Pharmacological Actions of Carbamate Insecticides at Mammalian Melatonin Receptors.

Integrated in silico chemical clustering and melatonin receptor molecular modeling combined with in vitro 2-[125I]-iodomelatonin competition binding were used to identify carbamate insecticides with affinity for human melatonin receptor 1 (hMT1) and human melatonin receptor 2 (hMT2). Saturation and kinetic binding studies with 2-[125I]-iodomelatonin revealed lead carbamates (carbaryl, fenobucarb, bendiocarb, carbofuran) to be orthosteric ligands with antagonist apparent efficacy at hMT1 and agonist apparent efficacy at hMT2 Furthermore, using quantitative receptor autoradiography in coronal brain slices from C3H/HeN mice, carbaryl, fenobucarb, and bendiocarb competed for 2-[125I]-iodomelatonin binding in the suprachiasmatic nucleus (SCN), paraventricular nucleus of the thalamus (PVT), and pars tuberalis (PT) with affinities similar to those determined for the hMT1 receptor. Carbaryl (10 mg/kg i.p.) administered in vivo also competed ex vivo for 2-[125I]-iodomelatonin binding to the SCN, PVT, and PT, demonstrating the ability to reach brain melatonin receptors in C3H/HeN mice. Furthermore, the same dose of carbaryl given to C3H/HeN mice in constant dark for three consecutive days at subjective dusk (circadian time 10) phase-advanced circadian activity rhythms (mean = 0.91 hours) similar to melatonin (mean = 1.12 hours) when compared with vehicle (mean = 0.04 hours). Carbaryl-mediated phase shift of overt circadian activity rhythm onset is likely mediated via interactions with SCN melatonin receptors. Based on the pharmacological actions of carbaryl and other carbamate insecticides at melatonin receptors, exposure may modulate time-of-day information conveyed to the master biologic clock relevant to adverse health outcomes. SIGNIFICANCE STATEMENT: In silico chemical clustering and molecular modeling in conjunction with in vitro bioassays identified several carbamate insecticides (i.e., carbaryl, carbofuran, fenobucarb, bendiocarb) as pharmacologically active orthosteric melatonin receptor 1 and 2 ligands. This work further demonstrated that carbaryl competes for melatonin receptor binding in the master biological clock (suprachiasmatic nucleus) and phase-advances overt circadian activity rhythms in C3H/HeN mice, supporting the relevance of circadian effects when interpreting toxicological findings related to carbamate insecticide exposure.

Molecular evolution of melatonin receptor genes (mtnr) in vertebrates and its shedding light on mtnr1c.

Melatonin receptors (MTNRs) play important roles in regulation of circadian rhythms and seasonal reproduction. However, their origin and evolution in vertebrates have not been investigated. Here, we performed a comprehensive examination by comparative genome mining of MTNRs in vertebrates. We successfully extracted 164 putative encoding sequences for MTNRs (including 57 mtnr1a, 59 mtnr1b and 48 mtnr1c) from 45 high-quality representative genomes. Interestingly, the putative expansions of mtnr1a and mtnr1b in zebrafish were also identified in other Cyprinifomes, but not in other orders of teleost. Using phylogenetic interference, we observed this expansion to be clustered into a primitive position of the Actinopterygii, which may be resulted from teleost-specific genome duplication. The C-terminal extension of MTNR1C, predicted to be proteoglycan 4 (PRG4), originated after the speciation of Monotremata or Marsupialia. Our present genomics survey provides novel insights into the evolution of MTNRs in vertebrates and updates our understanding of these proteins.

The five dimensions of receptor pharmacology exemplified by melatonin receptors: An opinion.

Receptology has been complicated with enhancements in our knowledge of G-protein-coupled-receptor (GPCR) biochemistry. This complexity is exemplified by the pharmacology of melatonin receptors. Here, we describe the complexity of GPCR biochemistry in five dimensions: (a) receptor expression, particularly in organs/tissues that are only partially understood; (b) ligands and receptor-associated proteins (interactome); (c) receptor function, which might be more complex than the known G-protein-coupled systems; (d) ligand bias, which favors a particular pathway; and (e) receptor dimerization, which might concern all receptors coexpressed in the same cell. Thus, receptor signaling might be modified or modulated, depending on the nature of the receptor complex. Fundamental studies are needed to clarify these points and find new ways to tackle receptor functionality. This opinion article emphasizes the global questions attached to new descriptions of GPCRs and aims to raise our awareness of the tremendous complexity of modern receptology.

