 mg/kg, without obvious effect on locomotor activity in OFT mice model. Five phase II (M1-M5) and one phase I (M6) metabolites of catechin were detected in mice plasma after intragastric (i.g.) administration. CONCLUSION: Catechin is a potent antidepressant candidate from U. rhynchophylla by targeting melatonin receptors. The main metabolic pathways of catechin in mice plasma are glucuronidation (M3) and methylated glucuronidation (M4 and M5). This study provides valuable information for understanding the antidepressant potency of Gouteng and its active constituents.

Neu-P11, a novel MT1/MT2 agonist, reverses diabetes by suppressing the hypothalamic-pituitary-adrenal axis in rats.

Excessive glucocorticoid (GC) in type 2 diabetes mellitus (T2DM) reduces insulin sensitivity, impairs ß-cell function, increases gluconeogenesis and glycogenolysis, impairs glucose uptake and metabolism, and reduces the insulinotropic effects of glucagon-like peptide 1. Melatonin, which serves as a physiological regulator of the hypothalamic-pituitary-adrenal (HPA) axis, has been suggested to have anti-diabetic effects. The objective of the present study was to investigate the effect of the MT1/MT2 melatonin agonist Neu-P11 on glucose and lipid metabolism in T2DM rats induced by a high fat diet combined with low doses of streptozotocin. T2DM rats were intragastrically administered melatonin (20mg/kg), Neu-P11 (20, 10, 5mg/kg), or a vehicle for 4 weeks. The results showed that the increased food intake, water consumption, hyperglycemia, glucose intolerance, and insulin resistance in T2DM rats were all improved by Neu-P11 treatment. Neu-P11 increased GC receptor expression and suppressed 11ß-hydroxysteroid dehydrogenase 1 activity in the hippocampus by enhancing GC sensitivity and HPA feedback, thus decreasing the high GC levels. Transcript levels of the glucose metabolism-related genes peroxisome proliferator-activated receptor-γ, glucose transporter type-4, and adiponectin in adipose tissue were significantly increased after Neu-P11 treatment, while leptin mRNA was significantly decreased. Furthermore, MT1 and MT2 protein levels were enhanced by Neu-P11. These data suggest that normalization of the hyperactivated HPA axis by melatonin and Neu-P11 in T2DM regulates metabolic profiles and insulin sensitivity, which may attenuate insulin resistance and glucose homeostasis. Because Neu-P11 has superior pharmacokinetics and a longer half-life than melatonin, it might be beneficial in treating obesity and T2DM.

(±)-Uncarilins A and B, Dimeric Isoechinulin-Type Alkaloids from Uncaria rhynchophylla.

(±)-Uncarilins A and B (1a/1b and 2a/2b), two pairs of unusual dimeric isoechinulin-type enantiomers with a symmetric four-membered core, were isolated from Uncaria rhynchophylla driven by LCMS-IT-TOF analyses. Their structures were elucidated by extensive 1D and 2D NMR spectra, X-ray diffraction, and ECD spectroscopic data. (-)-Uncarilin B (2a) showed activities on MT1 and MT2 receptors with agonistic rates of 11.26% and 52.44% at a concentration of 0.25 mM.

A Cluster Headache Responsive to Ramelteon, a Selective Melatonin MT1/MT2 Receptor Agonist.

Patients with cluster headaches occasionally fail to respond to conventional preventive treatments. We herein report a case of a patient with a cluster headache in which the symptoms were refractory to conventional preventive treatments except for high-dose glucocorticoids. The headache attacks occurred daily while sleeping, thus the patient suffered from insomnia. Ramelteon, a selective melatonin receptor agonist and a member of a new class of insomnia therapies, completely suppressed the attacks during sleep and provided rapid relief from insomnia. This is the first English case report to describe the efficacy of ramelteon as a preventive treatment for cluster headaches.

Lignans from the Fruits of Melia toosendan and Their Agonistic Activities on Melatonin Receptor MT1.

Investigation on the fruits of Melia toosendan afforded seven new lignans (1-7), along with seventeen known compounds (8-24). The structures of the new compounds, involving four neo-lignans (1-4), two sesquilignans (5-6), and a nor-lignan (7), were elucidated based on extensive spectroscopic analyses (high-resolution electrospray ionization mass spectra, ultraviolet, infrared, one-dimensional and two-dimensional nuclear magnetic resonance). Compound 24 exhibited activity on melatonin receptor type 1 with an agonistic rate of 57.77% at 1.02 mM according to the assay on HEK293 cell lines in vitro.

Tasimelteon: first global approval.

