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.

S32212, a novel serotonin type 2C receptor inverse agonist/α2-adrenoceptor antagonist and potential antidepressant: I. A mechanistic characterization.

Although most antidepressants suppress serotonin (5-HT) and/or noradrenaline reuptake, blockade of 5-HT(2C) receptors and α(2)-adrenoceptors likewise enhances monoaminergic transmission. These sites are targeted by the urea derivative N- [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-1,2-dihydro-3-H-benzo[e]indole-3-carboxamide (S32212). S32212 was devoid of affinity for monoamine reuptake sites, yet displayed pronounced affinity (pK(i), 8.2) for constitutively active human 5-HT(2CINI) (h5-HT(2CINI)) receptors, behaving as an inverse agonist in reducing basal Gα(q) activation, [(3)H]inositol-phosphate production, and the spontaneous association of h5-HT(2CINI)-Renilla luciferase receptors with ß-arrestin2-yellow fluorescent protein. Furthermore, upon 18-h pretreatment, S32212 enhanced the plasma membrane expression of h5-HT(2CINI) receptors as visualized by confocal microscopy and quantified by enzyme-linked immunosorbent assay. Its actions were prevented by the neutral antagonist 6-chloro-5-methyl-N-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl]indoline-1-carboxamide (SB242,084), which also impeded the induction by long-term exposure to S32212 of otherwise absent Ca(2+) mobilization in mouse cortical neurones. In vivo, S32212 blunted the inhibitory influence of the 5-HT(2C) agonist 2-(3-chlorobenzyloxy)-6-(1-piperazinyl)pyrazine (CP809,101) on ventrotegmental dopaminergic neurones. S32212 also blocked 5-HT-induced Gα(q) and phospholipase C activation at the h5-HT(2A) and, less potently, h5-HT(2B) receptors and suppressed the discriminative stimulus properties of the 5-HT(2A) agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane in rats. S32212 manifested marked affinity for human α(2A)- (pK(i) 7.2), α(2B)- (pK(i) 8.2), and α(2C)- (pK(i) 7.4) adrenoceptors, at which it abolished noradrenaline-induced recruitment of Gα(i3), Gα(o), adenylyl cyclase, and extracellular-regulated kinase1/2. Moreover, S32212 dose-dependently abolished the discriminative stimulus effects of the α(2)-adrenoceptor agonist (S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(1',2',3',4'-tetrahydronaphthalene)] (S18616). Finally, S32212 displayed negligible affinity for α(1A)-adrenoceptors, histamine H(1) receptors, and muscarinic M(1) receptors. In conclusion, S32212 behaves as an inverse agonist at h5-HT(2C) receptors and as an antagonist at human α(2)-adrenoceptors (and h5-HT(2A) receptors). Its promising profile in preclinical models potentially relevant to the treatment of depression is described in J Pharmacol Exp Ther 340:765-780, 2012.

Design, synthesis and pharmacological evaluation of novel naphthalenic derivatives as selective MT(1) melatoninergic ligands.

Novel heterodimer analogues of melatonin were synthesized, when agomelatine (1) and various aryl units are linked via a linear alkyl chain through the methoxy group. The compounds were tested for their actions at melatonin receptors. Several of these ligands are MT(1)-selective with nanomolar or subnanomolar affinity. In addition, while most of the derivatives behave as partial agonists on one or both receptor subtypes, N-[2-(7-{4-[6-(1-methoxycarbonylethyl)naphthalen-2-yloxy]butoxy}naphthalen-1-yl)ethyl]acetamide (36), a subnanomolar MT(1) ligand with an 11-fold preference over MT(2) receptors, is a full antagonist on both receptors. Our results also confirm that the selectivity seen for the MT(1) receptor arises predominantly from steric factors and is not a consequence of the bridging of melatonin receptor dimers.

Synthesis of new 8(S)-HETE analogs and their biological evaluation as activators of the PPAR nuclear receptors.

Structural modifications around 8-HETE (8-hydroxyeicosatetraenoic acid), a natural agonist of the PPAR (peroxisome proliferator-activated receptor) nuclear receptors have led previously to the identification of a promising analog, the quinoline S 70655. Series of novel quinoline or benzoquinoline derivatives were designed through the modification of this lead. Variations of the nature of the aromatic core and of the side chains were carried out. The SAR studies indicated the high sensitivity of the upper acid chain to modifications as well as the strong effect of the length and size of the lipophilic side chain. They afforded several new promising PPARalpha/gamma dual agonists with a high PPARalpha activity in vitro.

Design and synthesis of 3-phenyltetrahydronaphthalenic derivatives as new selective MT2 melatoninergic ligands. Part II.