Structural insights into melatonin receptors.

The long-anticipated high-resolution structures of the human melatonin G protein-coupled receptors MT1 and MT2 , involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti-insomnia and antidepression drugs using X-ray free-electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane-buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT2 from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT2 structure allowed accurate mapping of type 2 diabetes-related single-nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein-membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT1 and MT2 with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next-generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects.

Is There Sufficient Evidence that the Melatonin Binding Site MT3 Is Quinone Reductase 2?

In the 1980s, researchers used binding studies to show that there is a melatonin binding site in addition to the receptors described previously. It was first termed ML2 and then, in 1999, MT3 Purification efforts led to its identification as quinone reductase 2. Several lines of evidence support the notion that MT3 is the same as quinone reductase 2, including the detection and characterization of MT3 whenever quinone reductase 2 was added to various systems under various conditions. This evidence is discussed in this review, which summarizes the results of relevant cellular and animal experiments. The recent discovery that the quinone reductase 2 enzyme can be partly membrane-associated may unite the current body of evidence and allow us to conclude definitively that the third melatonin binding site, MT3 , is indeed quinone reductase 2.

Characterization of the Mel1c melatoninergic receptor in platypus (Ornithorhynchus anatinus).

Melatonin is a neurohormone produced in both animals and plants. It binds at least three G-protein-coupled receptors: MT1 and MT2, and Mel1cGPR. Mammalian GPR50 evolved from the reptilian/avian Mel1c and lost its capacity to bind melatonin in all the therian mammal species that have been tested. In order to determine if binding is lost in the oldest surviving mammalian lineage of monotremes we investigated whether the melatonin receptor has the ability to bind melatonin in the platypus (Ornithorhynchus anatinus), and evaluated its pharmacological profile. Sequence and phylogenetic analysis showed that platypus has in fact retained the ancestral Mel1c and has the capacity to bind melatonin similar to other mammalian melatonin receptors (MT1 and MT2), with an affinity in the 1 nM range. We also investigated the binding of a set of melatoninergic ligands used previously to characterize the molecular pharmacology of the melatonin receptors from sheep, rats, mice, and humans and found that the general profiles of these compounds make Mel1c resemble human MT1 more than MT2. This work shows that the loss of GPR50 binding evolved after the divergence of monotremes less than 190MYA in therian mammals.

Update on melatonin receptors: IUPHAR Review 20.

Melatonin receptors are seven transmembrane-spanning proteins belonging to the GPCR superfamily. In mammals, two melatonin receptor subtypes exist - MT1 and MT2 - encoded by the MTNR1A and MTNR1B genes respectively. The current review provides an update on melatonin receptors by the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology. We will highlight recent developments of melatonin receptor ligands, including radioligands, and give an update on the latest phenotyping results of melatonin receptor knockout mice. The current status and perspectives of the structure of melatonin receptor will be summarized. The physiological importance of melatonin receptor dimers and biologically important and type 2 diabetes-associated genetic variants of melatonin receptors will be discussed. The role of melatonin receptors in physiology and disease will be further exemplified by their functions in the immune system and the CNS. Finally, antioxidant and free radical scavenger properties of melatonin and its relation to melatonin receptors will be critically addressed.

Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles.

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

Amyloid ß peptide directly impairs pineal gland melatonin synthesis and melatonin receptor signaling through the ERK pathway.

Melatonin is the hormone produced by the pineal gland known to regulate physiologic rhythms and to display immunomodulatory and neuroprotective properties. It has been reported that Alzheimer disease patients show impaired melatonin production and altered expression of the 2 G protein-coupled melatonin receptors (MTRs), MT1 and MT2, but the underlying mechanisms are not known. Here we evaluated whether this dysfunction of the melatonergic system is directly caused by amyloid ß peptides (Aß(1-40) and Aß(1-42)). Aß treatment of rat pineal glands elicited an inflammatory response within the gland, evidenced by the up-regulation of 52 inflammatory genes, and decreased the production of melatonin up to 75% compared to vehicle-treated glands. Blocking NF-κB activity prevented this effect. Exposure of HEK293 cells stably expressing recombinant MT1 or MT2 receptors to Aß lead to a 40% reduction in [(125)I]iodomelatonin binding to MT1. ERK1/2 activation triggered by MTRs, but not by the ß2-adrenergic receptor, was markedly impaired by Aß in HEK293 transfected cells, as well as in primary rat endothelial cells expressing endogenous MTRs. Our data reveal the melatonergic system as a new target of Aß, opening new perspectives to Alzheimer disease diagnosis and therapeutic intervention.

