Efficient axonal transport of endolysosomes relies on the balanced ratio of microtubule tyrosination and detyrosination.

In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated/detyrosinated MT for proper AIS length and axonal transport processes.

Mathematical modeling of the microtubule detyrosination/tyrosination cycle for cell-based drug screening design.

Microtubules and their post-translational modifications are involved in major cellular processes. In severe diseases such as neurodegenerative disorders, tyrosinated tubulin and tyrosinated microtubules are in lower concentration. We present here a mechanistic mathematical model of the microtubule tyrosination cycle combining computational modeling and high-content image analyses to understand the key kinetic parameters governing the tyrosination status in different cellular models. That mathematical model is parameterized, firstly, for neuronal cells using kinetic values taken from the literature, and, secondly, for proliferative cells, by a change of two parameter values obtained, and shown minimal, by a continuous optimization procedure based on temporal logic constraints to formalize experimental high-content imaging data. In both cases, the mathematical models explain the inability to increase the tyrosination status by activating the Tubulin Tyrosine Ligase enzyme. The tyrosinated tubulin is indeed the product of a chain of two reactions in the cycle: the detyrosinated microtubule depolymerization followed by its tyrosination. The tyrosination status at equilibrium is thus limited by both reaction rates and activating the tyrosination reaction alone is not effective. Our computational model also predicts the effect of inhibiting the Tubulin Carboxy Peptidase enzyme which we have experimentally validated in MEF cellular model. Furthermore, the model predicts that the activation of two particular kinetic parameters, the tyrosination and detyrosinated microtubule depolymerization rate constants, in synergy, should suffice to enable an increase of the tyrosination status in living cells.

Mapping the Melatonin Receptor. 8. Selective MT2 Agonists Derived from 5,6-Dihydroindolo[2,1-a]isoquinolines and Related Systems.

A series of substituted indolo[2,1-a]isoquinolines and indolo[1,2-a]benzoxazines have been prepared, as melatonin analogues, to investigate the nature of the binding site of the melatonin receptor. Agonist and antagonist potency of all the analogues was measured using the [35S]GTPγS binding assay protocol. The binding affinity of the analogues were measured by competition binding studies against the human MT1 (hMT1) and MT2 (hMT2) receptors stably transfected in Chinese Hamster Ovarian (CHO) cells, using 2-[125 I]-iodomelatonin, as a ligand. N-Acetyl 2-(10-methoxy-5,6-dihydroindolo[2,1-a]isoquinolin-12-yl)propyl-1-amine (12 a) binds strongly to both the hMT1 and hMT2 receptors, and shows a preference for the hMT2, as does its propanamido counterpart 12 b. The introduction of two methyl groups into their side chain, analogues 15 a and 15 b, leads to antagonism, in the case of the former, and drastically diminishes its hMT1 binding; an analogous profile is seen for 15 b, which, however, is a partial agonist. Introduction of chlorine or methoxy groups into ring 4 gives compounds, that are weakly binding, with a preference for MT2. Substitution of oxygen for carbon at position 5 gives the indolo[1,2-c]benzoxazines 33, 36 a and b, that bind strongly to the human receptors, 33, 36 b being potent agonists at the melatonin receptors, but do not discriminate between hMT1 and hMT2.

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease.

Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions that go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase (TTL). Here we show that TTL heterozygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density and both synaptic plasticity and memory deficits. We further report decreased TTL expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harbouring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid-ß peptide toxicity and that expression of TTL, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid-ß peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid-ß peptide-induced synaptic damage and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease.

Synthesis and SAR Studies of Isoquinoline and Tetrahydroisoquinoline Derivatives as Melatonin Receptor Ligands.

