MT1 and MT2 Melatonin Receptors - American Chemical Society

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MT1 and MT2 Melatonin Receptors: Ligands, Models, Oligomers, and Therapeutic Potential Darius. P. Zlotos,*,† Ralf Jockers,‡,§,∥ Erika Cecon,⊥ Silvia Rivara,# and Paula A. Witt-Enderby∞ †

Department of Pharmaceutical Chemistry, The German University in Cairo, New Cairo City, 11835 Cairo, Egypt Inserm, U1016, Institut Cochin, Paris, France § CNRS UMR 8104, Paris, France ∥ Univ. Paris Descartes, Sorbonne Paris Cite, Paris, France ⊥ Department of Physiology, Institute of Bioscience, University of Sao Paulo, Sao Paulo 05508-090, Brazil # Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy ∞ Division of Pharmaceutical Sciences, School of Pharmacy, Duquesne University, 421 Mellon Hall, Pittsburgh, Pennsylvania 15282, United States ‡

S Supporting Information *

ABSTRACT: Numerous physiological functions of the pineal gland hormone melatonin are mediated via activation of two G-proteincoupled receptors, MT1 and MT2. The melatonergic drugs on the market, ramelteon and agomelatine, as well as the most advanced drug candidates under clinical evaluation, tasimelteon and TIK-301, are high-affinity nonselective MT1/MT2 agonists. A great number of MT2-selective ligands and, more recently, several MT1-selective agents have been reported to date. Herein, we review recent advances in the field focusing on high-affinity agonists and antagonists and those displaying selectivity toward MT1 and MT2 receptors. Moreover, the existing models of MT1 and MT2 receptors as well as the current status in the emerging field of melatonin receptor oligomerization are critically discussed. In addition to the already existing indications, such as insomnia, circadian sleep disorders, and depression, new potential therapeutic applications of melatonergic ligands including cardiovascular regulation, appetite control, tumor growth inhibition, and neurodegenerative diseases are presented.

1. INTRODUCTION Melatonin (Figure 1) is a hormone that is widely distributed in a variety of organisms, such as bacteria, unicellular algae, fungi, plants, vertebrates, and mammalians, including humans. In mammals, it is primarily produced by the pineal gland and released into blood circulation according to a circadian rhythm with high plasma levels at night. In 1917, McCord and Allen showed that extracts of the bovine pineal gland caused blanching of the skin of tadpoles.1 In 1958, Lerner et al. isolated the active compound from bovine pineal glands and named it melatonin because its skin-lightening effect resulted from promoting the aggregation of dark melanin granules in the dermal melanophore cells.2 One year later, the same research group elucidated the structure of melatonin as N-acetyl-5methoxytryptamine.3 The biosynthetic precursor of melatonin is tryptophan which is taken up from the blood into pinealocytes. Aromatic hydroxylation to 5-hydroxytryptophan followed by decarboxylation gives serotonin. In the rate-limiting step, the latter is acetylated to give N-acetylserotonin, which is finally converted to melatonin by O-methylation. The biosynthetic pathway © XXXX American Chemical Society

including the names of the enzymes involved is depicted in Figure 1. Melatonin is classified as a dietary supplement in the USA and in other countries and is often used to alleviate the symptoms of jet lag and as a sleep-promoting agent. However, it shows an unfavorable pharmacokinetic profile, such as high first pass metabolism and rapid elimination (half-life of 20−30 min), limiting its efficacy for many possible treatment purposes.4 Circulating melatonin is metabolized primarily in the liver by aromatic C6-hydroxylation and subsequent sulfate conjugation. A minor metabolic reaction is demethylation to Nacetylserotonin.5 In extrahepatic tissues, enzymatic N-deacetylation to 5-methoxytryptamine can occur. Moreover, nonenzymatic and enzymatic oxidative transformations are also possible. For instance, in the brain, an oxidative ring cleavage occurs leading to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) which is further deformylated to N1-acetyl-5methoxykynuramine (AFK).6 A possible intermediate for the Received: September 1, 2013

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dx.doi.org/10.1021/jm401343c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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melatonin via activation of distinct melatonin receptors, MT1 and MT2, produces diverse physiological responses in the body entraining sleep/wake,11 modulating the immune system,12 and modulating the cardiovascular system;11 however, other emerging areas include a role for melatonin and melatonin receptors in cancer protection,13−15 glucose regulation,16 bone physiology,17 and neurodegenerative disorders.11,18 These newer areas will be the focus of this review describing the potential for novel melatonergic ligands as future therapeutic agents for treating cardiovascular disease, cancer, neurodegenerative diseases, and obesity.

2. G-PROTEIN-COUPLED MELATONIN RECEPTORS 2.1. Melatonin Receptor Binding and Functional Assays. Melatonin receptors are predominantly coupled to heterotrimeric Gi/o proteins. The affinities for the different members of this subfamily (Gαi1, Gαi2, Gαi3, Gαo1, Gαo2) are currently unknown, and the precise coupling profile will also depend on the relative expression levels of these G proteins in a given cellular context. Coupling to other G proteins has been reported. Among the different reports, coupling of the MT1 receptor to Gq/11 proteins has been most consistently observed. In HEK293 cells, MT1 specifically couples to Gαi2, Gαi3, and Gαq/11 proteins.19 In addition, MT1 and MT2 receptors bind to β-arrestins 1 and 2.20−22 Binding and activation of G proteins and β-arrestins to GPCRs are typically monitored by coimmunoprecipitation or BRET experiments.23 Both G proteins and β-arrestins are contributing to the binding of melatonin with high affinity upon formation of a ternary ligand−receptor−G protein (β-arrestin) (L−R−G) complex. The assembly of this complex can be directly measured in radioligand binding experiments typically using the melatonin receptor-specific 2-[ 125 I]iodomelatonin ([125I]MLT) radioligand.24,25 The first melatonin receptor specific radioligand was [3H]melatonin, which was, however, only used in a limited number of studies because of its low specific activity.26 More recently, three novel iodinated radioligands, 1 (SD6), 2 (S70254), and 3 (DIV880), have been developed27 (Figure 2). Compounds 1 and 2 are melatonin derivatives, and both are labeled with an iodine atom at the acetyl group. 3 has a completely different structure and has been identified as an MT2-specific ligand in a high throughput screen. Whereas 1 is a full agonist with high affinity for MT1 and MT2, 2 and 3 are MT2-selective partial agonists. Wide application of these new tools in radioligand binding and autoradiography studies is expected to expand our knowledge about melatonin receptors in the near future. Interestingly, Bmax values determined in [125I]MLT and [125I]1 saturation binding experiments were similar in cells expressing MT2 receptors but diverged by a factor of 2 in cells expressing MT1 receptors, indicating that both radioligands detect different ternary complexes. Despite major efforts, MT1-selective radioligands are currently not available. The fact that all available radioligands are agonists somehow renders the pharmacological analysis of the results more complex, as high-affinity binding of agonists depends not only on the binding of the agonist to the receptor but also on the activation of the G protein or β-arrestin and ternary complex formation. To measure only the binding event of ligands to the receptor, radiolabeled antagonists would be needed, as antagonists are unable to trigger G protein (β-arrestin) activation. Unfortunately such compounds are currently not available for melatonin receptors. Binding of antagonists is

Figure 1. Biosynthesis and metabolism of melatonin: T-5M, tryptophan 5-monooxygenase; AAA-D, aromatic amino acids decarboxylase; AA-NAT, arylalkylamine N-acetyltransferase; HIOMT, hydroxyindole O-methyltransferase; AFMK, N1-acetyl-N2-formyl-5methoxykynuramine; AMK, N1-acetyl-5-methoxykynuramine.

formation of AFMK is cyclic 3-hydroxymelatonin, a direct product of scavenging two hydroxyl radicals by melatonin.7 The biosynthesis and biotransformations of melatonin are shown in Figure 1. Numerous physiological effects of melatonin are mediated via activation of the high-affinity G-protein-coupled receptors (GPCRs) named MT1 and MT2. Both receptor subtypes have been found in mammals, including humans, and were cloned in the mid-1990s.8 Additionally, a low-affinity melatonin binding site MT3 has been characterized as a melatoninsensitive form of the human enzyme quinone reductase 2.9 Melatonin receptors are located in many regions of the body in the central nervous system (CNS) and peripheral tissues (Supporting Information, Table 1).10 As expected, the effect of B



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all of them displayed dissociation constants (Kd) in the nanomolar range,30 which is higher than the subnanomolar affinity of melatonin determined on cloned MT1 and MT2 receptors several years later.31 Because [3H]melatonin has a low specific activity, the development of an iodinated derivative, [125I]MLT, represented an important progress in the field to confirm the nature and distribution of melatonin binding sites in tissues. Within the CNS, autoradiographic and binding assays using the [125I]MLT radioligand suggested the presence of melatonin receptors in different areas, with prominent labeling observed in the suprachiasmatic nuclei (SCN) of the hypothalamus and the pars tuberalis of the pituitary gland in mammals, with Kd values in the subnanomolar range (Supporting Information, Table 1). High affinity [125I]MLT binding sites have also been shown in the parietal cortex, hippocampus, thalamus, striatum, pons-medulla, area postrema, cerebellum, and retina within the picomolar range. Analysis of [125I]MLT binding in peripheral tissues revealed the presence of melatonin receptors in organs and cells of the immune system, such as thymus, spleen, lymphocytes, and neutrophils, in the gastrointestinal tract, kidney, lung, liver, adipocytes, pancreas, vas deferens, prostate, uterus, mammary, and adrenal gland, and in blood vessels. Kd values and total number of binding sites (Bmax) obtained by scatchard analysis of [125I]MLT binding in different studies are summarized in Supporting Information, Table 1. [125I]MLT binding sites in the subnanomolar range are compatible with known affinities of cloned MT1 and MT2 receptors. Highest densities of these binding sites (60 fmol/ mg) are found in the pituitary pars tuberalis, a major target for melatonin modulatory actions on reproduction and lactation, and in the retina, which also produces melatonin rhythmically.5 [125I]MLT binding sites in the nanomolar range (*, Table 1, Supporting Information) are described in some peripheral tissues and could correspond to the MT3 (QR2) protein; however, it cannot be excluded that these binding sites might correspond to a low-affinity state of the cloned MT1 and MT2 receptors that are stabilized by the expression of still unknown receptor interacting proteins. Additional techniques, such as in situ hybridization, RT-PCR, and immunohistochemistry, were also employed after melatonin receptors were cloned allowing further confirmation of those data on melatonin binding sites. In general, most of the brain areas that were positively stained with [125I]MLT were later shown to express MT1 receptors. On the other hand, MT2 receptor expression seems not to correlate with the number of [125I]MLT binding sites as its expression levels are typically very low and often undetectable in the [125I]MLT binding assay. This is nicely illustrated in some brain areas, such as hypothalamic suprachiasmatic nuclei,32 where targeted deletion of the MT1 receptor in mice completely abolishes any [125I]MLT binding despite the presence of MT2 receptors. Notwithstanding, the identification of the MT2 receptor commonly relies on the use of its partial agonist, 4-P-PDOT, as illustrated in endothelial and pancreatic cells.33,34 Application of the newly designed MT2-selective radioligands 2 and 3 might help to overcome these technical limitations.27 In spite of technical improvements in the identification of melatonin binding sites, it is worthwhile to mention some intrinsic features of these receptors that might interfere in their detection. First, the tissue-specificity expression of melatonin receptors might vary depending on the species. This is the case of cerebellar granule cells, which show exclusively MT1

Figure 2. Structures of radioligands used to determine binding affinity for MT1 and MT2 receptors.

typically determined indirectly in [125I]MLT competition assays or in functional assays competing with the effect of melatonin. Available functional assays able to monitor melatonin receptor activation at the cellular level can be divided into three categories, those measuring [35S]GTPγS incorporation into heterotrimeric G proteins, those determining second messengers levels such as cAMP, cGMP, intracellular Ca2+, or inositol phosphate, and those relying on the activation of ion channels.23,28 Furthermore, physiological readouts for melatonin action can be used in animals or tissues. The reader is referred to recent expert reviews on this issue for more details.10 2.2. Melatonin Binding Sites. The expression profile of melatonin receptors has been determined either at the mRNA level using in situ hybridization or RT-PCR experiments or at the protein level using immunohistochemical or radioligand binding techniques.10 Successful detection of melatonin receptors at the protein level has proven difficult because of the very low expression levels and the lack of specific highaffinity antibodies in particular for rodent receptors. Therefore, most studies relied on the [125I]MLT radioligand, which has been abundantly used for radioligand binding experiments with cells or membrane preparations and for autoradiography with tissue slices. Before that, binding studies were performed with [3H]melatonin right after the discovery of the pineal hormone and aimed to detect putative target organs of melatonin action by accessing the uptake of this molecule by different tissues. This analysis promptly suggested the widespread distribution of melatonin targets as all tissues analyzed rapidly concentrated the molecule, including brain, kidney, uterus, liver, and pituitary, thyroid, adrenal, and pineal glands.29 Evidence of specific melatonin binding sites was also provided by [3H]melatonin experiments through the analysis of its binding to brain tissue extracts, which detected binding sites in the hypothalamus, hippocampus, striatum, and midbrain; however, C



