Differences in Binding Sites of Two Melatonin Receptors Help to Explain Their Selectivity to Some Melatonin Analogs: A Molecular Modeling Study

Abstract

Numerous diseases have been linked to the malfunction of G-protein coupled receptors (GP-CRs). Their adequate treatment requires rational design of new high-affinity and high-selectivity drugs targeting these receptors. In this work, we report three-dimensional models of the human MT₁ and MT₂ melatonin receptors, members of the GPCR family. The models are based on the X-ray structure of bovine rhodopsin. The computational approach employs an original procedure for optimization of receptor-ligand structures. It includes rotation of one of the transmembrane α-helices around its axis with simultaneous assessment of quality of the resulting complexes according to a number of criteria we have developed for this purpose. The optimal geometry of the receptor-ligand binding is selected based on the analysis of complementarity of hydrophobic/hydrophilic properties between the ligand and its protein environment in the binding site. The elaborated “optimized” models are employed to explore the details of protein-ligand interactions for melatonin and a number of its analogs with known affinity to MT₁ and MT₂ receptors. The models permit rationalization of experimental data, including those that were not used in model building. The perspectives opened by the constructed models and by the optimization procedure in the design of new drugs are discussed.     

Biological basis and possible physiological implications of melatonin receptor-mediated signaling in the rat epididymis

The mammalian epididymis plays an important role in sperm maturation, an important process of male reproduction. Specific high-affinity 2-[(125)I]iodomelatonin binding sites, satisfying the pharmacokinetic properties of specific receptors, have been found in the rat corpus epididymis, suggesting a direct melatonin action on epididymal physiology. Subsequent molecular and cell biology studies have identified these 2-[(125)I]iodomelatonin binding sites to be mt(1) (MEL(1A)) and MT(2) (MEL(1B)) melatonin receptor subtypes. Changes in the binding characteristics of these receptors in the rat corpus epididymis in response to castration and steroid hormones like testosterone and hydrocortisone indicated that these membrane melatonin receptors are biologically functional receptors, whose activities are differentially regulated by testosterone and hydrocortisone. These melatonin receptors are coupled to pertussis toxin (PTX)-sensitive G(i) protein and probably participate in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions. Furthermore, rat corpus epididymal epithelial cell proliferation was stimulated by melatonin, whose action was dependent on the concentration and duration of exposure to the hormone. Interestingly, an MT(2) receptor ligand (4-phenyl-2-propionamidotetraline, 4-P-PDOT) induced a stimulatory effect on epididymal epithelial cell proliferation similar to that produced by melatonin. In contrast, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxo-thiazolidine-2-ylidene]-4-methyl-thiosemi-car bazone , CGP52608) and 8-bromo-cAMP inhibited epididymal epithelial cell proliferation. Taken together, our data lead us to postulate that one of the possible physiological functions of melatonin on the rat epididymis is the stimulation of mt(1) and MT(2) melatonin receptors resulting in the inhibition of cAMP signaling and an increase in epithelial cell proliferation.     

Molecular determinants for the differential coupling of Galpha(16) to the melatonin MT1, MT2 and Xenopus Mel1c receptors

The pineal neurohormone melatonin modulates a variety of physiological processes through different receptors. It has recently been reported that the cloned melatonin receptors (MT1, MT2 and Mel1c) exhibit differential abilities to stimulate phospholipase C (PLC) via G(16). Here we examined the molecular basis of such differences in melatonin receptor signaling. Coexpression of MT1 or MT2 with the alpha subunit of G(16) (Galpha(16) ) allowed COS-7 cells to accumulate inositol phosphates in response to 2-iodomelatonin. In contrast, Mel1c did not activate Galpha(16) even though its expression was demonstrated by radioligand binding and agonist-induced inhibition of adenylyl cyclase. As Mel1c possesses an exceptionally large C-terminal tail, we further asked if this structural feature prevented productive coupling to Galpha(16). Eleven chimeric melatonin or mutant receptors were constructed by swapping all or part of the C-terminal tail between MT1, MT2 and Mel1c. All chimeras were fully capable of binding 2-[(125) I]iodomelatonin and inhibiting adenylyl cyclase. Chimeras containing the full-length Mel1c tail were incapable of activating Galpha(16), while those that contained the complete C-terminal region of either MT1 or MT2 stimulated PLC. Incorporation of the extra portion of the C-terminal tail of Mel1c to either MT1 or MT2 completely abolished the chimeras' ability to stimulate PLC via Galpha(16). In contrast, truncation of the C-terminal tail of Mel1c allowed interaction with Galpha(16). Our results suggest that Galpha(16) can discern structural differences amid the three melatonin receptors and provide evidence for functional distinction of Mel1c from MT1 and MT2 receptors.     