In our constant search for new successors of agomelatine, we report herein a new series of compounds resulting from bioisosteric modulation of the naphthalene ring. The isoquinoline and tetrahydroisoquinoline derivatives were synthesized and pharmacologically evaluated. This isosteric replacement of the naphthalene group of agomelatine has led to potent agonist and partial agonist compounds with nanomolar melatonergic binding affinities. Overall, the presence of a nitrogen atom was accompanied with a decrease in the binding affinity toward both MT1 and MT2 and the loss of 5HT2C response, especially for tetrahydroisoquinoline in comparison with the parent compound. Interestingly, due to the presence of this nitrogen atom, a notable improvement in the pharmacokinetic properties was observed for all compounds.

A putative new melatonin binding site in sheep brain, MTx: preliminary observations and characteristics.

In mammals, MT1 and MT2 melatonin receptors are high affinity G protein-coupled receptors and are thought to be involved in the integration of the melatonin signaling throughout the brain and periphery. In the present study, we describe a new melatonin binding site, named MTx, with a peculiar pharmacological profile. This site had a low affinity for 2-[125I]-melatonin in saturation assays in hypothalamus and retina (pKD = 9.13 {plus minus} 0.05, Bmax = 1.12 {plus minus} 0.11 fmol/mg protein and pKD = 8.81 {plus minus} 0.50, Bmax = 7.65 {plus minus} 2.64 fmol/mg protein, respectively) and a very high affinity, in competition assays, for melatonin (pKi = 13.08 {plus minus} 0.18), and other endogenous compounds. Using autoradiography, we showed a preferential localization of the MTx in periventricular areas of the sheep brain, with a density 3 to 8 times higher than those observed for ovine MT1 In addition, using a set of well-characterized ligands, we showed that this site did not correspond to any of the following receptors: MT1, MT2, MT3 , D1, D2, noradrenergic, nor 5-HT2 Based on its affinity for melatonin, MTx did not seem to be implicated in the integration of cerebral melatonin concentration variations since they were saturating for MTx. Nevertheless, it remained of prime importance because of its periventricular distribution, in close contact with the CSF, and its peculiar pharmacological profile responding to both melatoninergic and serotoninergic compounds. Significance Statement Herein a putative new melatonin binding site is described in sheep brain parts in close contact with the 3rd ventricle. The characteristics of the pharmacological profile of this site is different from anything previously reported in the literature. The present work forms the basis of future full pharmacological characterization.

Negative regulation of melatonin secretion by melatonin receptors in ovine pinealocytes.

Melatonin (MLT) is a biological modulator of circadian and seasonal rhythms and reproduction. The photoperiodic information is detected by retinal photoreceptors and transmitted through nerve transmissions to the pineal gland, where MLT is synthesized and secreted at night into the blood. MLT interacts with two G protein-coupled receptors, MT1 and MT2. The aim of our work was to provide evidence for the presence of MLT receptors in the ovine pineal gland and define their involvement on melatonin secretion. For the first time, we identified the expression of MLT receptors with the specific 2-[125I]-MLT agonistic radioligand in ovin pinealocytes. The values of Kd and Bmax are 2.24 ± 1.1 nM and 20 ± 6.8 fmol/mg. MLT receptors are functional and inhibit cAMP production and activate ERK1/2 through pertussis toxin-sensitive Gi/o proteins. The MLT receptor antagonist/ inverse agonist luzindole increased cAMP production (189 ± 30%) and MLT secretion (866 ± 13%). The effect of luzindole on MLT secretion was additive with the effect of well-described activators of this pathway such as the ß-adrenergic agonist isoproterenol and the α-adrenergic agonist phenylephrine. Co-incubation of all three compounds increased MLT secretion by 1236 ± 199%. These results suggest that MLT receptors are involved in the negative regulation of the synthesis of its own ligand in pinealocytes. While adrenergic receptors promote MLT secretion, MLT receptors mitigate this effect to limit the quantity of MLT secreted by the pineal gland.

Primary role for melatonin MT2 receptors in the regulation of anhedonia and circadian temperature rhythm.