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benefit of the formation of GPCR oligomers. First, GPCR oligomerization has been shown to be part of the general quality control system for membrane proteins operating in the endoplasmatic reticulum, which guarantees that only properly folded proteins reach the cell surface. Successful assembly of GPCR oligomers is part of this quality system.53 Once at the cell surface, intercommunication between the different receptor protomers (subunits) of the complex may occur. This phenomenon can be observed at the level of ligand binding where the activation of one protomer inhibits or facilitates ligand binding to the second protomer. Such negative or positive allosteric effects can be achieved by ligand-induced conformational switches between the transmembrane domains of the two protomers. The current literature on melatonin receptors does not report any allosteric effects between the two binding sites of receptor dimers. BRET experiments and classical radioligand binding studies demonstrated that occupancy of the first binding site in protomer 1 has no influence on the binding of ligands to the protomer 2 (unpublished observation, RJ).51 Within GPCR dimers, two distinct G protein activation modes can be identified. If the same protomer binds the ligand and the G protein, then this is called a cis-activation mode. The second protomer, which apparently does not participate in the signal transduction, can, however, fulfill further functions such as scaffolding of regulatory proteins into the complex as described below. If ligand binding and G protein coupling are divided between the two protomers, then this is referred to as the trans-activation mode. Previous studies on MT1 receptors have shown that this receptor can operate in the cis-activation mode by forming a complex between an MT1 dimer and Gi and RGS20 proteins.23 In this configuration, protomer 1 binds the ligand and the G protein and protomer 2 the RGS20, a protein that regulates the speed of G protein signaling. Indeed, binding of RGS20 to MT1 dramatically slows the decay time of G protein inactivation, thus participating in prolonged signal transduction; however, these observations do not exclude a possible trans-activation mode of melatonin receptors under other circumstances. The example of the MT1/Gi/RGS20 complex and the asymmetric binding of GPCR-associated proteins to the different protomers of the dimer point toward another important feature of GPCR oligomerization, namely, to provide a large scaffolding platform for GPCR associated interacting partners to fine-tune GPCR signaling in a way that cannot be ensured by receptor monomers.54 As mentioned above, MT1 and MT2 receptors can also engage into heteromers. Formation of heteromers is a general phenomenon observed for many other GPCRs.49 Of interest, in many but not in all cases, GPCR heteromerization has been shown to have a profound impact on receptor function such as ligand binding, receptor internalization, and signal transduction. 52 These GPCR heteromers can therefore be considered as new and unique pharmacological targets as discussed under therapeutic applications. Several reports suggest that this might also be the case for MT1/MT2 heteromers. Indeed, a specific pharmacological profile of human MT1/MT2 heteromers has been established using a BRET-based assay that measures ligand-promoted conformational changes of the heterodimer.51 The melatonin receptor antagonist luzindole showed the highest selectivity with a preference of more than 100 times for heterodimers compared to MT2 homodimers. More recently, we determined the

expression in human tissue while the same cells in mouse express both MT1 and MT2 receptors.35 Additionally, melatonin receptors show different expression patterns during development and aging. The study by Thomas and co-workers has shown that human fetus brain expresses only MT1 receptors, while adult brains express both receptors.36 Changes in MT1 and MT2 receptor expression have been reported in different tissues from aged individuals 37 and in some pathological conditions, such as in Alzheimer’s disease,38 Huntington’s disease,39 Parkinson’s disease,40 depressed patients,41 preeclampsia,42 and human breast cancer.43 Finally, circadian variations must be taken into account when analyzing melatonin receptors. Rhythms in melatonin binding sites have been described since the first studies, and their profile varies depending on the tissue and species analyzed.44 In addition, the expression of melatonin receptors may also vary depending on the activation state of the cell, as suggested by the data on lymphocytes (Supporting Information, Table 1) As new molecular tools and techniques will push our detection limits even further, new melatonin binding sites and new cellular contexts expressing MT1 and MT2 receptors are expected. In addition, further studies are required in order to fulfill the gap between the changes in the expression of melatonin receptors and the development of certain pathologies. 2.3. Melatonin Receptor Oligomerization. The minimal functional unit of GPCRs appears to be a monomer. This conclusion is consistent with the current model where agonist binding to the receptor induces a conformational change that remodels the cytoplasmatic receptor surface to provide a docking site for heterotrimeric G proteins. Upon formation of the ternary ligand−receptor−G protein (L−R−G) complex, high-affinity agonist binding is observed. By comparing the structure of the β2-adrenoceptor (β2AR) obtained in the presence of the high-affinity agonist BI-167107 and the Gs protein with β2AR structures obtained in the presence of different ligands (inverse agonist, antagonist, agonist) and the structures of the Gs protein in its inactive and active state, the authors showed that the presence of the Gs protein and the formation of the L−R−G complex is indispensable to reach the fully active conformation of the receptor.45 These data are consistent with earlier in vitro studies with nanoscale lipid bilayers containing strict monomeric GPCRs showing that these receptors are fully functional in terms of G protein coupling and β-arrestin binding.46−48 Collectively, these observations demonstrate that GPCR monomers are sufficient to fulfill the basic GPCR functions. Intriguingly, an abundant literature reports that most GPCRs have the capacity to exist as dimers or higher order oligomeric complexes, even those being functional when forced to be strict monomers.49 MT1 and MT2 receptors were among the first receptors for which homodimerization and heterodimerization have been demonstrated in transfected cells at physiological expression levels.50 Interestingly, the propensity for homomer and heteromer formation does not seem to be identical. Whereas the propensity of human MT1/MT2 heteromer and MT1 homomer formation is similar, that of MT2 homomer formation is 3- to 4-fold lower, suggesting that the MT2 receptor preferentially exists as a heteromeric complex with MT1.51 The fact that GPCR monomers are fully functional in terms of G protein activation questions the functional significance of GPCR oligomerization.52 Several recent reports have addressed this issue, shedding light on the potential D



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with an acidic amino acid in TM6, the so-called “ionic lock” involved in the stabilization of the inactive conformation of GPCRs. In TM6 the CWXP motif can be recognized, which is of fundamental importance for the conformational changes triggering receptor activation. In TM7 the conserved NPXXY motif is substituted by the NAIXY motif. Since the crystal structures of MT1 and MT2 receptors are not yet available, in the past 2 decades a number of threedimensional (3D) receptor models were built by homology modeling. However, modeling of melatonin receptors has to cope with particular limitations and drawbacks. First, a suitable template structure is missing. While bovine rhodopsin was the only crystallized GPCR until 2007, since then a number of crystal structures of other GPCRs started to become available.63 However, all these structures share a poor similarity with MT1 and MT2 receptors. Sequence identity within TM domains is lower than 30%, and even lower if the binding-site region is considered, limiting the reliability of receptor models. In fact, it has been suggested that templates having more than 30−35% sequence identity with the target protein are required to produce accurate receptor models and to correctly predict binding modes.64 The low degree of sequence identity can, in fact, reflect differences in structural organization, such as helix arrangement and loop architecture, affecting the ligand binding site. For example, ECL2 of rhodopsin is deeply inserted within the helix bundle, sealing the binding site and contacting the ligand. In different melatonin receptor models, based on the rhodopsin structure, the conformation of ECL2 prevented the accommodation of ligands into the binding site. ECL2, then, had to be removed or rebuilt, with no experimental information driving its geometrical optimization. A further limitation toward the definition of accurate binding schemes is related to the physicochemical nature of the endogenous ligand and of the amino acids surrounding its binding site. Melatonin is a lipophilic molecule and lacks centers ionizable near physiological pH values, which could anchor the ligand to the binding site residues by charge−charge interactions. This peculiar property is reflected in the amino acid composition of the TM regions delimiting its putative binding site, which are more lipophilic than those of other GPCRs. For example, while aminergic receptors have a conserved acidic amino acid at position 3.32 (Ballesteros−Weinstein numbering65), a methionine is present in the corresponding position of MT1 and MT2 receptors. Ser5.42, interacting with the hydroxyl groups of isoprenaline in the β1 receptor, is replaced by a valine in melatonin receptors. Therefore, it is likely that the high binding affinity of melatonin for MT1 and MT2 receptors is mostly due to the sum of a number of hydrophobic and weak interactions, making it more difficult to be reproduced by a modeled structure. This lipophilic character could be one of the causes of the third limitation experienced by melatonin receptor modeling, that is, the lack of conclusive information about the ligand binding interactions from mutagenesis studies. Indeed, a number of mutagenesis experiments had been made on both MT1 and MT2 receptors,10 leading to results that can be roughly divided into two groups. In fact, while mutation of some amino acids led to a very limited decrease in binding affinity (usually no more than 10 times compared to wild type), other residues caused a complete loss of radioligand binding when mutated. In the first case, it is difficult to undoubtedly ascribe the subtle effect to an alteration in the binding pattern, while mutations that fully abolish binding affinity are, in principle, much more informative even if this may be an

signaling profile of murine MT1/MT2 heteromers and showed in transfected HEK293 cells and in the retina that physiological melatonin concentrations specifically activate the PLC/PKC pathway through MT1/MT2 heteromers.55 Additional improvements were observed for the Gi/cAMP pathway, with higher efficacy in cells expressing MT1/MT2 heteromers. In addition to MT1/MT2 heteromers, heteromerization events have been reported between MT1 or MT2 receptors and GPR50, another member of the melatonin receptor subfamily.20 In contrast to MT1 and MT2 receptors, GPR50 does not bind melatonin or any other known ligand, classifying GPR50 as an orphan seven transmembrane-spanning protein. Particularly striking effects have been observed for MT1/ GPR50 heteromers. In this heteromer, the long carboxylterminal tail of GPR50 prevents the recruitment of G proteins and β-arrestins to the MT1 protomer, most likely by sterical hindrance, thus preventing the formation of the ternary L−R− G complex and high affinity [125I]MLT binding. Formation of such complexes has been observed in immortalized human endothelial cerebral hCMEC/D3 cells where silencing of GPR50 recovered high-affinity [125I]MLT binding and signaling through the Gi/cAMP pathway.20 Despite the fact that coexpression of MT1 and MT2 receptors has been reported in many melatonin-sensitive tissues, the formal demonstration of MT1/MT2 heteromer formation is still in its infancy.56 Tissues of potential interest are the hypothalamic SCN,57 the hippocampus,58 the retina,59 the pancreas,16 and the adipose tissue.60 Furthermore, in situ hybridization experiments in cultured chicken astrocytes showed that some 25% of the cells coexpressed the Mel1c and MT1, which is in agreement with the formation of heterodimeric complexes.61 Recently, evidence for MT1/MT2 heteromer formation has been obtained in the mouse retina demonstrating for the first time that MT 1 /MT 2 heteromers form under natural, physiological conditions.55 Indeed, deletion of either receptor in the mouse retina resulted in the same phenotype, that is, a complete loss of melatonin-dependent rod photoreceptor light sensitivity. The same phenotype was observed in mice overexpressing a dominant-negative mutant melatonin receptor that competes with the formation of functional MT1/MT2 heteromers in photoreceptor cells. 2.4. Receptor Structure and Models. Human MT1 and MT2 receptors were cloned in the mid-1990s and they are 350 and 362 amino acids long, respectively, the MT2 subtype having a longer N-terminal portion.8 Their amino acid identity is about 55%, reaching 69% within the transmembrane domains. MT1 and MT2 receptors belong to the GPCR superfamily and have a general structural motif consisting of seven transmembrane (TM) helices spanning the membrane lipid bilayer, connected by three intracellular (ICL) and three extracellular (ECL) loops. They belong to the class A family, the members of which are characterized by a binding site for their natural ligand surrounded by the TM helices, approximately where retinal had been found in the crystal structure of rhodopsin.62 Both receptors exhibit the pattern of highly conserved sequences and motifs typical of class A GPCRs, with some peculiarities. A disulfide bridge connects two cysteines located at the Nterminus of TM3 and in ECL2 and contributes to the stabilization of the receptor extracellular portion. The conserved D(E)RY motif at the C-terminus of TM3 of class A GPCRs is substituted by a NRY sequence in both MT1 and MT2 receptors; the arginine residue forms an ionic interaction E