Melatonin enhances chondrogenic differentiation of human mesenchymal stem cells.

Intramembranous ossification and endochondral ossification are two ways through which bone formation and fracture healing occur. Accumulating amounts of evidence suggests that melatonin affects osteoblast differentiation, but little is known about the effects of melatonin on the process of chondrogenic differentiation. In this study, the effects of melatonin on human mesenchymal stem cells (MSCs) undergoing chondrogenic differentiation were investigated. Cells were induced along chondrogenic differentiation via high-density micromass culture in chondrogenic medium containing vehicle or 50 nm melatonin. Histological study and quantitative analysis of glycosaminoglycan (GAG) showed induced cartilage tissues to be larger and richer in GAG, collagen type II and collagen type X in the melatonin group than in the untreated controls. Real-time RT-PCR analysis demonstrated that melatonin treatment significantly up-regulated the expression of the genes involved in chondrogenic differentiation, including aggrecan (ACAN), collagen type II (COL2A1), collagen type X (COL10A1), SRY (sex-determining region Y)-box 9 (SOX9), runt-related transcription factor 2 (RUNX2) and the potent inducer of chondrogenic differentiation, bone morphogenetic protein 2 (BMP2). And the expression of melatonin membrane receptors (MT) MT1 and MT2 were detected in the chondrogenic-induced-MSCs by immunofluorescence staining. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to increase the size and GAG synthesis of the induced cartilage tissues, as well as to completely reverse the effect of melatonin on the gene expression of ACAN, COL2A1, COL10A1, SOX9 and BMP2 after 7 days of differentiation. These findings demonstrate that melatonin enhances chondrogenic differentiation of human MSCs at least partially through melatonin receptors.

A molecular and chemical perspective in defining melatonin receptor subtype selectivity.

Melatonin is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle. In addition to its intrinsic antioxidant property, the neurohormone has renowned regulatory roles in the control of circadian rhythm and exerts its physiological actions primarily by interacting with the G protein-coupled MT1 and MT2 transmembrane receptors. The two melatonin receptor subtypes display identical ligand binding characteristics and mediate a myriad of signaling pathways, including adenylyl cyclase inhibition, phospholipase C stimulation and the regulation of other effector molecules. Both MT1 and MT2 receptors are widely expressed in the central nervous system as well as many peripheral tissues, but each receptor subtype can be linked to specific functional responses at the target tissue. Given the broad therapeutic implications of melatonin receptors in chronobiology, immunomodulation, endocrine regulation, reproductive functions and cancer development, drug discovery and development programs have been directed at identifying chemical molecules that bind to the two melatonin receptor subtypes. However, all of the melatoninergics in the market act on both subtypes of melatonin receptors without significant selectivity. To facilitate the design and development of novel therapeutic agents, it is necessary to understand the intrinsic differences between MT1 and MT2 that determine ligand binding, functional efficacy, and signaling specificity. This review summarizes our current knowledge in differentiating MT1 and MT2 receptors and their signaling capacities. The use of homology modeling in the mapping of the ligand-binding pocket will be described. Identification of conserved and distinct residues will be tremendously useful in the design of highly selective ligands.

Melatonin receptor genes in vertebrates.

Melatonin receptors are members of the G protein-coupled receptor (GPCR) family. Three genes for melatonin receptors have been cloned. The MT1 (or Mel1a or MTNR1A) and MT2 (or Mel1b or MTNR1B) receptor subtypes are present in humans and other mammals, while an additional melatonin receptor subtype, Mel1c (or MTNR1C), has been identified in fish, amphibians and birds. Another melatonin related orphan receptor, GPR50, which does not bind melatonin, is found exclusively in mammals. The hormone melatonin is secreted primarily by the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone acts systemically in numerous organs. In the brain, it is involved in the regulation of various neural and endocrine processes, and it readjusts the circadian pacemaker, the suprachiasmatic nucleus. This article reviews recent studies of gene organization, expression, evolution and mutations of melatonin receptor genes of vertebrates. Gene polymorphisms reveal that numerous mutations are associated with diseases and disorders. The phylogenetic analysis of receptor genes indicates that GPR50 is an outgroup to all other melatonin receptor sequences. GPR50 may have separated from a melatonin receptor ancestor before the split between MTNR1C and the MTNR1A/B ancestor.