Circadian rhythms disturbance is widely observable in patients with major depression (MD) and is also associated with depression vulnerability. Of them, disturbed melatonin secretion rhythm is particularly relevant to MD and is strongly phase-locked to core body temperature (CBT) rhythm. Here we aim to study the specific role of each melatonin receptor (MT1 and MT2) subtype in melatonin regulation of circadian CBT and its possible relationship with depressive-like behaviors. MT1-/- , MT2-/- and WT (C57BL/6) mice were used.  Anhedonia, using the sucrose intake test, circadian CBT, environmental place preference (EPP) conditioning and vulnerability to chronic social defeat stress (CSDS) procedure were studied. Moreover, the antidepressant effects of reboxetine (15 mg/kg/day, i.p.) for three weeks or ketamine (15 mg/kg i.p. every four days, 4 doses in total) were studied. Further, exposure to ultra-mild stress induced by individual housing for several weeks was also studied in these mice. MT2-/- mice showed anhedonia and lower CBT compared to WT and MT1-/-. In addition, while reward exposure raised nocturnal CBT in WT this increase did not take place in MT2-/- mice. Further, MT2-/- mice showed an enhanced vulnerability to stress-induced anhedonia and social avoidance as well as an impaired acquisition of novelty seeking behavior. Both reboxetine and ketamine reverted anhedonia and induced a clear anti-helpless behavior in the tail suspension test (TST). Reboxetine raised CBT in mice and reverted ultra-mild stress-induced anhedonia. Our findings show a primary role for MT2 receptors in the regulation of circadian CBT as well as anhedonia and suggest that these receptors could be involved in depressive disorders associated to disturbed melatonin function.

Pharmacological evidence for transactivation within melatonin MT2 and serotonin 5-HT2C receptor heteromers in mouse brain.

Association of G protein-coupled receptors into heterodimeric complexes has been reported for over 50 receptor pairs in vitro but functional in vivo validation remains a challenge. Our recent in vitro studies defined the functional fingerprint of heteromers composed of Gi -coupled melatonin MT2 receptors and Gq -coupled serotonin 5-HT2C receptors, in which melatonin transactivates phospholipase C (PLC) through 5-HT2C . Here, we identified this functional fingerprint in the mouse brain. Gq protein activation was probed by [35 S]GTPγS incorporation followed by Gq immunoprecipitation, and PLC activation by determining the inositol phosphate levels in brain lysates of animals previously treated with melatonin. Melatonin concentration-dependently activated Gq proteins and PLC in the hypothalamus and cerebellum but not in cortex. These effects were inhibited by the 5-HT2C receptor-specific inverse agonist SB-243213, and were absent in MT2 and 5-HT2C knockout mice, fully recapitulating previous in vitro data and indicating the involvement of MT2 /5-HT2C heteromers. The antidepressant agomelatine had a similar effect than melatonin when applied alone but blocked the melatonin-promoted Gq activation due to its 5-HT2C antagonistic component. Collectively, we provide strong functional evidence for the existence of MT2 /5-HT2C heteromeric complexes in mouse brain. These heteromers might participate in the in vivo effects of agomelatine.

Quinazoline and phthalazine derivatives as novel melatonin receptor ligands analogues of agomelatine.

For further development of successors of Agomelatine through modulation of its pharmacokinetic properties, we report herein the design, synthesis and pharmacological results of a new family of melatonin receptor ligands. Issued from the introduction of quinazoline and phthalazine scaffolds carrying an ethyl amide lateral chain and a methoxy group as bioisosteric ligands analogues of previously developed Agomelatine. The biological activity of the prepared analogues was compared with that of Agomelatine. Quinazoline and phthalazine rings proved to be a versatile scaffold for easy feasible MT1 and MT2 ligands. Potent agonists with sub-micromolar binding affinity were obtained. However, the presence of two nitrogen atoms resulted in compounds with lower affinity for both MT1 and MT2, in comparison with the parent compound, balanced by the exhibition of good pharmacokinetic properties.

GPR50-Ctail cleavage and nuclear translocation: a new signal transduction mode for G protein-coupled receptors.

Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.

Quantitative assessment of oligomeric amyloid ß peptide binding to α7 nicotinic receptor.