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indirect effect, caused by improper folding of some portions of the receptor or the inability to reach the high-affinity agonist binding state of the receptor. While the limitations discussed are worthy of attention, mutagenesis studies have identified three main amino acids in the MT1 receptor that appear to be involved in melatonin binding. Mutation of His1955.46, highly conserved within the melatonin receptor subfamily, led to a decrease in binding affinity for melatonin and other ligands in both sheep66 and human67 receptors. In the human MT1 receptor, His195Ala mutation gave a 2- to 3-fold reduction in binding affinity and potency in the GTPγS assay for melatonin, suggesting that this amino acid may be involved in both ligand binding and receptor activation. His195 was proposed to interact with the methoxy oxygen of melatonin because binding affinity of a derivative, lacking the methoxy group, was not affected by this mutation. Replacement of Ser1103.35 and Ser1143.39 with Ala led to a 4- to 9-fold reduction in melatonin binding affinity, while it did not affect binding affinity of the antagonist luzindole.68 This gave rise to the hypothesis that agonists and antagonists interact with different binding sites that are only partially overlapping. Mutation of the same amino acids was evaluated on MT2 receptors, and in this case, mutation of His2085.46 led to a 4-fold decrease in binding affinity for melatonin, but no effect was observed when Ser1233.35 and Ser1273.39 were replaced by Ala.69 Another relevant mutation at the MT2 receptor was Asn1754.60Ala, leading to a 4-fold decrease in binding affinity for melatonin but not for ligands lacking the methoxy group. This suggested a role for this amino acid in interacting with the methoxy group. Mazna and co-workers reported that mutation of Val2045.42, Asn2686.52, Leu2726.56, Ala2756.59, Val2917.36, Leu2957.40, Tyr2987.43, Tyr188 (ECL2) resulted in no specific [ 125 I]MLT binding at the MT 2 receptor.70,71 Taken together, these data suggest that the putative MT1 and MT2 binding sites span from TM3 to TM7 in the upper portion of the helical bundle, probably making contacts with some residues of the extracellular loops (Figure 3). The first homology models of melatonin receptors, reported in the mid-1990s, were based on the crystallographic structure of bacteriorhodopsin72,73 or on the low-resolution structure of rhodopsin74 and only reproduced the transmembrane portions of the MT1 or the Xenopus laevis melatonin receptors. Nonetheless, these models pointed out a number of amino acids, including Ser3.35, Ser3.39, Asn4.60, and His5.46, as those likely to interact with melatonin and gave the first indications for the mutagenesis experiments that were performed some years later. Receptor models based on the high-resolution crystal structure of bovine rhodopsin came after year 2000.75 Melatonin and other ligands were docked according to binding hypotheses based mainly on mutagenesis data, which focused on key contacts that were either imposed as restraints or applied as selection criteria in the modeling procedures. The first MT1 receptor model was reported by researchers from Takeda, who docked ramelteon into a receptor model composed of TM segments and ECL2 only.76 The conformation of ECL2, which initially resembled that found in rhodopsin, prevented the accommodation of the ligand. Thus, a simulated annealing protocol was applied to obtain a more favorable geometry. In the proposed binding mode, the furan oxygen of ramelteon is hydrogen-bonded to His1955.46 and its adjacent carbon interacts with Val1925.43 while the amide group is hydrogen-bonded to Tyr175 and Ser182 on ECL2.

Figure 3. Schematic representation of amino acids for which mutagenesis data on melatonin receptors are discussed in the text. On the 7 TM backbone of a crystallized GPCR structure (β2 adrenoceptor, PDB code 2RH1), the positions corresponding to Ser3.35 and Ser3.39 of MT1 are highlighted by purple rings and those corresponding to Asn4.60, Val5.42, Asn6.52, Leu6.56, Ala6.59. Val7.36, Leu7.40 and Tyr7.43 of MT2 by blue rings. The position corresponding to His5.46, common to both receptors, is represented by a yellow ring. The approximate location of the putative binding site is shown by the cyan volume of melatonin, manually superposed on the cocrystallized β2-ligand carazolol.

Three other MT1 models were built based on the same binding hypothesis, supposing an interaction of the methoxy oxygen of melatonin with His1955.46 and of the amide group with the two serines, Ser1103.35 and Ser1143.39, in TM3. Interestingly, the same authors also proposed MT2 receptor models based on the same binding scheme (even if mutagenesis data had not confirmed the relevance of the two serines for this receptor subtype) and evaluated the differences in binding sites conferring differential affinity to subtype-selective ligands. In particular, the research group of Zefirov described MT1 and MT2 receptor models in which melatonin is accommodated into very similar receptor pockets.77,78 The acyl group of the amide side chain is positioned close to the extracellular portion of TM2, where a lipophilic pocket can accommodate groups no bigger than the propionyl one, consistent with the structure− activity relationship (SAR) data. Substituents in position 2 of the indole ring interact with amino acids in TM6, such as Trp6.48 of the CWXP motif, and in TM7. Selectivity for the MT2 receptor, shown by compounds carrying bulky substituents in position 1 or 2 of the indole ring, is justified by the presence of a smaller amino acid in the MT2 receptor (Val1283.40) compared to the MT1 one (Ile1153.40). Next, Chugunov et al. proposed models of the active state of MT1 and MT2 receptors.79 As the crystal structure of a GPCR in its active state was not available, TM3 was rotated to optimize the polar interactions between melatonin and the helix residues. In particular, helix rotation modified the positions of Ser1103.35 and Ser1143.39, located outside the binding site in the original alignment, and allowed their interaction with the amide group of melatonin. During the building procedure, melatonin was fixed in its putative bioactive conformation, obtained from a preliminary pharmacophore analysis. MT1 and MT2 binding sites demonstrated some differences, for example, the orientation of Trp6.48, that were claimed to provide an explanation for the MT2 selectivity exhibited by some compounds. Selectivity was also discussed in terms of F



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methoxy oxygen is bound to Tyr2987.43 and the indole ring undertakes hydrophobic interactions with Val2045.42 and Leu2726.56. The amide fragment is hydrogen-bonded to Tyr188 in ECL2, and the acetyl group in the side chain points toward the extracellular portion of TM7. Antagonist compounds did not share the same binding scheme as melatonin, but they were bound mainly through unspecific hydrophobic interactions. The amino acids proposed to interact with melatonin were mutated to evaluate their role. Interestingly, receptors expressing mutations of many of these residues revealed a complete loss of [125I]MLT binding (Val2045.42, Asn2686.52, Leu2726.56, Ala2756.59, Val2917.36, Leu2957.40, Tyr2987.43, Y188 (ECL2)). Zefirova et al. exploited the active form of rhodopsin to build an agonist-bound conformation of the MT2 receptor, limiting the model to its TM portions.82 Automated docking was performed that, interestingly, gave a binding mode for melatonin similar to that previously proposed by Farce and co-workers;80 the methoxy oxygen interacts with His2085.46, the indole ring with Val1243.36, the amide oxygen with Asn1754.60, and the amide nitrogen with the backbone carbonyl of Ala1173.29. Another MT2 receptor model was employed to investigate the structural determinants for MT2 receptor selectivity.83 Indeed, many MT2-selective antagonists are characterized by a bulky lipophilic substituent in a position corresponding to 1 or 2 of the indole ring of melatonin, characterized by an out-ofplane arrangement.84 The MT2 receptor model was based on the structure of rhodopsin, even if TM5 was rebuilt to avoid the presence of a bulge in correspondence of His2085.46. The binding pocket was adapted, by simulated annealing, around a potent and selective MT2 antagonist kept in its putative bioactive conformation. In the resulting binding scheme, His2085.46 formed a T-shaped interaction with the aromatic indole ring and the amide oxygen was hydrogen-bonded to Tyr183 in ECL2. A protein counterpart for the methoxy oxygen was not found, consistent with the observation that melatonergic antagonists often lack this group. Interestingly, a set of topologically different antagonists could be docked into the receptor model, sharing the same binding scheme. The outof-plane substituent was accommodated into a hydrophobic pocket delimited by amino acids from TM3, TM5, and TM6, where the interaction with Trp2646.48 of the CWXP motif may be related to the antagonist behavior shown by these compounds. By comparison of the amino acids delimiting this additional pocket with the corresponding ones in the MT1 receptor, it is possible to identify two amino acids with smaller side chains (MT2, Val1283.40, Ile2135.51; MT1, Ile1153.40, Met2005.51). The smaller cavity in the MT1 receptor hampers a correct accommodation of the compounds, thus suggesting an explanation for the MT2 selectivity conferred by the out-ofplane substituent. Although the majority of MT1 and MT2 3D models were built using bovine rhodopsin as the template, the receptor models described so far demonstrate a heterogeneous scenario in terms of proposed binding schemes and ligand conformations. This is particularly evident for the MT2 receptor. This heterogeneity highlights the limited ability of available techniques to reproduce the key elements of molecular recognition at MT1 and MT2 receptors, which raises doubts about the potential utility of homology models to drive structure-based lead discovery. As stated before, multiple elements contribute to the limits of MT1 and MT2 models,

complementarity between lipophilic/hydrophilic portions of the compounds and the corresponding regions of the MT1 and MT2 receptors. A further attempt to model the active state of the MT1 receptor was made by Farce et al. who adopted a similar technique to generate the active conformation of the MT1 and MT2 receptors.80 Indeed, TM3 and TM5 of the MT1 receptor and TM4 of the MT2 receptor were rotated to optimize their interactions with melatonin. Contrary to the previously cited models, in this case melatonin interacts with the two receptor subtypes according to two different binding schemes. In the MT1 receptor it is bound to His1955.46, Ser1103.35, Ser1143.39, and Trp2516.48, which is consistent with the previous models. To build the MT2 receptor, melatonin was manually docked in proximity of TM4 and TM5, interacting with His2085.46 through its methoxy oxygen, and with Asn1754.60 and Thr191 in ECL2 with the amide group. While this was justified by information from mutagenesis, the resulting MT2 binding site is positioned higher, more toward the extracellular portion of the TM bundle compared to the MT1 one. Moreover, the conformation of melatonin is different in the two receptor−ligand models. The ability of these models to reproduce known SARs for melatonin receptor ligands was assessed by molecular docking of different compounds in both MT1 and MT2 models. Luzindole could not be docked into the two receptor models, which is consistent with the hypothesis that active conformations had been reproduced. Compounds with scaffolds different from the indolylethylamine one of melatonin could only be docked with different patterns of interaction. This was ascribed either to the necessity of more advanced modifications of the binding sites or to the possibility that several binding modes could exist, depending on the nature of the ligands. The most recent MT1 receptor model differs from the previous ones in that it has been built on the active form of the β2 adrenoceptor.81 The agonist 2-phenylmelatonin was docked using an induced-fit procedure taking into account receptor flexibility, while the ligand was rigidly maintained into its putative bioactive conformation, obtained from ligand-based pharmacophore analysis. In this model, the methoxy group is close to His1955.46 with the oxygen atom interacting with Tyr1875.38 and the amide oxygen hydrogen-bonded to Tyr2987.43. The 2-phenyl ring was accommodated in an additional hydrophobic cavity lined by amino acids from TM6 and TM7. The complex was submitted to a molecular dynamics simulation into a solvated lipid bilayer, which allowed reshaping of the binding site around the ligand. This receptor model was used for docking of MT1-selective agonists carrying a bulky lipophilic substituent in the position corresponding to that of the methoxy group in melatonin. Interestingly, such a lipophilic substituent, which confers selectivity for the MT1 subtype, could be accommodated between the tips of TM3 and TM4, toward the extracellular space, in a region where MT1 receptor has amino acids with smaller side chains than MT2 receptor (e.g., Gly1043.29 in MT1 vs Ala1173.29 in MT2). Besides the three MT2 receptor models by Zefirov,78 Chugunov,79 and Farce80 described in the previous paragraph with their cognate MT1 models, other MT2 homology models were reported, proposing different binding schemes for melatonin. In 2004 Mazna et al. described the first model of the MT2 receptor, which was built based on the rhodopsin template.70,71 Different from the docking protocols already discussed, manual docking of melatonin was not driven by mutagenesis data and led to a binding scheme in which the G



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selection and of knowledge-based structural refinement in model building.90 On a data set of 29 potent melatonergic ligands and 2500 decoys with similar physicochemical properties, the enrichment factors for different MT2 receptor models built from a wide set of templates were calculated, both before and after ligand-guided adaptation of the binding sites. Binding site reshaping was performed with selected ligands, docked in poses consistent with both mutagenesis information and pharmacophore models. Ligand-adapted models showed significant improvements in screening efficiency compared to unrefined models. The screening performance strongly varied with the choice of template structure and reshaping ligand; the best-performing model, built from the crystal structure of β2 adrenoceptor and reshaped by a tetralin ligand, correctly retrieved 24 of the 29 active compounds among the first 52 scored hits. Moreover, being built on the active form of β2 adrenoceptor, it also showed some ability to discriminate melatonergic agonists from antagonists. However, the best combinations of template and reshaping ligand could not be predicted a priori. Moreover, screening the same database on shape similarity with the ligands gave high enrichment factors, generally higher than receptor models. This may be due to the high structural and physicochemical similarity between melatonin receptor ligands, but it also points out that making useful predictions with homology-based melatonin receptor models is still a challenging task. Nevertheless, reliable receptor models are needed to find novel melatonin receptor ligand classes, which cannot be obtained with a ligand-based screening approach. In the future, until the first crystal structures of melatonin receptors become available, the availability of better structural templates and of additional biophysical, biochemical, and ligand based information could lead to more reliable melatonin receptors, hopefully allowing for the unraveling of the intimate bases of ligand recognition, subtype selectivity, and receptor activation.