Synthesis of new N-(arylcyclopropyl)acetamides and N-(arylvinyl)acetamides as conformationally-restricted ligands for melatonin receptors.

N-(Arylcyclopropyl)acetamides and N-(arylvinyl)acetamides or methyl ureas have been prepared as constrained analogues of melatonin. The affinity of these new compounds for chicken brain melatonin receptors and recombinant human MT(1) and MT(2) receptors was evaluated using 2-[(125)I]-iodomelatonin as radioligand. Strict ethylenic or cyclopropyl analogues of the commercialized agonist agomelatine (Valdoxan®) were equipotent to agomelatine in binding bioassays. However, the ethylenic analogue was more effective than the cyclopropyl one in the melanophore aggregation bioassay, but was still less potent than the disubstituted 2,7-dimethoxy-naphtalenic compounds.

Synthesis and configuration determination of all enantiopure stereoisomers of the melatonin receptor ligand 4-phenyl-2-propionamidotetralin using an expedient optical resolution of 4-phenyl-2-tetralone.

An efficient and practical approach for the synthesis of all four stereoisomers of the MT(2) melatonin receptor ligand 4-phenyl-2-propionamidotetralin (4-P-PDOT), each in enantiomerically pure form (ee > 99.9%), was developed. The strategy involved an optical resolution procedure of the key precursor (±)-4-phenyl-2-tetralone with the unusual resolving agent (S)-mandelamide, through the formation of four dihydronaphtalene-spiro-oxazolidin-4-one diastereomers. Interestingly, NMR experimental observations in combination with geometric calculations, provided unambiguous configuration assignments of all stereocenters of the key spiro stereoisomers. Cleavage of each single spiro diastereomer under acidic conditions gave enantiopure (R)- or (S)-4-phenyl-2-tetralone, which were then converted to each 4-P-PDOT single enantiomer by using stereoselective reactions.

Bivalent ligand approach on N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide: synthesis, binding affinity and intrinsic activity for MT(1) and MT(2) melatonin receptors.

We report the synthesis, binding properties and intrinsic activity at MT(1) and MT(2) melatonin receptors of new dimeric melatonin receptor ligands in which two units of the monomeric agonist N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide (1) are linked together through different anchor points. Dimerization of compound 1 through the methoxy substituent leads to a substantial improvement in selectivity for the MT(1) receptor, and to a partial agonist behavior. Compound 3a, with a trimethylene linker, was the most selective for the MT(1) subtype (112-fold selectivity) and compound 3d, characterized by a hexamethylene spacer, had the highest MT(1) binding affinity (pK(iMT1)=8.47) and 54-fold MT(1)-selectivity. Dimerization through the aniline nitrogen of 1 abolished MT(1) selectivity, leading to compounds with either a full agonist or an antagonist behavior depending on the nature of the linker.

Design and synthesis of naphthalenic derivatives as new ligands at the melatonin binding site MT3.

Naphthalenic analogs of MCA-NAT (5-methoxycarbonylamino-N-acetyltryptamine) have been synthesized and evaluated as melatonin receptor ligands. Introduction of a methoxycarbonylamino substituent at the C-7 position of the naphthalenic nucleus yields MT3 selective ligands. This selectivity can be modulated with suitable variations of the C-7 position and the acyl group on the C-1 side chain. We identified new series of compounds with affinity for the MT3 binding site in the nanomolar range, and singled out a selective ligand, (N-[2-(7-methylsulfamoyl-naphth-1-yl)ethyl]acetamide (17), with a Ki of 4.9 nM and selectivity of 1024 and 2040 versus MT1 and MT2 receptors respectively.

International Union of Basic and Clinical Pharmacology. LXXV. Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors.