Therapeutic potential of melatonin ligands

Melatonin is a neurohormone that is believed to be involved in a wide range of physiological functions. In humans, appropriate clinical trials confirm the efficacy of melatonin or melatoninergic agonists for the MT1 and MT2 receptor subtypes in circadian rhythm sleep disorders only. Nevertheless, preclinical animal model studies relevant to human pathologies involving validated reference compounds lead to other therapeutic possibilities. Among these is a recently developed treatment concept for depression, which has been validated by the clinical efficacy of agomelatine, an agent having both MT1 and MT2 agonist and 5-HT2C antagonist activity. A third melatonin binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). The physiological role of this enzyme is not yet known. Recent results obtained by different groups suggest: (1) that inhibition of QR2 may lead to "protective" effects and (2) that over-expression of this enzyme may have deleterious effects. The inhibitory effect of melatonin on QR2 observed in vitro may explain the protective effects reported for melatonin in different animal models, such as cardiac or renal ischemia-effects that have been attributed to the controversial antioxidant properties of the hormone. The development of specific ligands for each of these melatonin binding sites is necessary to link physiological and/or therapeutic effects.     

Synthesis and biological activity of new melatonin dimeric derivatives

A new series of melatonin (MLT) dimers were obtained by linking together two melatonin units with a linear alkyl chain through the MLT acetamido group or through a C-2 carboxyalkyl function. The binding properties of these ligands were evaluated in in vivo experiments on cloned human MT(1) and MT(2) receptors expressed in NIH3T3 rat fibroblast cells. The class of 2-carboxyalkyl dimers was the most interesting one with compounds having good MT(1)/MT(2) nanomolar affinity. The data obtained suggest that the spacer length is crucial for optimal interaction at both receptor subtypes as well as to determine functional activity of the resulting dimers.     

Design of subtype selective melatonin receptor agonists and antagonists.

Studies of the physiological actions of melatonin have been hindered by the lack of specific, potent and subtype selective agonists and antagonists. In the present study, we describe the utility of a melanophore cell line from Xenopus laevis for exploring structure-activity relationships among novel melatonin analogues and report a novel MT2-selective agonist (IIK7) and MT2-selective receptor antagonist (K185). IIK7 is a potent melatonin receptor agonist in the melanophore model, and in NIH3T3 cells expressing human mt1 and MT2 receptor subtypes. In radioligand binding experiments IIK7 is 90-fold selective for the MT2 subtype. K185 is devoid of agonist activity, but acts as a competitive melatonin antagonist in melanophores. A low concentration (10(-9) M) antagonizes melatonin inhibition of forskolin stimulation of cyclic AMP in NIH3T3 cells expressing human MT2 receptors, but has no effect in cells expressing mt1 receptors. In binding assays, K185 is 140-fold selective for the MT2 subtype.     

Physiological and metabolic functions of melatonin

Melatonin is a lipophilic hormone, mainly produced and secreted at night by the pineal gland. Melatonin synthesis is under the control of postganglionic sympathetic fibers that innervates the pineal gland. Melatonin acts via high affinity G protein-coupled membrane receptors. To date, three different receptor subtypes have been identified in mammals: MT1 (Mel 1a) and MT2 (Mel 1b) and a putative binding site called MT3. The chronobiotic properties of the hormone for resynchronization of sleep and circadian rhythms disturbances has been demonstrated both in animal models or in clinical trials. Several other physiological effects of melatonin in different peripheral tissues have been described in the past years. In this way, it has been demonstrated that the hormone is involved in the regulation of seasonal reproduction, body weight and energy balance. This contribution has been focused to review some of the physiological functions of melatonin as well as the role of the hormone in the regulation of energy balance and its possible involvement in the development of obesity.     

Chimeric melatonin mt1 and melatonin-related receptors. Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt1 receptor

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt(1) receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[(125)I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[(125)I]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt(1) receptor sequences identified specific 2-[(125)I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt(1) receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.     

Ligand binding to the human MT2 melatonin receptor: the role of residues in transmembrane domains 3, 6, and 7

To better understand the mechanism of interactions between G-protein-coupled melatonin receptors and their ligands, our previously reported homology model of human MT2 receptor with docked 2-iodomelatonin was further refined and used to select residues within TM3, TM6, and TM7 potentially important for receptor-ligand interactions. Selected residues were mutated and radioligand-binding assay was used to test the binding affinities of hMT2 receptors transiently expressed in HEK293 cells. Our data demonstrate that residues N268 and A275 in TM6 as well as residues V291 and L295 in TM7 are essential for 2-iodomelatonin binding to the hMT2 receptor, while TM3 residues M120, G121, V124, and I125 may participate in binding of other receptor agonists and/or antagonists. Presented data also hint at possible specific interaction between the side-chain of Y188 in second extracellular loop and N-acetyl group of 2-iodomelatonin.     

The expression of MT1 and MT2 melatonin receptor mRNA in several rat tissues

The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT(1) and MT(2) melatonin receptors are expressed in certain tissues of the rat. The expression of MT(1) and MT(2) melatonin receptor mRNA was determined using a real-time quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT(1) and MT(2) melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT(2) mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT(1) mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT(1) and MT(2) receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT(1) melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT(2) receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism.     