including the availability of only remotely related templates and the inconclusive nature of mutagenesis data. On the other hand, an improvement in the predictive ability of homology models can be obtained integrating information available from ligand SARs and pharmacophore models. Indeed, when appropriate templates are not available, the importance of combining modeling techniques with a knowledge-based refinement has been demonstrated by several studies.64b,85 For example, fixing the ligands in their putative bioactive conformations helps in discarding alternative solutions, focusing on those consistent with ligand-based information. Several pharmacophore models for melatonin receptor ligands have been reported since the mid-1990s, based mainly on the analysis of conformationally constrained compounds. Two different pharmacophore analyses86,87 proposed that the acylaminoethyl side chain of melatonin, in its bioactive conformation, is perpendicular to the indole ring, with an anti conformation of the carbon− carbon bond of the ethylene portion. The evaluation of experimental data for stereoselective ligands led to unambiguous definition of the spatial arrangement of pharmacophoric elements for both agonists and MT2-selective antagonists, concluding that the active conformation is characterized by the arrangement of the amide side chain below the plane of the indole ring, when melatonin is represented with the methoxy group on the left (Figure 4).88 The pharmacophore model for

3. MELATONERGIC DRUGS Melatonin is a popular treatment of sleep problems related to circadian rhythm like those caused by jet lag, shift work, and delayed sleep phase syndrome. However, unrestricted use of melatonin products available over the counter in the U.S. is questionable because their purity, safety, and potency are not regulated by the U.S. Food and Drug Administration.91,92 In 2007, a prolonged-release formulation of melatonin (Circadin, Neurim Pharmaceuticals, Lundbeck) was approved in Europe by the European Medicines Agency for the treatment of insomnia in patients aged >55 years.93 Other obstacles for the clinical use of melatonin are the unfavorable pharmacokinetic profile, such as rapid elimination, limiting its efficacy for many possible treatment purposes, and lack of selectivity for the different biological targets including MT1 and MT2 receptors. These drawbacks have led to the development of melatonergic ligands with improved properties. Numerous patents claiming the use of melatonergic agonists, mostly for the treatment of sleep disorders, have been filed in the past decade. Advances in the field have been summarized in several recently published review articles.94−96 The first synthetic melatonergic agent on the market is the nonselective high-affinity MT1/MT2 agonist ramelteon (Rozerem, Takeda Pharmaceuticals Inc.), approved for the treatment of insomnia. Ramelteon was introduced in the U.S. in 2005. It is characterized by a half-life of 0.83−1.93h that is indeed longer than that of melatonin (20−30 min).97 Ramelteon

Figure 4. Bioactive conformations proposed for melatonin, corresponding to model B (left, yellow carbons) by Jansen et al.86 and to model B (right, orange carbons) by Spadoni et al.87 The antagonist 4P-PDOT (green carbons) is superposed to the structure on the right, showing the arrangement of the additional phenyl ring conferring MT2 selectivity.

MT2-selective antagonists shares the same arrangement of common elements with that of agonists, with an additional outof-plane substituent oriented opposite the amide side chain (Figure 4).89 Ligand-based information has been included in the protocols applied for some of the MT1 and MT2 models previously described. Chugunov et al. defined the extent of TM3 helix rotation in both MT1 and MT2 models, based on the optimization of amino acid interactions with melatonin, docked in its bioactive conformation.79 The ligand binding sites in the MT1 and MT2 models proposed by Rivara et al.81,83 were adapted around selected ligands maintained in fixed conformations, consistent with the outcomes of pharmacophore analysis. Recently, the ability of MT2 receptor models to retrieve known melatonergic ligands in a virtual screening campaign was tested, evaluating the influence of template H



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removing the acetyl group from the acetamide moiety exhibits no affinity for melatonin receptors. (2) Relocation of the methoxy group from C5 to C4, C6, or C7 leads to a dramatic loss of affinity. (3) Replacement of the 5-methoxy substituent by H, OH, halogen, or bulkier alkoxy groups reduces receptor affinity. The 5-chloro- and 5-bromo analogues are full agonists with ∼10 times lower binding than melatonin. (4) Increasing the length of the alkyl substituent attached to the amide carbonyl group from CH3 to C3H7 enhances affinity. Larger substituents are detrimental for binding. Cyclopropyl and cyclobutyl groups mostly reverse intrinsic activity toward antagonism. Conversion of the acylaminoethyl side chain to an ethyl ester reduces binding. (5) N1-Substitution with methyl is well tolerated. Bulkier groups decrease both binding and intrinsic activity. (6) Substitutions at the 2-position with a halogen, methyl, or a phenyl group generate agonists with ∼10fold increased binding. (7) 6-Chloromelatonin and 6fluoromelatonin demonstrate comparable affinity to melatonin. 6-Hydroxymelatonin and 6-methoxymelatonin display 25-fold and 100-fold reduced binding, respectively. (8) Replacement of the melatonin indole scaffold with benzimidazole, i.e., the formal replacement of C3 with N, dramatically reduces binding affinity. (9) The nature of the aromatic ring is not crucial for melatonin receptor recognition, as the exchange of indole in melatonin by various aromatic scaffolds, such as naphthalene, benzofuran, benzothiophene, indane, tetralin, and quinoline, maintains high binding and agonist potency. Melatonin, the natural agonist at both MT1 and MT2 receptors, displays equal subnanomolar affinity toward both MT1 and MT2. The binding constants Ki determined in either 2-[125I]iodomelatonin or [3H]melatonin displacement assays range between 0.15 and 1.00 nM depending on the cell lines used for receptor expression and on the research laboratory.

undergoes extensive liver metabolism. The major metabolite is a product of the ω-1 hydroxylation in the propionamide side chain.98 Although the activity of the major metabolite is 30-fold lower than that of ramelteon, its serum levels are 20- to 100fold higher than those of the parent drug, suggesting that it may significantly contribute to the overall clinical effect of ramelteon.99 Another nonselective MT1/MT2 agonist agomelatine (Valdoxan, Servier) was approved in Europe for the treatment of major depression in 2009. The antidepressant effect of agomelatine is thought to be caused by the combination of its antagonist behavior at 5-HT2C serotonin receptors and its agonistic effect on MT1 and MT2 receptors.100 Tasimelteon (VEC-162)101 and 4 (TIK-301, PD-6735, LY156735, β-methyl-6-chloromelatonin),102 both nonselective MT1/MT2 agonists, are the most advanced melatonergic drug candidates undergoing clinical trials for the treatment of sleep disorders. In the phase III study, tasimelteon was demonstrated to improve sleep latency, sleep efficiency, and wake after sleep onset.103 Interestingly, in May 2013, Vanda Pharmaceuticals submitted a new drug application to the Food and Drug Administration for tasimelteon for the treatment of non-24-h sleep−wake disorder in subjects suffering total blindness.104 4 has been reported to act as an antagonist at the serotonin receptor subtypes 5-HT 2C and 5-HT 2B , opening new perspectives for its possible antidepressant action.105 The structures of ramelteon, agomelatine, tasimelteon, and 4 are displayed in Figure 5.

5. NONSELECTIVE MT1/MT2 RECEPTOR LIGANDS Numerous ligands displaying high binding affinity but no selectivity toward MT1 or MT2 receptors have been reported (Figure 6 and Figure 7). Their structures include substituted melatonins, such as 2-iodomelatonin (MT1, Ki = 0.068 nM; MT2, Ki = 0.22 nM; COS-7 cells),110 6-chloro-2-methylmelatonin (MT1, Ki = 1.34 nM; MT2, Ki = 2.2 nM; COS-7 cells),110 β-methyl- and β,β-dimethylmelatonin 5a,b111 and bioisosteric melatonin analogues obtained by exchange of the indole nucleus with other ring scaffolds 6,1127a,b,112,113 8,114 9,115 10a,b,116 11a,b,117 12,116 13a−d,118 14−16,119 17,120 18;121 ring-opened derivatives 19,122 20,123 21,124 22 and 23,125 24 and 25;126 and conformationally restricted ligands 26a−c,111 27a,b,127 28−29a,b,128 30,129 31130 32a,b131 33a−c,132 34,133 35,121 36.134 Methyl monosubstitution and disubstitution in the β-position of the melatonin side chain maintain high binding affinity and agonist activity at both receptor subtypes. Racemic βmethylmelatonin 5a (MT1, Ki = 1.67 nM; MT2, Ki = 2.94 nM) and β,β-dimethylmelatonin 5b (MT1, Ki = 1.12 nM; MT2, Ki = 2.75 nM) show a slightly reduced binding at MT1 and MT2 receptors expressed in NIH3T3 cells compared to melatonin (MT1, Ki = 0.39 nM; MT2, Ki = 0.35 nM). Interestingly, both 5a and 5b are more potent agonists than melatonin in the Xenopus laevis melanophore assay. A further increase in bulk and/or rigidisation is much better tolerated at MT2 than at MT1 receptors. For instance, in the series of N1methyl substituted melatonin analogues with a cyclopropane, cyclobutane, or cyclopentane ring attached to the β-position of

Figure 5. Structures of melatonergic drugs on the market and in clinical studies.

4. SARS OF MELATONIN A great number of melatonin receptor ligands belonging to different structural classes and their affinities toward melatonin receptors determined in [125I]MLT competition binding studies were reported in the 1980s and early 1990s.106,107 Although various native tissue preparations, such as chicken brain and retina, hamster brain, rabbit retina, ovine pars tuberalis, quail optic tecta, Xenopus melanophore, each containing no clearly defined receptor subtypes (see Supporting Information, Table 1), were used by different research groups, the following common SARs for melatonin could be concluded.73,108 Later studies on recombinant MT1 and MT2 receptors confirmed that these principles generally apply to both MT1 and MT2 subtypes.109 (1) The methoxy group and the amide side chain of melatonin in an appropriate relative position are essential for high receptor affinity and intrinsic activity. Removal of the methoxy group leading to Nacetyltryptamine results in over a 1000-fold lower affinity and partial agonist activity. 5-Methoxytryptamine obtained by I



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Figure 6. Structures of nonselective MT1/MT2 receptor ligands (part 1).

MT2 affinity as well as in intrinsic activity (EC50 = 0.09 nM). A similar increase in MT1- and MT2-binding was observed for 2phenyl-4-azamelatonin 10b (MT1, Ki = 0.04 nM; MT2, Ki = 0.20 nM). In contrast, 2-phenyl-3a-aza-melatonin 12 is characterized by Ki(MT1) = 28 nM and Ki(MT2) = 8 nM, indicating that carbon−nitrogen exchange in position 3a of melatonin is detrimental for binding affinity at MT1 and MT2 receptors. In compounds 13−16, the ether oxygen in a position equivalent to the 5-methoxy group of melatonin is incorporated into a dihydrofuran ring. This structural motif, which is also present in ramelteon (MT1, Ki = 0.014 nM; MT2, Ki = 0.112 nM; CHO cells), is responsible for increased MT1/MT2 binding. In a series of indene analogues 13a−d, the direct melatonin bioisostere 13a shows 5−10 times higher affinities at both subtypes than melatonin. Introduction of a bromine, a phenyl, or a 3-furyl group in a position equivalent to C2 of melatonin further enhanced binding generating melatonergic ligands with the highest affinity known to date, 13b (MT1, Ki = 0.0087 nM; MT2, Ki = 0.014 nM), 13c (MT1, Ki = 0.0082 nM; MT2, Ki = 0.0065 nM), and 13d (MT1, Ki = 0.0065 nM; MT2, Ki = 0.0096 nM), respectively. In order to improve in vivo activity and metabolic stability of the highly lipophilic ligand 13c (log D = 3.49), three more hydrophilic diaza analogues 14 (log D = 1.66), 15 (log D = 1.39), and 16a (log D = 2.04) were investigated. The more hydrophilic agents, 14 and 15, are more stable against oxidative metabolism, showing a much slower clearance than 16a and 13c in rat hepatic microsomes. However, 14 (MT1, Ki > 100 nM; MT2, Ki = 6.1 nM) and 15 (MT1, Ki = 20 nM; MT2, Ki = 4.5 nM) display much lower binding affinities than 16a (MT1, Ki = 0.082 nM; MT2, Ki =

the side chain 26a−c, the cyclopropane derivative 26a shows 28-fold binding preference for the MT2 subtype (MT1, Ki = 212 nM; MT2, Ki = 7.5 nM). While the homologous cyclobutane analogue 26b displays increased affinity for both MT1 (Ki = 10.6 nM) and MT2 (Ki = 0.86 nM), a further ring extension giving the cyclopentane homologue 26c leads to dramatic reduction of affinity at both receptors (MT1, Ki = 589 nM; MT2, Ki = 85.1 nM). In the forskolin-stimulated cAMP release assays, 26b and 26c behave as MT1 antagonists. Interestingly, at the MT2 receptors, 26b is an agonist equipotent to melatonin, whereas 26c shows no receptor activation representing a functionally MT1-selective antagonist. Among the melatonin analogues obtained by bioisosteric replacement of the indole scaffold with other aromatic rings, the naphthalene, tetralin, benzofuran, and benzothiophene derivatives agomelatine, 8, 7a, and 6, respectively, are equipotent to melatonin in terms of MT1/MT2 receptor affinity and intrinsic activity. The quinoline analogue 9 has only been investigated in radioligand binding studies showing 5-fold lower affinities for MT1 and MT2 than melatonin. The effect of replacing one carbon atom of the indole nucleus of melatonin by nitrogen is dependent on the ring position. While 4azamelatonin 10a shows MT1 (Ki = 0.24 nM) and MT2 (Ki = 0.36 nM) binding affinities identical to those of melatonin for receptors expressed in HEK293 cells, 7a-azamelatonin 11a displays reduced binding at MT1 (Ki = 12.3 nM) and MT2 (Ki = 4.0 nM) receptors expressed in NIH3T3 cells, behaving as a much less potent agonist (EC50 = 10.2 nM) than melatonin (EC50 = 0.02 nM) in Xenopus laevis melanophore assay. As expected, 2-phenyl substitution of 11a to give 11b (MT1, Ki = 0.58 nM; MT2, Ki = 0.40 nM) caused an increase in MT1 and J



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Figure 7. Structures of nonselective MT1/MT2 receptor ligands (part 2).