The hormone melatonin (5-methoxy-N-acetyltryptamine) is synthesized primarily in the pineal gland and retina, and in several peripheral tissues and organs. In the circulation, the concentration of melatonin follows a circadian rhythm, with high levels at night providing timing cues to target tissues endowed with melatonin receptors. Melatonin receptors receive and translate melatonin's message to influence daily and seasonal rhythms of physiology and behavior. The melatonin message is translated through activation of two G protein-coupled receptors, MT(1) and MT(2), that are potential therapeutic targets in disorders ranging from insomnia and circadian sleep disorders to depression, cardiovascular diseases, and cancer. This review summarizes the steps taken since melatonin's discovery by Aaron Lerner in 1958 to functionally characterize, clone, and localize receptors in mammalian tissues. The pharmacological and molecular properties of the receptors are described as well as current efforts to discover and develop ligands for treatment of a number of illnesses, including sleep disorders, depression, and cancer.

Mutation screening of melatonin-related genes in patients with autism spectrum disorders.

BACKGROUND: One consistent finding in autism spectrum disorders (ASD) is a decreased level of the pineal gland hormone melatonin and it has recently been demonstrated that this decrease to a large extent is due to low activity of the acetylserotonin O-methyltransferase (ASMT), the last enzyme in the melatonin synthesis pathway. Moreover, mutations in the ASMT gene have been identified, including a splice site mutation, that were associated with low ASMT activity and melatonin secretion, suggesting that the low ASMT activity observed in autism is, at least partly, due to variation within the ASMT gene. METHODS: In the present study, we have investigated all the genes involved in the melatonin pathway by mutation screening of AA-NAT (arylalkylamine N-acetyltransferase), ASMT, MTNR1A, MTNR1B (melatonin receptor 1A and 1B) and GPR50 (G protein-coupled receptor 50), encoding both synthesis enzymes and the three main receptors of melatonin, in 109 patients with autism spectrum disorders (ASD). A cohort of 188 subjects from the general population was used as a comparison group and was genotyped for the variants identified in the patient sample. RESULTS: Several rare variants were identified in patients with ASD, including the previously reported splice site mutation in ASMT (IVS5+2T>C). Of the variants affecting protein sequence, only the V124I in the MTNR1B gene was absent in our comparison group. However, mutations were found in upstream regulatory regions in three of the genes investigated, ASMT, MTNR1A, and MTNR1B. CONCLUSIONS: Our report of another ASD patient carrying the splice site mutation IVS5+2T>C, in ASMT further supports an involvement of this gene in autism. Moreover, our results also suggest that other melatonin related genes might be interesting candidates for further investigation in the search for genes involved in autism spectrum disorders and related neurobehavioral phenotypes. However, further studies of the novel variants identified in this study are warranted to shed light on their potential role in the pathophysiology of these disorders.

MT3/QR2 melatonin binding site does not use melatonin as a substrate or a co-substrate.

Quinone reductase 2 (QR2, E.C. 1.10.99.2) is implicated in cell reactive oxygen species production. The catalytic activity of this enzyme is inhibited by 1 microM of melatonin. QR2 was identified as the third melatonin binding site (MT3). It is of major importance to understand the exact roles of melatonin and QR2 in oxidative stress. A fascinating possibility that melatonin could serve as a co-substrate or substrate of QR2 was hypothesized recently. In the current investigation, nuclear magnetic resonance studies of the QR2 catalytic reaction were performed, the results led us to conclude that, whatever the conditions, melatonin is not cleaved off to form N1-acetyl-N2-formyl-5-methoxykynurenine by a catalytically active QR2, very strongly indicating that melatonin is neither a substrate nor a co-substrate of this enzyme. Further studies are needed in order to better understand the relationship between MT3/QR2, melatonin and redox status of the cells, in order to better explain the anti-oxidant activities of melatonin at pharmacological concentrations (>1 microM).

A novel quantitative structure-activity relationship method to predict the affinities of MT3 melatonin binding site.

The linear regression (LR) and non-linear regression methods--grid search-support vector machine (GS-SVM) and projection pursuit regression (PPR) were used to develop quantitative structure-activity relationship (QSAR) models for a series of derivatives of naphthalene, benzofurane and indole with respect to their affinities to MT3/quinone reductase 2 (QR2) melatonin binding site. Five molecular descriptors selected by genetic algorithm (GA) were used as the input variables for the LR model and two non-linear regression approaches. Comparison of the results of the three methods indicated that PPR was the most accurate approach in predicting the affinities of the MT3/QR2 melatonin binding site. This confirmed the capability of PPR for the prediction of the binding affinities of compounds. Moreover, it should facilitate the design and development of new selective MT3/QR2 ligands.