Tasimelteon (HETLIOZ™) is an orally bioavailable agonist of the melatonin MT1 and MT2 receptors that has been approved in the US for the treatment of non-24-hour sleep-wake disorder. It is the first US FDA-approved medication for this orphan indication. Melatonin is thought to play a role in governing the body's natural sleep-wake cycle through physiological processes regulated in the suprachiasmatic nucleus of the hypothalamus. The hormone is secreted by the pineal gland, with onset typically occurring when daylight begins to dim. In healthy, sighted individuals, the endogenous circadian period is a little over 24 hours, but is entrained to the 24-hour day through exposure to environmental cues, such as light and darkness. In the absence of these cues, synchronisation is lost and the circadian rhythm follows the intrinsic non-24-hour clock, resulting in disorders like non-24-hour sleep-wake disorder. Because the rhythm of endogenous melatonin is considered to be a measure of the human circadian phase, the carefully timed administration of melatonin analogues, such as tasimelteon, can potentially promote circadian readjustment. This article summarizes the milestones in the development of tasimelteon leading to this first approval for the treatment of non-24-hour sleep-wake disorder.

Circadian rhythms, melatonin and depression.

The master biological clock situated in the suprachiasmatic nuclei of the anterior hypothalamus plays a vital role in orchestrating the circadian rhythms of multiple biological processes. Increasing evidence points to a role of the biological clock in the development of depression. In seasonal depression and in bipolar disorders it seems likely that the circadian system plays a vital role in the genesis of the disorder. For major unipolar depressive disorder (MDD) available data suggest a primary involvement of the circadian system but further and larger studies are necessary to conclude. Melatonin and melatonin agonists have chronobiotic effects, which mean that they can readjust the circadian system. Seasonal affective disorders and mood disturbances caused by circadian malfunction are theoretically treatable by manipulating the circadian system using chronobiotic drugs, chronotherapy or bright light therapy. In MDD, melatonin alone has no antidepressant action but novel melatoninergic compounds demonstrate antidepressant properties. Of these, the most advanced is the novel melatonin agonist agomelatine, which combines joint MT1 and MT2 agonism with 5-HT(2C) receptor antagonism. Adding a chronobiotic effect to the inhibition of 5-HT(2C) receptors may explain the rapid impact of agomelatine on depression, since studies showed that agomelatine had an early impact on sleep quality and alertness at awakening. Further studies are necessary in order to better characterize the effect of agomelatine and other novel melatoninergic drugs on the circadian system of MDD patients. In summary, antidepressants with intrinsic chronobiotic properties offer a novel approach to treatment of depression.

ACS chemical neuroscience molecule spotlight on Valdoxen.

A new and novel non-SSRI potential treatment option for major depressive disorders is Valdoxen (agomelatine) which is currently in phase III clinical studies. Valdoxen is a norepinephrine disinhibitor (NNDI) and is also an antagonist of the 5-HT(2C) receptor.

Managing jet lag: Some of the problems and possible new solutions.

Jet lag is due to the misalignment of the internal circadian clock(s) with external time cues. For short stopovers (1-2 days) adapting the circadian system is not advised, and at present immediate circadian adaptation is virtually impossible. The use of short-term measures such as judicious naps, caffeine and short acting hypnotics to maintain alertness and sleep is preferred. For intermediate length stays (3-5 days) a phase position with the circadian nadir situated within the sleep period is desirable but difficult to achieve. For longer stays (more than 4-5 days) strategies to hasten adaptation include timed exposure to and avoidance of light. The use of artificial light enriched with short wavelengths may be beneficial. The American Academy of Sleep Medicine recommends the timed use of the chronobiotic melatonin to hasten adaptation. Large individual differences in rate and direction of adaptation make timing treatment according to individual circadian phase difficult. Individual differences in tolerance to the sleep deprivation of jet lag may relate to a length polymorphism in the human clock gene PER3. The maximum efficacy for jet lag avoidance is by pre-flight adaptation, however, this requires time and commitment.

Molecular mechanisms of melatonin anticancer effects.