Phenylalkylamide 19 represents the minimal structure required for the ligand recognition by melatonin receptors. It shows a 100 times lower binding affinity than melatonin in chicken brain assay. Linking the C1 methoxy oxygen of 19 to C2 through 2,3-dihydro-1,4-dioxine ring seems to increase binding. The corresponding butyramide 20 displays just 24 times lower affinity (Ki = 2.4 nM) than melatonin for chicken brain membranes acting as partial agonist in the cAMP assay. The 2,3-dihydro-1,4-benzoxathiine analogue 21 derived from 20 by oxygen−sulfur exchange and replacement of the amide group with a retroamide moiety shows 10-fold reduced affinity toward MT1 (IC50 = 2.3 nM) and MT2 (IC50 = 6.7 nM) receptors expressed in HEK293 cells relative to melatonin. In a series of phenoxyalkylamides and phenylthioalkylamides formally obtained from 19 by an isosteric exchange of the benzylic methylene group with oxygen and sulfur and branching the side chain by introduction of one or two methyl groups, the (S)-enantiomers behave as eutomers at both receptor subtypes. The highest stereoselectivity was observed for the thioanalogues. For example, the acetamide 22 shows a ratio of Ki eutomer (5.75 nM) to Ki distomer (2754 nM) of 480 for MT1 receptors. The (S)-enantiomer of the N-cyclopropyl substituted phenoxyalkyl derivative (S)-23 displays the highest binding affinity of the whole series with Ki(MT1) = 0.72 nM and Ki(MT2) = 4.4 nM, behaving as full MT1/MT2 agonist as assessed through [35S]GTPγS binding analysis. The structurally related (anilinoethyl)amide 24 displays similar binding for MT1 (Ki = 0.81 nM) and enhanced affinity for MT2 (Ki = 0.65 nM) at receptors expressed in NIH3T3

0.085 nM). Moreover, the ligand lipophilicity efficiency values LLE (LLE is a parameter estimating the potential of the binding interaction without the contribution of lipophilicity, defined as pKi − log D) for 14 (MT1, LLE < 5.3; MT2, LLE = 6) and 15 (MT1, LLE = 6.3; MT2, LLE = 7) are much lower than those for 13c (MT1, LLE = 7.6; MT2, LLE = 7.7), suggesting that factors other than lipophilicity contribute to their reduced binding affinity. On the contrary, 16a shows LLE = 8.0 at both receptor subtypes, being as high as the LLE for 13c. Consequently, the 7a-azanalogue of melatonin 16a has been chosen for further structure optimization. To improve the metabolic clearance, the phenyl group of 16a has been replaced by the less lipophilic ethyl moiety. The resulting compound 16b (MT1, Ki = 0.062 nM; MT2, Ki = 0.420 nM) is a full agonist in the cAMP assay equipotent to melatonin. The oral bioavailability of 16b in rats is 19.7%, and it is able to penetrate the blood−brain barrier after iv administration (B/P = 0.28, 15 min after iv administration to rats). 16b significantly decreases the percentage of wakefulness and increases the percentage of slow wave sleep in freely moving cats, making it a promising candidate for further clinical evaluation. Compound 17 is designed by simultaneous relocation of the 5-methoxy group of melatonin to position 6 and of the amide side chain to N1. 17 was reported to display 5-fold higher binding affinity for quail optic tecta membranes than melatonin. Compound 18, the 7-aza analogue of 17, exhibits Ki(MT1) = 1.4 nM and Ki(MT2) = 0.6 nM at receptors expressed in HEK293 cells acting as MT1 and MT2 agonist as revealed through [35S]GTPγS binding analysis. K



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Figure 8. Structures of MT2-selective ligands.

displays considerably lower binding (MT1, Ki = 130 nM; MT2, Ki = 31 nM) and acts as MT1/MT2 full antagonist. A recently reported constrained analogue of agomelatine with the side chain incorporated into a cyclopropane ring 28 (MT1, Ki = 0.3 nM; MT2, Ki = 0.7 nM; HEK293 cells) displays binding affinities similar to those of agomelatine with an agonist potency 40 times less than melatonin in the melanophore aggregation assay. The trans stereochemistry is essential for high binding. An extremely high affinity is generated by a trans double bond introduced into the ethylamido side chain of agomelatine. The resulting ligand 29a (MT1, Ki = 0.04 nM; MT2, Ki = 0.03 nM) is a 6 times more potent agonist than melatonin. Introduction of a second methoxy group to give 29b retains high MT1 and MT2 binding and causes a 500-fold increase in agonist activity in the melanophore aggregation assay. The tricyclic analogues, derived from melatonin, 30, and 31 and from agomelatine 32a,b, are also potent MT1/MT2 agonists displaying binding affinities similar to those of melatonin. In order to estimate the bioactive conformation of the acetylaminoethyl side chain of melatonin, the racemic mixture of the constrained agonist (±)-31 was resolved and the structure of the levorotatory enantiomer was determined by X-

cells also acting as full MT1/MT2 agonist as revealed through [35S]GTPγS binding analysis. Side chain elongation by one carbon to give 25 is tolerated at the MT1 (Ki = 0.83 nM), whereas it causes reduced MT2 binding (Ki = 20.0 nM), generating 24-fold preference for the MT1 receptor. 25 behaves as full MT1/MT2 agonist. Replacement of the N-methyl group in 24 by phenyl or naphthyl generates MT2-selective ligands discussed later in this chapter. Melatonergic ligands with the N-acylaminoethyl side chain partly incorporated into a ring have been frequently reported. For many of these conformationally restricted compounds, their action is stereoselective, and the eutomers retain high binding and agonistic action at MT1 and MT2 receptors. For example, the S enantiomer of N-phenylpiperidinecyclopropanecarboxamide (S)-27a is a potent nonselective partial to full agonist displaying Ki(MT1) = 9.9 nM and Ki(MT2) = 2.7 nM, while (R)-27a is a low affinity ligand (MT1, Ki = 1 μM; MT2, Ki = 95 nM) acting as an MT2 antagonist and a weak partial MT1 agonist using the FLIPR assay on rat MT1 and MT2 receptors. Introduction of a chlorine at the position adjacent to the methoxy substituted carbon of (S)-27a generates a potent MT1/MT2 partial agonist (S)-27b with increased affinity (MT1, Ki = 1.2 nM; MT2, Ki = 2.1 nM). The enantiomer (R)-27b L



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ray analysis, assigning its absolute configuration as R.88 (+)-(S)31 is the eutomer displaying ∼500 times higher affinity for both receptors expressed in NIH3T3 cells (MT1, Ki = 0.18 nM; MT2, Ki = 0.29 nM) and acting as a full MT1 and MT2 agonist. Attaching a six-membered N-methylpiperazine ring to the N1−C2 bond of melatonin to give 33a leads to a 10-fold decrease of binding for both MT1 and MT2. The corresponding butyramide 33b displays similarly moderate affinity at MT1 (Ki = 6.6 nM) and MT2 (Ki = 6.9 nM), acting as a partial agonist at MT1 and an antagonist at MT2 as assessed using cAMP assays. Expansion of the piperazine ring of 33a to the seven-membered 1,4-diazepane produced a dramatic drop in binding affinity at both subtypes (compound 33c; MT1, Ki =5,620 nM; MT2, Ki = 883 nM). A similar effect was observed by extending the N1− C2 substituent of 33a to a bulkier indoline moiety (compound 34; MT1, Ki =1,800 nM; MT2, Ki = 410 nM). Both ligands 33c and 34 display a slight preference for MT2. Other rigid tricyclic ligands with a hydrocarbon chain bridging the positions equivalent to N1 and C2 of melatonin are also better tolerated at the MT2 subtype. For example, compound 35 bearing a cyclohexene ring is a high-affinity MT2 ligand (Ki = 0.68 nM) exhibiting 13-fold preference for MT2 and agonist activity at both receptor subtypes. The rigid tricyclic dihydrodibenzocycloheptene analogue 36 shows Ki(MT1) = 27 nM and Ki(MT2) = 1.3 nM at receptors expressed in NIH3T3 cells acting as antagonist at both subtypes assessed through [35S]GTPγS binding analysis.

dependent release of dopamine.110Another MT2-selective ligand behaving as an antagonist in the same assay is the melatonin analogue GR128107 39110 with the acetamide side chain incorporated into a piperidine ring (MT1, Ki = 90.4 nM; MT2, Ki = 0.8 nM). 4-P-PDOT (4-phenyl-2-propionamidotetralin) is a highly MT2-selective antagonist displaying about 330 times higher affinity for the MT2 (Ki = 1.5 nM) than for the MT1 (Ki = 501 nM) receptors expressed in COS-7 cells.110 Although four stereoisomers of 4-P-PDOT exist, a pair of cis and a pair of trans enantiomers, the majority of reports on 4-P-PDOT did not specify the composition of the mixtures employed. Recently, all four single stereoisomers have been separated136 and pharmacologically evaluated at receptors expressed in NIH3T3 cells.89 The eutomer for (±)-cis-4-P-PDOT (MT1, Ki = 76 nM; MT2, Ki = 0.69 nM) is the (+)-(2S,4S) enantiomer 4089 displaying ∼170-fold higher affinity for MT2 (Ki = 0.55 nM) than for MT1 (Ki = 95 nM) and 15% and 45% intrinsic activity at MT1 and MT2 relative to melatonin in the [35S]GTPγS assay. The (−)-(2R,4R) mirror image shows MT1 (Ki = 257 nM) and MT2 (Ki = 98 nM) exhibiting ∼3 times lower agonist potency than 40. For the less MT2-selective (±)-trans-4-P-PDOT (MT1, Ki = 223 nM; MT2, Ki = 8.7 nM), the eutomer is the (+)-(2R,4S) enantiomer exhibiting just a 13fold MT2 binding preference (MT1, Ki = 129 nM; MT2, Ki = 9.5 nM). Introduction of a methoxy group in position 8 of the racemic mixtures of 4-P-PDOT leads to an increased MT1 and MT2 affinity. The cis analogue 41 (MT1, Ki = 14.8 nM; MT2, Ki = 0.06 nM) is characterized by ∼250-fold MT2 selectivity and shows 6-fold higher affinity for the MT2 receptors than melatonin, displaying 20% and 40% intrinsic activity relative to melatonin at MT1 and MT2 receptors, respectively. These pharmacological data have been used to postulate a superposition model for MT2 selective antagonists suggesting that MT1/MT2 agonists and MT2 antagonists can share the same arrangement of their common pharmacophoric elements. In the series of tetrahydroisoquinoline analogues bearing a bulky lipophilic substituent in position 1, ligand 42a displaying MT2 selectivity greater than 100-fold (MT1, IC50 > 1000 nM; MT2, IC50= 9.7 nM) is substituted with 2,2-diphenylethyl group.137 42a is a full MT2 antagonist as assessed in the cAMP assay. Removal of one of phenyl rings in the side chain led to a decrease in MT2 receptor affinity (42b; MT1, IC50 > 1000 nM; MT2, IC50= 76 nM). In the series of 2-benzylbenzofuran derivatives, the unsubstituted analogue 43a displays just a 23-fold preference for MT2 receptors (MT1, Ki = 1.27 nM; MT2, Ki = 0.05 nM; HEK293 cells).113 Introduction of OMe, Cl, F, and CF3 in different positions of the benzyl group of 43a modulated binding affinity for MT1 receptors with the m-OMe benzyl analogue 43b (MT1, Ki = 40.6 nM; MT2, Ki = 0.33 nM) showing the highest MT2 selectivity (123-fold) and acting as MT1/MT2 antagonist ([35S]GTPγS assay). The allyl analogue 43c (MT1, Ki = 21.6 nM; MT2, Ki = 0.11 nM) is characterized by the highest MT2 selectivity (192) acting as MT1/MT2 partial agonist. 2-Phenyltetralin analogues 44a−c are highly MT2-selective ligands showing selectivity ratios between 66 and 426.138 All reported compounds are (±)-cis racemates. The butyramide 44a displays the highest MT2 affinity (Ki = 0.1 nM) and 350fold MT2 selectivity (MT1, Ki = 35 nM) in receptors expressed in HEK293 or CHO cells behaving as MT1 agonist and MT2 partial agonist. The bromomethyl derivative 44b displays