Following our studies of the melatoninergic receptors, we have developed new tetrahydronaphthalenic derivatives of melatonin that have been tested as selective melatonin receptors ligands. Regarding the role of the phenyl substituent to obtain selective ligands, modulation of selectivity and activity have been achieved by modifications of the acyl group and substitutions on the phenyl ring. Ten of the seventeen evaluated derivatives have MT(2) receptor affinity similar to that of melatonin. Moreover, we have achieved remarkable MT(2) selectivity over MT(1) (selectivity >100) and have been able to further extend the RSA of the tetrahydrophthalenic series. However, the compounds presented here display partial agonist or antagonist behavior instead of full agonist.

S33138 [N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]-benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenylacetamide], a preferential dopamine D3 versus D2 receptor antagonist and potential antipsychotic agent: I. Receptor-binding profile and functional actions at G-protein-coupled receptors.

The novel, potential antipsychotic, S33138 (N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenylacetamide), displayed approximately 25-fold higher affinity at human (h) dopamine D(3) versus hD(2L) (long isoform) and hD(2S) (short isoform) receptors (pK(i) values, 8.7, 7.1, and 7.3, respectively). Conversely, haloperidol, clozapine, olanzapine, and risperidone displayed similar affinities for hD(3), hD(2L), and hD(2S) sites. In guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]-GTPgammaS) filtration assays, S33138 showed potent, pure, and competitive antagonist properties at hD(3) receptors, displaying pK(B) and pA(2) values of 8.9 and 8.7, respectively. Higher concentrations were required to block hD(2L) and hD(2S) receptors. Preferential antagonist properties of S33138 at hD(3) versus hD(2L) receptors were underpinned in antibody capture/scintillation proximity assays (SPAs) of Galpha(i3) recruitment and in measures of extracellular-regulated kinase phosphorylation. In addition, in cells cotransfected with hD(3) and hD(2L) receptors that assemble into heterodimers, S33138 blocked (pK(B), 8.5) the inhibitory influence of quinpirole upon forskolin-stimulated cAMP formation. S33138 had low affinity for hD(4) receptors (<5.0) but revealed weak antagonist activity at hD(1) receptors (Galphas/SPA, pK(B), 6.3) and hD(5) sites (adenylyl cyclase, 6.5). Modest antagonist properties were also seen at human serotonin (5-HT)(2A) receptors (Galpha(q)/SPA, pK(B), 6.8, and inositol formation, 6.9) and at 5-HT(7) receptors (adenylyl cyclase, pK(B), 7.1). In addition, S33138 antagonized halpha(2C) adrenoceptors ([(35)S]GTPgammaS, 7.2; Galpha(i3)/SPA, 6.9; Galpha(o)/SPA, 7.3, and extracellular-regulated-kinase, 7.1) but not halpha(2A) or halpha(2B) adrenoceptors (<5.0). Finally, in contrast to haloperidol, clozapine, olanzapine, and risperidone, S33138 displayed negligible affinities for multiple subtypes of alpha(1)-adrenoceptor, muscarinic, and histamine receptor. In conclusion, S33138 possesses a distinctive receptor-binding profile and behaves, in contrast to clinically available antipsychotics, as a preferential antagonist at hD(3) versus hD(2) receptors.

Synthesis of 3-phenylnaphthalenic derivatives as new selective MT(2) melatoninergic ligands.

A series of naphthalenic analogues of melatonin were prepared and evaluated as melatonin receptor MT(2) selective ligands. Activity and MT(2) selectivity can be modulated with suitable variations of the C-3 phenyl and the acyl group on the C-1 side chain. Surprisingly, in contrast with what had been previously described in other series (2-benzylindoles, 2-benzylbenzofurans and 3-phenyltetralins), the presence of a C-3 phenyl with a functional group on the meta position seems to be primordial for MT(2) affinity and selectivity. Indeed, N-[2-(3-(3-hydroxymethylphenyl)-7-methoxynaphth-1-yl)ethyl]acetamide (21) is one of the best MT(2) selective ligands described until now and behaves as an antagonist.

Mechanistic characterization of S 38093, a novel inverse agonist at histamine H3 receptors.