BACKGROUND AND PURPOSE: Progressive dysfunction of cholinergic transmission is a well-known characteristic of Alzheimer's disease (AD). Amyloid ß (Aß) peptide oligomers are known to play a central role in AD and are suggested to impair the function of the cholinergic nicotinic ACh receptor α7 (α7nAChR). However, the mechanism underlying the effect of Aß on α7nAChR function is not fully understood, limiting the therapeutic exploration of this observation in AD. Here, we aimed to detect and characterize Aß binding to α7nAChR, including the possibility of interfering with this interaction for therapeutic purposes. EXPERIMENTAL APPROACH: We developed a specific and quantitative time-resolved FRET (TR-FRET)-based binding assay for Aß to α7nAChR and pharmacologically characterized this interaction. KEY RESULTS: We demonstrated specific and high-affinity (low nanomolar) binding of Aß to the orthosteric binding site of α7nAChR. Aß binding was prevented and reversed by the well-characterized orthosteric ligands of α7nAChR (epibatidine, α-bungarotoxin, methylylcaconitine, PNU-282987, S24795, and EVP6124) and by the type II positive allosteric modulator (PAM) PNU-120596 but not by the type I PAM NS1738. CONCLUSIONS AND IMPLICATIONS: Our TR-FRET Aß binding assay demonstrates for the first time the specific binding of Aß to α7nAChR, which will be a crucial tool for the development, testing, and selection of a novel generation of AD drug candidates targeting Aß/α7nAChR complexes with high specificity and fewer side effects compared to currently approved α7nAChR drugs. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C-terminal domain.

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non-24-hour sleep-wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT1 and MT2 , belonging to the G protein-coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT1 and MT2 . Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT1 and MT2 by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT1 or MT2 . None of the mABs cross-reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR-KO mice as control. MT2 expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta-cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.

Melatonin receptors limit dopamine reuptake by regulating dopamine transporter cell-surface exposure.

Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT1 and MT2 receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT1 or MT2 receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT1 and MT2) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.

Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2.

For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT1 and MT2) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT1, MT2, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT1 receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (Phodopus sungorus) expresses a non-functional MT2. We observed that unlike this hamster, the European hamster (Cricetus cricetus) does not have a stop codon in the MT2 sequence. Thus, we undertook the tedious task of cloning the MT2 receptor. We partially succeeded, sequencing the complete exon 2 and a fragment of exon 1 (from putative amino acids 12 to 38 and 77 to 323), after several years of efforts. In order to show that the protein parts we cloned were capable to sustain some binding capacities, we designed a chimeric MT2 receptor using a consensus sequence to replace the unknown amino acids, based on other small rodent MT2 sequences. This chimeric construct could bind melatonin in the nanomolar range. This work is meant to be the basis for attempts from other laboratories of the community to determine the complete natural sequence of the European hamster MT2 receptor. The present work is the first to show that, among the hamsters, if the Siberian is a natural knockout for MT2, the European one is not.

First evidence of neuronal connections between specific parts of the periaqueductal gray (PAG) and the rest of the brain in sheep: placing the sheep PAG in the circuit of emotion.

The periaqueductal gray (PAG) is a mesencephalic brain structure organised in subdivisions with specific anatomical connections with the rest of the brain. These connections support the different PAG functions and especially its role in emotion. Mainly described in territorial and predatory mammals, examination of the PAG connections suggests an opposite role of the ventral and the dorsal/lateral PAG in passive and active coping style, respectively. In mammals, the organisation of PAG connections may reflect the coping style of each species. Based on this hypothesis, we investigated the anatomical connections of the PAG in sheep, a gregarious and prey species. Since emotional responses expressed by sheep are typical of active coping style, we focused our interest on the dorsal and lateral parts of the PAG. After injection of fluorogold and fluororuby, the most numerous connections occurred with the anterior cingulate gyrus, the anterior hypothalamic region, the ventromedial hypothalamic nucleus and the PAG itself. Our observations show that the sheep PAG belongs to the neuronal circuit of emotion and has specific parts as in other mammals. However, unlike other mammals, we observed very few connections between PAG and either the thalamic or the amygdalar nuclei. Interestingly, when comparing across species, the PAG connections of sheep were noticeably more like those previously described in other social species, rabbits and squirrel monkeys, than those in territorial species, rats or cats.