6. MT2-SELECTIVE LIGANDS According to the recommendations of the International Union of Basic and Clinical Pharmacology, a selective ligand should display at least 50−100 times higher binding affinity and/or potency for one receptor subtype relative to the other.135 By application of these criteria, compounds with less than 50-fold selectivity are considered to be nonselective. Selectivity toward MT2 is easier to accomplish than toward MT1, and many series of MT2-selective ligands are known. It has been hypothesized that MT2 receptors possess a lipophilic pocket in an area close to the N1−C2 binding region of melatonin which is not present at the MT1 receptor. This hydrophobic pocket is positioned out-of-plane of the indole nucleus of melatonin, and when occupied, the corresponding ligands show reduced intrinsic activity. Accordingly, most MT2-selective antagonists or partial agonists bear a flexible bulky hydrophobic substituent in a position topologically equivalent to C2 or N1 of melatonin. The importance of this structural element has been revealed by 3D-QSAR and in docking experiments within the putative receptor model.83 Moderate MT 2 selectivity can also be achieved by introduction of Cl and OCH3 at C6 of melatonin. 6Chloromelatonin 37a110 (MT1, Ki = 11.4 nM; MT2, Ki = 0.20 nM; COS-7 cells) and 6-methoxymelatonin 37b110 (MT1, Ki = 207 nM; MT2, Ki = 3.5 nM; COS-7 cells) display 60-fold selectivity toward MT2, both behaving as MT1/MT2 agonists110 (Figure 8). The first melatonin receptor antagonists used to discriminate the role of MT1 and MT2 receptors in melatonin-mediated effects is luzindole 38a.110 While luzindole shows just a 15-fold preference for the MT2 subtype (MT1, Ki = 158 nM; MT2, Ki = 10.2 nM), the corresponding 5-methoxy analogue 38b110 is 130 times more selective for MT2 than for MT1 receptors (MT1, Ki = 32.7 nM; MT2, Ki = 0.25 nM) acting as partial agonist in an assay based on rabbit retina mediating inhibition of the calciumM



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become an interesting future target for the treatment of sleep disorders. Introduction of a methoxy group in the meta position of the phenyl ring of 48a increases MT2 selectivity. The resulting ligand 48b126 is characterized by very high MT2 binding (0.036 nM) and MT2 selectivity (527-fold), behaving as an MT1 partial agonist. Replacement of the phenyl group in 48a by a bulkier 2-naphthyl moiety to give 48c greatly reduces MT1 binding and MT2 intrinsic activity. 48c (MT1, Ki = 132 nM; MT2, Ki = 0.11 nM) exhibits the highest, 1200-fold, MT2 selectivity in whole series, acting as MT1 partial agonist and MT2 antagonist.126 Benzyloxy substituted 3-(3-methoxyphenyl)propylamides exemplified by 49a have been initially reported to exhibit extraordinarily high affinity for MT2 receptors in the subpicomolar range and MT2 selectivities up to ∼1 000 000fold in a binding assay on melatonin receptors expressed in CHO cells using [3H]melatonin as the radioligand.143 In this assay, melatonin displays Ki(MT1) = 0.296 nM and Ki(MT2) = 0.429 nM, which is in the same concentration range as the binding constants obtained using [125I]MLT. The binding data of 49a have been recently revised by the same research group to Ki(MT1) = 121 nM and Ki(MT2) = 0.291 nM, resulting in 417-fold selectivity toward MT2.144 In order to reduce the undesirable high metabolic clearance of 49a, the metabolically labile OCH2 linkage has been replaced by −CH2CH2− (49b) and −CC− (49c).144 While 49b showed no improvement in metabolic stability, 49c exhibited indeed a longer in vitro halflife, 2−3 times that of 49a. Moreover, the triple bond linked ligand 49c displays higher binding affinity and selectivity for MT2 (MT1, Ki = 38.2 nM; MT2, Ki = 0.073 nM; 527-fold selectivity) than the ethylene linked compound 49b (MT1, Ki = 22.2 nM; MT2, Ki = 0.55 nM; 40-fold selectivity). Surprisingly, 49a, 49b, and 49c are full MT2 agonists in Ca2+-based FLIPR assay, showing 181-, 679-, and 77-fold selectivity for MT2, and 12, 25, and 5 times higher agonist potency than melatonin at MT2 receptors, respectively. Another highly potent MT2-selective full agonist, 50a, was obtained by introduction of a cyclohexylmethyl group at C7 of the nonselective MT1/MT2 agonist 13a.118 Compound 50a displays 799-fold MT2 selectivity with Ki(MT1) = 9.2 nM and Ki(MT2) = 0.012 nM exhibiting 99% agonist potency relative to 1 μM melatonin in the forskolin-induced cAMP accumulation assay. In ICR (imprinting control region) mice, 50a showed reentrainment effects to a new light/dark cycle, indicating the involvement of MT2 receptors in the regulation of chronobiotic activity. Replacement of the cyclohexyl ring in 50a by a phenyl to give 50b has little effect on MT1 binding (Ki = 2.7 nM) and causes a 7-fold increase in MT2 affinity (Ki = 0.0085 nM) resulting in decreased selectivity toward MT2 (316-fold). The MT2/MT1 selectivity ratio is further reduced to 132 for the 3thienyl analogue 50c (MT1, Ki = 0.88 nM; MT2, Ki = 0.0067 nM).118 The highest MT2-selective ligand in this series is the 3isopropylphenyl substituted analogue 51 displaying 1200-fold lower affinity for MT1 (Ki = 13 nM) than for MT2 (Ki = 0.011 nM). 50b and 51 are partial MT2 receptor agonists. The MT2-selective antagonist 52 (MT1, Ki = 1290 nM; MT2, Ki = 8.7 nM) is a melatonin analogue obtained by simultaneous translocation of the 5-methoxy group to C4 and the shortened methylamido side chain from C3 to C2.83 The structural feature responsible for MT2 selectivity is the 4-chlorobenzyl substituent at N1.

dramatically reduced MT1 binding (Ki = 809 nM), while its MT2 affinity (Ki = 1.9 nM) is only moderately decreased resulting in the highest, 426-fold MT2 selectivity. The acetamide 44c with the m-OMe substituted phenyl group is 278 times more selective for MT2 than for MT1 receptors (MT1, Ki = 247 nM; MT2, Ki = 0.89 nM). Remarkable MT2 selectivity was achieved by introduction of meta-substituted phenyl groups in position 3 of the nonselective agonist agomelatine (MT1, Ki = 0.06 nM; MT2, Ki = 0.27 nM). 3-Phenylagomelatine 45a retains high binding for MT2 (Ki = 0.37 nM), whereas the MT1 affinity (Ki = 53 nM) is 140 times reduced leading to 132-fold MT2 selectivity.139 Introduction of a hydroxymethyl group in meta position generates the most favorable ligand in this series with 45b displaying high MT2 affinity (Ki = 0.36 nM) and 763-fold selectivity toward MT2 (MT1, Ki = 275 nM) and behaving as an MT2 antagonist. Bioisosteric replacement of the hydroxyl group in 45b by an amino moiety to give 45c leads to similar reduction in both MT1 (Ki = 1390 nM) and MT2 (Ki = 3.4 nM) binding maintaining the high MT2 selectivity (409-fold). Replacement of the hydroxymethyl group in 45b by an aldehyde function (45d) has no effect on MT2 (0.42 nM) and slightly enhances MT1 binding (137 nM) maintaining high MT2 selectivity (326-fold). The high selectivity toward MT2 receptors in this series is rather surprising because most melatonergic ligands bearing a phenyl ring in the equivalent position, such as 7b, 10b, 11b, 12, 13c, 15, and 16a are highaffinity agonists showing no subtype selectivity. Moreover, the C3-phenyl ring is coplanar with the naphthalene ring, and consequently, the structures of 45a−d do not satisfy the pharmacophore model for MT2 antagonists requiring a bulky lipophilic substituent arranged out of plane of the core ring. Remarkable MT2 selectivity can also be achieved by introduction of alkyl groups bulkier than propyl in position 3 of agomelatine.140 The isoamyl-substituted analogue 46a (MT1, Ki = 72 nM; MT2, Ki = 0.01 nM) shows an extremely high 7200-fold binding selectivity toward MT2 receptors behaving as partial MT1/MT2 agonist with intrinsic activity 900 times higher at MT2 than at MT1 receptors in the [35S]GTPγS binding assay. Further increase in bulk exemplified by the methylcyclobutyl derivative 46b (MT1, Ki = 119 nM; MT2, Ki = 0.48 nM) is detrimental for MT2 binding, leading to reduced MT2-selectivity. Introduction of a phenyl ring at the benzylic position of compound 19 to give 47 (MT1, Ki = 2187 nM; MT2, Ki = 5.4 nM) generates 405-fold selectivity toward MT2.141 Compound 47 acts as MT1/MT2 partial agonist. To increase water solubility of the highly lipophilic agent 47, the benzylic carbon was exchanged by nitrogen. The resulting ligand 48a (UCM765)126 displays extremely high affinity (Ki = 0.066 nM) and 64-fold selectivity for the MT2 receptors, behaving as an MT1/MT2 partial agonist. 48a has been recently reported to promote nonrapid eye movement sleep (NREMS) in rats and mice.142 This effect is nullified by the pharmacological blockage or genetic deletion of MT2 receptors. Interestingly, the effects of 48a on sleep are different from those of nonselective MT1/ MT2 agonists. In particular, the structurally related MT1/MT2 agonist 24 (UCM793) slightly decreases sleep onset without having an effect on NREMS maintenance, similar to ramelteon, suggesting that dual MT1/MT2 agonistic activity accounts for the effect on sleep onset, whereas selectivity for MT2 receptors has an additional effect on NREMS maintenance. Because of the selective promotion of NREMS, MT2 receptors could N



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corresponding allylcarboxamide 53b, the MT1/MT2 affinity ratio is increased to 220 (MT1, Ki = 52.8 nM; MT2, Ki = 0.24 nM). The tetracyclic melatonin analogues with the 2-phenyl substituent linked through the ortho position to the indole nitrogen by methylene (54a; MT1, Ki = 4.47 nM; MT2, Ki = 0.05 nM) and propylene (54b; MT1, Ki = 66.1 nM; MT2, Ki = 0.50 nM) groups show 89- and 132-fold selectivity for the MT2 receptors expressed in NIH3T3 cells, respectively.146 Remarkably, while compound 54a, in which the 2-phenyl substituent is coplanar with the indole ring, behaves as full agonist at MT1 and MT2 receptors, compound 54b possessing a bulkier sevenmembered ring attached to N1-C2 and the C2-phenyl arranged out-of-plane of the indole nucleus is an MT2 antagonist in both melanophore aggregation and cAMP assays. Melatonin analogues substituted at C2 with conformationally flexible N-methylindoline (55; MT1, Ki = 115 nM; MT2, Ki = 1.1 nM)147 and N-methylisoindoline groups (56; MT1, Ki = 282 nM; MT2, Ki = 2.3 nM)148 are also MT1/MT2 full antagonists (cAMP assay) displaying ∼100-fold selectivity toward MT2 in receptors expressed in CHO cells. Expansion of the indoline ring in 55 by insertion of a methylene group between the indolic nitrogen and the benzene ring leads to reduced MT1 (251 nM) and MT2 (21 nM) binding. 5-Me, 5Br, 6-NO2, and 6-NH2 substitution of the indoline ring of 55 also causes substantial reduction in MT2 affinity and selectivity.

The structural requirements for MT2-selective antagonists exemplified by compounds 48c and 52 are illustrated in Figure 9.

Figure 9. Model of compounds 48c (orange carbons) and 52 (yellow carbons) within the MT2 receptor binding site.83 The out-of-plane group of MT2-selective antagonists is accommodated within a lipophilic pocket close to Trp264 of the CWXP motif. In the MT1 receptor, Val128 is replaced by the bulkier isoleucine and Ile213 by a methionine.