The authors have shown that, via activation of its MT1 receptor, melatonin modulates the transcriptional activity of various nuclear receptors and the proliferation of both ER alpha+ and ER alpha- human breast cancer cells. Employing dominant-negative (DN) and dominant-positive (DP) G proteins, it was demonstrated that G alpha i2 proteins mediate the suppression of estrogen-induced ER alpha transcriptional activity by melatonin, whereas the G alpha q proteins mediate the enhancement of retinoid-induced RAR alpha transcriptional activity by melatonin. In primary human breast tumors, the authors' studies demonstrate an inverse correlation between ER alpha and MT1 receptor expression, and confocal microscopic studies demonstrate that the MT1 receptor is localized to the caveoli and that its expression can be repressed by estrogen and melatonin. Melatonin, via activation of its MT1 receptor, suppresses the development and growth of breast cancer by regulation of growth factors, regulation of gene expression, regulation of clock genes, inhibition of tumor cell invasion and metastasis, and even regulation of mammary gland development. The authors have previously reported that the clock gene, Period 2 (Per2), is not expressed in human breast cancer cells but that its reexpression in breast cancer cells results in increased expression of p53 and induction of apoptosis. The authors demonstrate that melatonin, via repression of ROR alpha transcriptional activity, blocks the expression of the clock gene BMAL1. Melatonin's blockade of BMAL1 expression is associated with the decreased expression of SIRT1, a member of the Silencing Information Regulator family and a histone and protein deacetylase that inhibits the expression of DNA repair enzymes (p53, BRCA1 & 2, and Ku70) and the expression of apoptosis-associated genes. Finally, the authors developed an MMTV-MT1-flag mammary knock-in transgenic mouse that displays reduced ductal branching, ductal epithelium proliferation, and reduced terminal end bud formation during puberty and pregnancy. Lactating female MT1 transgenic mice show a dramatic reduction in the expression of beta-casein and whey acidic milk proteins. Further analyses showed significantly reduced ER alpha expression in mammary glands of MT1 transgenic mice. These results demonstrate that the MT1 receptor is a major transducer of melatonin's actions in the breast, suppressing mammary gland development and mediating the anticancer actions of melatonin through multiple pathways.

Agomelatine: a novel mechanism of antidepressant action involving the melatonergic and the serotonergic system.

The clinical finding that depressive disorders are often associated with desynchronization of internal rhythms has encouraged the idea that resetting normal circadian rhythms may have antidepressant potential. Agomelatine, a naphthalene analog of melatonin, is both an agonist of human cloned melatonergic MT1 and MT2 receptors and a serotonin 5-HT2C receptor antagonist. Agomelatine combines zeitgeber (synchroniser of the circadian system) activity with neurotransmitter augmentation properties (enhances the levels of dopamine and noradrenaline in frontal cortex). The efficacy of agomelatine in treating depression has been shown in three short-term, pivotal, randomized, placebo-controlled studies. These studies have demonstrated agomelatine to be efficacious in Major Depressive Disorder at the standard dose of 25mg/day, with the possibility of increasing doses to 50mg/day in those patients with insufficient improvement. The number of adverse events during the treatment period was comparable to placebo. Four studies have shown the positive effect of agomelatine on sleep continuity and quality and shortening of sleep latency. Despite these promising data, further studies are needed to examine agomelatine's efficacy over a longer treatment period.

Pharmacology of a new antidepressant: benefit of the implication of the melatonergic system.

The limitations of current antidepressant medications merit the exploration of alternative agents with novel antidepressant mechanisms of action. The established clinical finding that desynchronization of internal rhythms plays an important role in the pathophysiology of depressive disorders has stimulated the idea that resetting normal circadian rhythms may have antidepressant potential. Recent experiments using the novel melatonin receptor agonist and serotonin 2 (5-HT2c) receptor antagonist agomelatine (S20098; N[2-(7-methoxy-1-naphthyl)ethyl]- acetamide) revealed a notable chronobiotic activity and clear antidepressant-like effects in a variety of preclinical models. Binding studies performed in vitro proved that agomelatine is a high-affinity agonist at both the melatonin MT1 and MT2 receptor types. In addition, these studies revealed that agomelatine, in contrast to melatonin, blocks 5-HT2c receptors with significant affinity. Antagonism of 5-HT2c receptors is reported for various established antidepressant compounds. The antidepressant properties of agomelatine are thus based on its melatonergic actions and 5-HT2c receptor antagonism.

Bicyclic melatonin receptor agonists containing a ring-junction nitrogen: Synthesis, biological evaluation, and molecular modeling of the putative bioactive conformation.

Employing 1,3-dipolar cycloaddition for the synthesis of the 7a-azaindole nucleus, analogues of melatonin have been synthesized and tested against human and amphibian melatonin receptors. Introducing a phenyl substituent in position 2 of the heterocyclic moiety significantly increased binding affinity to both the MT1 and MT2 receptors. Shifting the methoxy group from position 5 to 2 of the 7a-azaindole ring led to a substantial reduction of MT1 binding when MT2 recognition was maintained. We theoretically investigated the hypothesis whether the 2-methoxy function of the azamelatonin analogue 27 is able to mimic the 5-methoxy group of the neurohormone by directing its 2-methoxy function toward the methoxy binding site. DFT calculations and experimental binding differences of analogue compounds indicate that the energy gained by forming the methoxy-specific hydrogen-bond interaction should exceed the energy required for adopting an alternative conformation.