The orphan GPR50 receptor promotes constitutive TGFß receptor signaling and protects against cancer development.

Transforming growth factor-ß (TGFß) signaling is initiated by the type I, II TGFß receptor (TßRI/TßRII) complex. Here we report the formation of an alternative complex between TßRI and the orphan GPR50, belonging to the G protein-coupled receptor super-family. The interaction of GPR50 with TßRI induces spontaneous TßRI-dependent Smad and non-Smad signaling by stabilizing the active TßRI conformation and competing for the binding of the negative regulator FKBP12 to TßRI. GPR50 overexpression in MDA-MB-231 cells mimics the anti-proliferative effect of TßRI and decreases tumor growth in a xenograft mouse model. Inversely, targeted deletion of GPR50 in the MMTV/Neu spontaneous mammary cancer model shows decreased survival after tumor onset and increased tumor growth. Low GPR50 expression is associated with poor survival prognosis in human breast cancer irrespective of the breast cancer subtype. This describes a previously unappreciated spontaneous TGFß-independent activation mode of TßRI and identifies GPR50 as a TßRI co-receptor with potential impact on cancer development.

Tetrahydroquinoline Ring as a Versatile Bioisostere of Tetralin for Melatonin Receptor Ligands.

A new family of melatonin receptor ligands, characterized by a tetrahydroquinoline (THQ) scaffold carrying an amide chain in position 3, was devised as conformationally constrained analogs of flexible N-anilinoethylamides previously developed. Molecular superposition models allowed to identify the patterns of substitution conferring high receptor binding affinity and to support the THQ ring as a suitable scaffold for the preparation of melatonin ligands. The biological activity of 3-acylamino-THQs was compared with that of the corresponding tetralin derivatives. The THQ ring proved to be a versatile scaffold for easy feasible MT1 and MT2 ligands, which resulted as more polar bioisosteres of their tetralin analogs. Potent partial agonists, with subnanomolar binding affinity for the MT2 receptor, were obtained, and a new series of THQ derivatives is presented. The putative binding mode of potent THQs and tetralines was discussed on the basis of their conformational equilibria as inferred from molecular dynamics simulations and experimental NMR data.

Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50.

BACKGROUND AND PURPOSE: Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT1 receptor and in the inactivation of the closely related orphan receptor GPR50. EXPERIMENTAL APPROACH: Chimeric MT1 -GPR50 receptors were generated and functionally analysed in terms of 2-[125 I]iodomelatonin binding, Gi /cAMP signalling and ß-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations. KEY RESULTS: MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of the MT1 receptor (ii) the most suitable features for melatonin binding in MT1 receptors and (iii) major predicted rearrangements upon MT1 receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 receptors and GPR50 respectively. CONCLUSIONS AND IMPLICATIONS: Our work demonstrated the crucial role of the E2 loop for MT1 receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Synthesis and biological evaluation of new naphtho- and quinolinocyclopentane derivatives as potent melatoninergic (MT1/MT2) and serotoninergic (5-HT2C) dual ligands.

We recently reported a series of naphthofuranic compounds as constrained agomelatine analogues. Herein, in order to explore alternative ethyl amide side chain rigidification, naphthocyclopentane and quinolinocyclopentane derivatives with various acetamide modulations were synthesized and evaluated at both melatonin (MT1, MT2) and serotonin (5-HT2C) receptors. These modifications has led to compounds with promising dual affinity and high MTs receptors agonist activity. Enantiomeric separation was then performed on selected compounds allowing us to identify levogyre enantiomers (-)-17g and (-)-17k as the highest (MT1, MT2)/5-HT2C dual ligands described nowadays.