The benzothiophene analogue 53a derived from melatonin by replacement of N1 with S, exchange of 5-OMe with F, and introduction of 4-fluorophenylthio moiety at C2 is an MT1/ MT2 partial agonist showing 65 times higher binding for MT2 (Ki = 0.87 nM) than for MT1 (Ki = 57 nM).145 For the

7. MT1-SELECTIVE LIGANDS Development of MT1-selective ligands remains a challenging task, and only few compounds with some preference for MT1

Figure 10. Structures of MT1-selective ligands. O



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in CHO cells acting as MT1/MT2 agonist.150 Monomeric ligands, such as the 4-phenylbutyl substituted benzoxazole analogue 63153 (MT1, Ki = 0.63 nM; MT2, Ki = 22 nM; CHO cells) and the dihydrobenzopyran derivative 64 (MT1 Ki = 3.4 nM, MT2 Ki = 21.4 nM, CHO cells; MT1 Ki = 1.2. nM, MT2 Ki = 29 nM, HEK cells)150 show only 35- and 25-fold preference for MT1 receptors, respectively. In an extensive series of melatonin analogues obtained by exchanging the ether methyl group with arylalkyl and aryloxyalkyl moieties of different chain lengths, the most MT1 selective compounds 65a and 65b are substituted with Ph(CH2)3 and PhO(CH2)3 groups, respectively, confirming the optimal spacer length generating MT1 selectivity to be (CH2)3.154 Although 65a (MT1, Ki = 3.9 nM; MT2, Ki = 49 nM; CHO cells) and 65b (MT1, Ki = 7.9 nM; MT2, Ki = 87 nM; CHO cells) show only ∼10-fold preference toward MT1 receptors, their MT1/MT2 binding ratio is higher than that of the dimeric agomelatine 57a that has been found to display just a 3-fold preference toward MT1 receptors (MT1, Ki = 112 nM; MT2, Ki = 355 nM) in the same study. These findings underscore the importance of running multiple reference compounds having ideally different binding profiles when conducting pharmacological analysis to minimize the variability of results between different laboratories. Compound 65b behaves as MT1/MT2 agonist in the cAMP assay. In a similar approach, N-(anilinoalkyl)amides bearing arylalkyloxy subtituents of different chain lengths have been examined on melatonin receptors expressed in NIH3T3 cells. The phenylbutyloxy analogue 66 displays the highest 78-fold selectivity toward MT1 (MT1, Ki = 1.17 nM; MT2, Ki = 91.2 nM), acting as partial agonist at both receptor subtypes.81 As proposed in an MT1 receptor model, the phenylbutyloxy substituent of 66 forms hydrophobic interactions with several amino acids such Gln101, Gln104, Phe105, Val159, Leu163, Leu168, Gly169, and Tyr175 (Figure 11). In the MT2 receptor model, some of the latter are replaced by bulkier amino acids, preventing an optimal accommodation of the phenylbutyloxy group and thus reducing binding. Substantial MT1 selectivity could be achieved by introduction of two fluorine atoms into the N-acetyl group of agomelatine.

have been reported so far. Moreover, while for MT2-selective agents Ki(MT1)/Ki(MT2) > 1000 can be achieved, ligands preferentially binding to MT1 reach maximally ∼100-fold higher affinity for MT1 than for MT2 receptors. A common structural feature conferring MT1 selectivity is a bulky, hydrophobic ether replacing the methoxy group in a position equivalent to C5 of melatonin. The first MT1-selective agents were obtained by connecting two agomelatine units via their ether oxygens by a polymethylene spacer.149 In a series of these (CH2)n-linked bivalent ligands (n = 2−10), the highest MT1 selectivity was achieved for n = 3 (57a) and n = 4 (57b) (Figure 10). Compound 57a displays 224 times higher affinity for the MT1 (Ki = 0.5 nM) than for the MT2 receptors (Ki = 112 nM) expressed in HEK cells, acting as an MT1/MT2 antagonist. In CHO cells (MT1, Ki = 3.9 nM; MT2, Ki = 149 nM) the Ki(MT2)/Ki(MT1) binding ratio was reduced to 38.150 The (CH2)4-linked agomelatine dimer 57b shows Ki(MT1) = 3.1 nM, Ki(MT2) = 167 nM in CHO cells (54-fold MT1 selectivity) and Ki(MT1) = 0.6 nM, Ki(MT2) = 73.2 nM in HEK cells (122-fold MT1 selectivity). An extensive series of heterodimer analogues of 57b obtained by replacement of one of its agomelatine head groups with various aryl moieties including the equally substituted indole (melatonin), tetralin, benzofuran, and benzothiophene has been evaluated at receptors expressed in CHO cells.151 Further structure modifications included variation of the amide substituent at one or both sides, exchange of one of the ethylamido side chains by acetic acid and methyl acetate, and replacement of one of the agomelatine units by a biphenylcarboxylic acid, the corresponding methyl ester, and alcohol. The most MT1-selective ligands are biphenylcarboxylic acid 58a (MT1, Ki = 0.55 nM; MT2, Ki = 51.3 nM) and the corresponding alcohol 58b (MT1, Ki = 0.09 nM; MT2, Ki = 6.53 nM) exhibiting 93-fold and 72-fold MT1 selectivity, respectively, and acting as partial MT1/MT2 agonists as assessed through [35S]GTPγS binding analysis. In contrast, the less MT1-selective ligand 59 (MT1, Ki = 0.37 nM; MT2, Ki = 4.22 nM) behaves as MT1/MT2 full agonist. As compounds 58a,b and 59 lack the amido side chain attached to an aromatic ring in one part of their structures, the findings indicate that a bivalent ligand is not necessary to achieve MT1 selectivity. Interestingly, replacement of one of the agomelatine units in the parent compound 57b by equally substituted indole, benzofuran, benzothiophene, and tetralin nuclei causes reduction of MT1 selectivity from 120-fold to 21-, 10-, 5-, and 26-fold, respectively, with the tetralin analogue 60 (MT1, Ki = 0.26 nM; MT2, Ki = 6.79 nM) displaying the highest 26-fold preference for MT1 receptors. Another series of bivalent ligands showing 5- to 102-fold MT1 selectivity is a product of linking two molecules of the nonselective MT1/MT2 agonist 24 via ether oxygens using (CH2)n spacers of different lengths (n = 3−6, 8, 10).152 Similar to the dimeric agomelatine series, in the most MT1-selective ligand 61a (MT1, Ki = 20.4 nM; MT2, Ki = 2089 nM; NIH3T3 cells), the head pharmacophores are separated by a (CH2)3 spacer. The corresponding (CH2)6 analogue 61b exhibits the highest MT1 (3.4 nM) affinity and is 54 times more selective for MT1 than for MT2 receptors. Both compounds behave as partial MT1/MT2 agonists as assessed through [35S]GTPγS binding analysis. A dimeric MT1-selective agent 62 results from a direct coupling of two agomelatine units via the aromatic C7 carbon atoms and displays Ki(MT1) = 5.2 nM and Ki(MT2) = 246 nM

Figure 11. Structural hypothesis for MT1 selectivity.81 Compound 66 (orange carbons) is represented within the MT1 binding site. The phenylbutyloxy substituent is accommodated in a lipophilic region, mainly defined by TM3 and TM4, pointing toward the extracellular space. Gly104, Val159, and Leu163 are replaced in the MT2 receptor by alanine, leucine, and phenylalanine, respectively. These bulkier amino acids hamper the accommodation of the phenylbutyloxy group. P



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The difluoroacetamide 67 (MT1, Ki = 4.3 nM; MT2, Ki = 0.03 nM) is 143 times more selective for MT1 than for MT2 receptors, acting as nonselective MT1/MT2 agonist in the [35S]GTPγS assay.140 The corresponding monofluoroacetamide is a nonselective MT1/MT2 agonist showing MT1 and MT2 affinity similar to that of agomelatine.140

or enhance recycling back to the plasma membrane in breast tumors may prove to be efficacious agents by enhancing the efficacy of the melatonin response similar to what is reported for somatostatin receptor SSTR2−SSTR5 dimers and their enhanced growth-inhibiting effects in GH-sensitive tumors.168 Receptor dimerization, especially dimers formed with μ opioid receptors (μRs), has been shown to enhance μR function probably by preventing μR internalization or promoting μR recycling back to the plasma membrane. For example, μR−δ opioid receptor (δR) dimers169 or μR− nociceptive receptor (NOPR) dimers have been shown to enhance the efficacy of μR agonists at μRs,170 activate μRs via δR activation,171 and modulate μR-mediated inhibition of Ntype calcium channels and μR desensitization by inducing NOPR internalization.172 This ability to maintain melatonin receptor expression at the plasma membrane could serve two important purposes: (1) to maintain receptor coupling to signal transduction cascades that underlie melatonin’s therapeutic efficacy (e.g., β-arrestin/MEK/ ERK1/2 coupling to maintain melatonin’s antiproliferative and prodifferentiation actions in cancer cells) and (2) to maintain therapeutic efficacy of melatonin in response to chronic agonist exposure by preventing melatonin receptor internalization. Receptor dimers may also be used to tone down a response especially as shown for HER2+ breast cancers where SSTR5 has been shown to modulate HER2-positive breast cancers through its ability to break up EGFR−ErbB2 heterodimers by driving ErbB2 into heteromers with SSTR 5.173 Assuming that melatonin receptors also heteromerize with ErbB2, ligands promoting this complex formation may prevent EGFR−ErbB2 heterodimer formation and be effective at treating HER2+ breast cancer. Besides stabilizing receptor complexes at the plasma membrane and maintaining their responsiveness to their respective agonist, bivalent melatonergic ligands could also be developed to direct receptor coupling to signal transduction cascades (i.e., β-arrestin/MEK/ERK1/2) that are known to slow cell growth and induce cellular differentiation.168 Coexpression of MT1 and MT2 receptors in other cellular systems has been shown to modulate receptor coupling to ERK1/2 and their ability to respond to melatonin over time.174 Recent data on MT1/MT2 heteromers showing modified signaling capacities of heteromers on the cAMP and PKC pathways support the existence of MT1/MT2 heteromerspecific properties that remain to be extended toward the ERK1/2 pathway.55 Many of the current melatonergic drugs on the market or in clinical development are indicated for the treatment of sleep disorders (ramelteon, tasimelteon, 4) and depression (agomelatine). As stated previously, the drugs that are showing some efficacy for treating sleep disorders and depression are those with dual action (i.e., agomelatine with its melatonin receptor agonist/5-HT2C receptor antagonist actions and 4 with its melatonin receptor agonist/5-HT2C and 5-HT2B receptor antagonist actions, respectively). Therefore, drug therapies targeted at multiple receptors rather than one to achieve similar therapeutic outcomes may show improved efficacy with better disease prognosis. For example, in the context of CNS disorders like depression, Alzheimer’s disease, and Parkinson’s disease, bivalent drugs designed to stabilize receptor heterodimers may show enhanced therapeutic efficacy either through synergistic interactions augmenting a desired physiological effect or by prolonging a desired drug effect at receptors through a

8. THERAPEUTIC PERSPECTIVES Over the years, many studies have been published demonstrating a wide variety of receptors comprising homo- and heteromeric complexes. The existence of such complexes has to be taken into account for future drug design and therapeutic applications. Importantly, the functional consequences of receptor oligomerization are diverse, which increases the potential for developing novel and unique therapeutic agents with clinical utility that covers a wide spectrum of conditions or diseases. For example, these types of drugs could be used to manage chronic conditions like pain and inflammation or diseases affecting the cardiovascular system, the CNS, or metabolism as well as cancer (see Table 2, Supporting Information). Many of these studies show that receptor oligomerization may alter receptor function (i.e., increase function or decrease function) depending on the identity of the oligomerization partner and which signal transduction processes are being affected (i.e., receptor coupling to Gproteins, receptor-mediated effects on intracellular proteins like MAPKs, or receptor internalization/recycling processes). Apart from forming new pharmacological entities with distinct properties, GPCR oligomerization also opens new therapeutic perspectives. Among these new tools will be compounds specifically targeting GPCR homomers and heteromers, which can be monovalent and bivalent. Bivalent ligands are typically designed with the idea to stabilize preexisting dimers or to obtain increased affinity by the binding of two pharmacophores to GPCR dimers. Bivalent ligands also offer the possibility of combining pharmacophores with agonistic and antagonistic properties on the respective homomers. For example, to treat hypertension, bivalent drugs could be developed to antagonize B2 bradykinin receptors (B2Rs) dimerized to angiotensin converting enzyme I (ACE) to inhibit ACE activity,155 lowering blood pressure. Bivalent drugs designed to stabilize ACE-B2R dimers (ACE-B2R)156 or angiotensin type II receptor (AT2R) and B2R complexes (AT2R-B2R)157 could be developed to increase NO production in blood vessels to increase blood flow in poorly perfused tissues. In a similar manner, novel melatonergic bivalent ligands could be developed to treat cardiovascular disease, since both MT1 and MT2 receptors have been detected in arteries and antagonism of one receptor (MT2) leads to enhanced activity of the other receptor, MT1.158 However, because the MT1 receptor mediates vasoconstriction and the MT2 receptor mediates vasodilation,158 a bivalent MT1 antagonist−MT2 agonist ligand may prove to be an efficacious antihypertensive drug. Many studies have shown melatonin receptor expression in cancer cell lines or tumor tissues where activation of MT1 and MT2 receptors by melatonin produces antiproliferative and prodifferentiating (cancer protective) effects.13,159−164 In some breast cancers, the expression patterns of MT1 and MT2 receptors are modified or down-regulated,165,166 which may be contributing to cancer proliferation and tumorigenesis. Overexpression of MT1 restores the antiproliferative actions of melatonin.167 Compounds that prevent receptor internalization Q