Binding affinity and biological activity of oxygen and sulfur isosteres at melatonin receptors as a function of their hydrogen bonding capability

Analogues of melatonin (1) and of N-acetyl 5-ethoxytryptamine (3) in which the oxygen atoms are replaced by sulfur have been prepared and tested against human and amphibian melatonin receptors. All sulfur analogues show a decreased binding affinity at human MT1 and MT2 receptors and a reduced potency as melatonin agonists on the Xenopus melanophore assay. The 5-methoxy oxygen of melatonin is significantly more important for receptor binding than the amide oxygen. N-Acetyl 5-ethoxytryptamine shows a decrease in both binding affinity and potency in comparison with melatonin. In this series, replacing either the ethoxy or amide oxygen by sulfur has a similar but smaller effect on both binding affinity and potency. Using K(B)(H) values from Abraham's equations we have assessed the possibility of estimating EC50 values for sulfur isosteres from the EC50 values of their oxygen analogues.     

The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.     

Studies of the melatonin binding site location onto quinone reductase 2 by directed mutagenesis

Melatonin is a neurohormone implicated in both biorhythm synchronization and neuroprotection from oxidative stress. Its functions are mediated by two G-protein-coupled-receptors (MT1 and MT2) and MT3, which corresponds to quinone oxidoreductase 2 (QR2). To determine the binding site of QR2 for melatonin, point mutations of residues crucial for the enzymatic activity of hQR2 were performed. The substitution of the hydrophobic residues Phe126, Ile128 and Phe178 by tyrosines at the active site significantly increased enzymatic activity and decreased the affinity of a structural analog of melatonin, the 2[¹²⁵I]iodo-MCANAT. The mutation of residues implicated in zinc chelating (His¹⁷³; His¹⁷⁷) had no effect on radioligand binding. Destabilisation of the cofactor FAD by mutation N18E showed that 2[¹²⁵I]iodo-MCANAT binding was closely linked to the conformational integrity of human QR2. Surprisingly, the mutations C222F and N161A, which are distant from the determined binding site of the ligand, increased the affinity of 2[¹²⁵I]iodo-MCANAT for hQR2. What seems to better explain the binding variations among the mutants are the activity recorded with BNAH and coenzyme Q1. Various hypotheses are discussed based on the various parameters used in the study: nature of the substrates and co-substrates and nature of the amino acid changes. This study, which constitutes the first structural analysis of hQR2, should enable to better understand the biological role of melatonin on this enzyme and particularly, the discrepancies between the pharmacologies of the melatonin binding site (MT3) and the QR2 catalytic activity.     

Methods for the evaluation of drug action at the human melatonin receptor subtypes

NIH3T3 fibroblast cells transfected with the full-length coding regions of the mt1 and MT2 human melatonin receptors stably expressed the receptor, coupled to a pertussis-toxin-sensitive G protein and exhibiting high affinity for melatonin. Both mt1 and MT2 melatonin receptors mediated the incorporation of [35S]GTPgammaS into isolated membranes via receptor-catalyzed exchange of [35S]GTPgammaS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [35S]GTPgammaS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [35S]GTPgammaS binding. Luzindole did not increase basal [35S]GTPgammaS binding but competitively inhibited melatonin-stimulated [35S]GTPgammaS binding, thus exhibiting antagonist action. Two other mt1 antagonists, 4P-PDOT and N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide, behaved as partial agonists at the MT2 subtype, with relative intrinsic activities of 0.37 and 0.39, respectively. For the first time, these findings show important differences in analogue intrinsic activity between the human mt1 and MT2 melatonin receptor subtypes.     

Sites and mechanisms of action of melatonin in mammals: the MT1 and MT2 receptors

The rhythmic secretion of melatonin by the pineal gland plays a key role in the synchronisation of circadian and seasonal functions with cyclic environmental variations. The biological effects of this neurohormone are relayed mainly by G-protein-coupled seven-transmembrane receptors. These receptors, known as MT1 and MT2, are present in a large number of central and peripheral structures in mammals, with considerable inter-species variations. However, only the suprachiasmatic nuclei of the hypothalamus, the site of the master circadian biological clock, and the pars tuberalis of the adenohypophysis contain melatonin receptors in the majority of species. Inhibition of the production of AMPc by a Gi/Go protein is one of the principal signalling pathways of the MT1 and MT2 receptors, although many other signal transduction pathways are also brought into play according to the cell type studied (PKC, Ca2+, K+ channels or GMPc in the case of MT2, etc.). Numerous factors or physiological stimuli are capable of influencing the number and functional status of the MT1 and MT2 receptors, such as melatonin, the photoperiod, the circadian clock or the phenomena of receptor dimerisation. Melatonin has numerous physiological effects for which the mechanisms of action and the specific role of the MT1 and MT2 receptors have not yet been clearly elucidated. However, selective pharmacological tools for each of the two receptor subtypes are currently being identified, notably in the Servier Group, for the purpose of furthering our knowledge of the functionality and physiological role of the MT1 and MT2 receptors in the central and peripheral structures.     

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

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