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(P2Y1R) ligands to treat Parkinson’s or Alzheimer’s disease,192 D1 dopamine receptor (D1R)−D2 dopamine receptor (D2R) bivalent ligands to treat schizophrenia,193 or D1R−D3R ligands to treat depression.194 However, whether or not the bivalent ligands should be dual agonists, dual antagonists, or agonist/ antagonist combinations needs to be considered in the context of the function of these receptors within a given tissue and the desired therapeutic outcome. One last area that should be targeted by use of bivalent ligands should be in appetite control. As shown in Table 2 (Supporting Information), there are many heterodimeric combinations that profoundly affect appetite and energy balance. These dimeric combinations include V1B vasopressin receptors (V1BR) and corticotropic releasing factor receptors (CRFR),195 metabotropic glutamate receptors (mGluR) and GABAB receptors,196 OX1 orexin receptors (OX1R) and CB1 cannabinoid receptors (CB1R),197 ghrelin receptors (GHSR) and MC3 melanocortin receptors (MC3R),198 and MT1 receptors and the orphan melatonin related receptor GPR50 protein.56 The fact that these receptors play significant roles in energy expenditure, appetite, and sleep has wide appeal for the development of bivalent drugs for the treatment of obesity, diabetes, and cancer cachexia. For example, a V1BR−CRFR dual agonist may be used to enhance insulin secretion in diabetes mellitus type 2. For the treatment of cachexia, a dual acting OX1R−CB1R (antagonist) ligand or GHSR−MC3R (agonist) ligand may be developed to stimulate appetite in cancer patients or a dual acting OX1R−CB1R (agonist) ligand or GHSR−MC3R (antagonist) ligand may be developed to suppress appetite for the treatment of obesity. The fact that MT2 receptor variants are associated with the development of type 2 diabetes199 and the fact that the orphan receptor GPR50 is highly expressed in the hypothalamus and involved in weight regulation,200 thermoregulation, and metabolism indicate a link between these receptors and metabolic diseases that could be the basis for future drug design.201

stabilization of these receptor complexes at the plasma membrane by preventing agonist-induced internalization and/ or down-regulation of receptors. Melatonin receptors are expressed in key brain regions involved in memory,175−177 mood,178 and locomotor activity,179 and their pattern of expression changes (mostly decreases) in disease states like Alzheimer’s disease,177 depression,178 and Parkinson’s disease.179 It has already been shown that melatonin receptor subtypes, MT1 and MT2, can heterodimerize with each other or with the melatonin related receptor, GPR50, and influence melatonin receptor function;50,180,181 and so perhaps changes in their expression patterns may contribute to disease onset, progression, and prognosis as reviewed.18 Chronic drug therapy given early enough could be used to prevent the loss of melatonin receptor expression or normalize melatonin receptor subtype expression in these regions of the brain to prevent, slow, or even reverse the course of the disease. As stated previously and in the context of anticancer therapies, melatonergic ligands may be used to prevent MT1−MT2 internalization by stabilizing plasma membranebound melatonin receptors. This would prevent melatonin receptor desensitization and maintain melatonin efficacy at melatonin receptors to improve therapeutic outcomes in individuals afflicted with cancer. These types of drugs may also prove to be beneficial in people afflicted with CNS disorders that affect mood, cognition, and motor coordination, since a loss of MT1 receptors was established in models thought to predict antidepressant effects in humans182 while a lack of MT2 receptors in rodent models may play a role in memory loss.183 Therefore, a gain in or maintenance of melatonin receptor expression by use of monovalent or bivalent melatonergic drugs may be effective as antidepressants. Chronic antidepressant treatment has been shown to increase MT1 mRNA levels and decrease MT2 mRNA levels in mouse hippocampus.184 New melatonergic ligands could also be developed to promote the coupling of melatonin receptors, MT1 and MT2, to signal transduction cascades thought to play significant roles in memory. Coexpression of MT1 and MT2 receptors in cerebellar granular cells has been shown to modulate melatonin receptor coupling to ERK1/2,174 a signal transduction pathway that may play a role in memory and neurogenesis.35,18 Besides melatonin receptors, heterodimerization of M2 muscarinic receptors (M2Rs) with GABAB2R has been proposed as a mechanism to prevent M2R downregulation; a loss of M2Rs may be one of the underlying causes of Alzheimer’s disease.185 Therefore, maintaining M2R expression, perhaps by the use of bivalent M2R−GABAB2R ligands or even MT1−M2R or MT2−M2R bivalent ligands, may prove to be efficacious for treating Alzheimer’s disease and warrant investigation. With respect to Parkinson’s disease, most studies186 report a beneficial effect of melatonin in preclinical and cell culture models of Parkinson’s disease. For many of the studies, melatonin treatment prevented dopamine neuronal loss or dopamine transporter down-regulation induced by rotenone,187 6-hydroxydopamine, 188 or 1-methyl-4-phenylpyridinium (MPP).189−191 Bivalent drugs coupling melatonin receptors to dopamine receptors or to the dopamine transporter may help to maintain dopamine receptor function in the CNS to prevent or treat Parkinson’s disease. Besides melatonin receptors, other bivalent drugs to consider for treating CNS disorders may include A1 adenosine receptor (A1R)-P2Y1 purinergic receptor

9. CONCLUSION The melatonin receptor family is composed of two high-affinity MT1 and MT2 receptors. The large expression profile and the low expression levels made it initially difficult to define clear therapeutic application for these receptors. Multiple nonselective MT1/MT2 agonists have been developed of which the first are now on the market for the treatment of insomnia, circadian sleep disorders, and depression. More recently subtype-selective ligands, in particular for the MT2 receptor, were developed. Although the structure of melatonin receptors has not been solved yet, major progress is expected from molecular modeling, which will also assist the development of new subtype-selective compounds. In addition, therapeutic fields of interest have to be clearly defined for these subtypeselective compounds. Type 2 diabetes might be one of these fields, as recent studies established an association between multiple rare loss-of-function MT2 mutants and type 2 diabetes risk. Future trends in melatonin receptor pharmacology are also likely to see an extension of the development of compounds with dual actions. This concept has been successfully applied to the development of the antidepressant agomelatine, which combines the agonistic effect on melatonin receptors with antagonistic effect on serotonin 5-HT2C receptors. This class of ligands is obviously of particular interest, as it has the potential to combine therapeutic effects of both receptors in a synergistic R



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manner. On the basis of the increasingly recognized in vivo relevance of melatonin receptor di(oligo)mers, the development of dimer-specific and particularly MT1/MT2 heteromerspecific monovalent and bivalent ligands will open new opportunities for drug design. Finally, further improvement and confirmation of our knowledge on the role of melatonin receptors in physiological and pathophysiological situations are likely to become new fields for melatonin receptor ligands such as neurodegenerative diseases and cancer.

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system, inhibitors of epidermal growth factor receptor, and histamine H3 receptor antagonists. Paula A. Witt-Enderby received her Bachelor of Science degree in Biochemistry in 1988 from the University of Illinois and her Ph.D. in Pharmacology and Toxicology in 1993 from the University of Arizona. She completed a postdoctoral fellowship from 1993 to 1996 at Northwestern University Medical School, IL, in the Department of Molecular Pharmacology and Biological Chemistry on a National Research Service Award. At present, she is a Professor of Pharmacology in the Division of Pharmaceutical Sciences at Duquesne University School of Pharmacy, PA. Her research interests focus on melatonin receptor-mediated signal transduction processes that modulate cellular differentiation with an application to anticancer and bone-stimulating therapies and in the development of novel melatonergic ligands.

ASSOCIATED CONTENT

S Supporting Information *

[125I]MLT binding sites in different tissues and cells (Table 1) and receptor dimers and their therapeutic potential (Table 2). This material is available free of charge via the Internet at http://pubs.acs.org.

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ABBREVIATIONS USED ACE, angiotensin converting enzyme; AFK, N1-acetyl-5methoxykynuramine; A1R, A1 adenosine receptor; AFMK, N1-acetyl-N2-formyl-5-methoxykynuramine; AT2R, angiotensin type II receptor; B2R, B2 bradykinin receptor; BRET, bioluminescence resonance energy transfer; cAMP, cyclic adenosine monophosphate; CB1R, CB1 cannabinoid receptor; CHO, Chinese hamster ovary; CRFR, cortocotropic releasing factor receptor; D1R, dopamine D1 receptor; D2R, dopamine D2 receptor; D3R, dopamine D3 receptor; δR, δ opioid receptor; ErbB2, avian erythroblastic leukemia viral oncogene homologue 2; ERK, extracellular signal-regulated kinase; GABAB2R, γ-aminobutyric acid receptor subtype B2; FLIPR, fluorescent imaging plate reader; GHSR, ghrelin receptor; GTPγS, guanosine 5′-O-(3-thiotriphosphate); HEK, human embryonic kidney; HER2, human epidermal growth factor receptor 2; 5-HT2b, 5-hydroxytryptamine (serotonin) receptor subtype 2b; 5-HT2c, 5-hydroxytryptamine (serotonin) receptor subtype 2c; LLE, ligand lipophilicity efficiency; mGluR, metabotropic glutamate receptor; OX1R, OX1 orexin receptor; P2Y1R, purinergic receptor P2Y1; PKC, protein kinase C; PLC, phospholipase C; [125I]MLT, 2-[125I]iodomelatonin; ECL, extracellular loop; ICL, intracellular loop; L−R−G complex, ligand−receptor−G protein complex; MAPK, mitogen-activated protein kinase; MC3R, MC3 melanocortin receptors; MPP, 1-methyl-4-phenylpyridinium; M2R, M2 muscarinic receptor; MT1, melatonin receptor subtype 1; MT2, melatonin receptor subtype 2; μR, mu opioid receptor; NOPR, nociceptive receptor; NREMS, nonrapid eye movement sleep; SAR, structure−activity relationship; 4P-PDOT, 4-phenyl-2propionamidotetralin; RT-PCR, reverse transcription polymerase chain reaction; SCN, suprachiasmatic nucleus; SSTR2, somatostatin receptor type 2; SSTR5, somatostatin receptor type 5; TM, transmembrane; V1BR, V1B vasopressin receptor

AUTHOR INFORMATION

Corresponding Author

*Phone: +20 2 2758 1041. Fax: +20 2 2758 1041. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Darius P. Zlotos studied chemistry in Bonn, Germany, and received his Ph.D. in Pharmaceutical Chemistry in 1997. After postdoctoral time in Wü r zburg, Germany (1998−2008, “Habilitation” in Pharmaceutical Chemistry in 2004), he was appointed Professor of Pharmaceutical Chemistry at The Germany University in Cairo, Egypt, in 2008. His recent research is focused on the development of subtypeselective ligands for melatonin, acetylcholine, and glycine receptors. Ralf Jockers studied in Cologne, Germany, and received a Ph.D. in Biotechnology and Biochemistry from the University of Braunschweig, Germany. For postdoctoral training he joined the laboratory of Dr. A. D. Strosberg at Cochin Institute, Paris, where he worked on the regulation of β-adrenoceptors. Since 1998 he holds a permanent position at INSERM, France. He published more than 100 scientific papers and delivered more than 80 invited conferences. His research centers on the regulation of membrane receptors such as G-proteincoupled receptor and cytokine receptors by receptor oligomerization and by receptor-associated protein complexes. For 10 years his group has been working on melatonin and leptin receptors. He recently discovered by large-scale exon sequencing multiple rare mutations of the melatonin MT2 receptors associated with type 2 diabetes. Erika Cecon received her degree in Biological Sciences in 2007 and her Masters degree in Physiology in 2010 from the University of Sao Paulo (Sao Paulo, Brazil). At present she is a Ph.D. student in Physiology in Sao Paulo. In 2013, a scholarship by FAPESP funding agency supported her stay in Dr. Ralf Jockers’ laboratory, France. Her main research interests concern the modulation of melatonin synthesis and melatonin receptor-mediated signaling in pathological conditions.

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MT1 and MT2 Melatonin Receptors - American Chemical Society

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