Histaminergic H3 inverse agonists, by stimulating central histamine release, represent attractive drug candidates to treat cognitive disorders. The present studies aimed to describe the mechanistic profile of S 38093 a novel H3 receptors inverse agonist. S 38093 displays a moderate affinity for rat, mouse and human H3 receptors (Ki=8.8, 1.44 and 1.2µM, respectively) with no affinity for other histaminergic receptors. In cellular models, the compound was able to antagonize mice H3 receptors (KB=0.65µM) and to suppress cAMP decrease induced by an H3 agonist via human H3 receptors (KB=0.11µM). The antagonism properties of the compound were confirmed by electrophysiological studies on rat hippocampal slices (from 0.1µM). In cells expressing a high H3 density, S 38093 behaved as a moderate inverse agonist at rat and human H3 receptors (EC50=9 and 1.7µM, respectively). S 38093 was rapidly absorbed in mouse and rat (Tmax=0.25-0.5h), slowly in monkey (2h), with a bioavailability ranging from 20% to 60% and t1/2 ranging from 1.5 to 7.4h. The compound was widely distributed with a moderate volume of distribution and low protein binding. The brain distribution of S 38093 was rapid and high. In mice, S 38093 significantly increased ex vivo N-tele-Methylhistamine cerebral levels from 3mg/kg p.o. and antagonized R-α-Methylhistamine-induced dipsogenia from 10mg/kg i.p. Taken together, these data suggest that S 38093, a novel H3 inverse agonist, is a good candidate for further in vivo evaluations, in particular in animal models of cognition.

Molecular tools to study melatonin pathways and actions.

Melatonin, an indoleamine neurohormone that is synthesized mainly in the pineal gland and derived from 5-HT, has many effects on a wide range of physio-pathological functions. Some of these effects are mediated by the interactions of melatonin with the two melatonin MT1 and MT2 receptors. Other effects are often suggested to be due to the chemical antioxidant nature of this indoleamine, and are observed at high, non-physiological concentrations. However, it is increasingly believed that some of these effects are due to interactions with other protein targets. In this review, we summarize the molecular pharmacology of melatonin, including the main enzymes involved in its synthesis and catabolism, and the proteins that mediate its actions. Furthermore, various compounds, mainly inhibitors and antagonists, that can be used to dissect these functions and pathways are presented.

Functional characterization of human neuropeptide Y receptor subtype five specific antagonists using a luciferase reporter gene assay.

Neuropeptide Y (NPY) has several receptors; one of them, the neuropeptide Y5 receptor (NPY5) seems involved in feeding behavior in mammals. Although this particular receptor has been extensively studied in the literature, the difficulties encountered to obtain a stable cell line expressing this recombinant receptor have impaired the development of tools necessary to establish its molecular pharmacology. We thus established a method for the functional study of new ligands. It is based upon the cotransfection in human melatonin receptor 1 (MT1)-overexpressing HEK293 cells of three plasmids encoding melanocortin receptor (MC5), neuropeptide Y5 receptor (NPY5) and a cyclic AMP response element-controlled luciferase. Once challenged with alphaMSH, the MC5 receptor activates the cyclic AMP response, through the coupling protein subunit G(s). In contrast, NPY5 agonists, through the NPY5 receptor which is negatively coupled to the same pathway, counteract the alphaMSH-mediated effect on cyclic AMP level. Using appropriate controls, this method can pinpoint compounds with antagonistic activity. Simple and straightforward, this system permits reproducible measurements of agonist or antagonist effects in the presence of neuropeptide Y, the natural agonist. This method has the advantage over already existing methods and beyond its apparent complexity, to enhance the cyclic AMP concentration at a 'physiological' level, by opposition to a forskolin-induced adenylate cyclase activation. Finally, to further validate this assay, we showed results from (1) a series of natural peptidic agonists that permitted the standardization and (2) a series of potent nonpeptidic antagonists (affinity >10(-9) M) that form a new class of active NPY5 receptor antagonists.

New ligands at the melatonin binding site MT(3).

The third melatonin binding site, MT3 is a non-classical one since it is not a seven transmembrane domains receptor, but an enzyme, quinone reductase 2. A major concern for the study of the physiological role of this site is the lack of specific ligands, permitting to more accurately dissect the pathways linked to the activation of MT3. Indeed, in the course of finding new ligands, we identified a new series of compounds with affinity to the binding site in the nM range, particularly 2,3-dimethoxy 7-hydroxy 10-methyl 5H 10H indeno(1,2-b)indol-10-one (DMHMIO), with a Ki of 190 pM. Based on slightly different and novel synthons compared to most of the compounds used in melatonin pharmacology studies, these compounds offer new perspective for the description of the melatonin pathways, so much more by not having any affinity towards the MT1 and MT2 'classical' melatonin receptors.

Ligand modulation of [35S]GTPgammaS binding at human alpha(2A), alpha(2B) and alpha(2C) adrenoceptors.

Affinities and efficacies of chemically diverse ligands--some of them used as clinical agents--were examined, employing [3H]RX821,002 and [35S]GTPgammaS binding assays, respectively, at human (h) cloned, halpha(2A), halpha(2B) and halpha(2C) adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK(B) and pK(i) values, which were highly correlated for each alpha(2)-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPgammaS binding by GTPgammaS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for alpha(2)-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of halpha(2A)-, halpha(2B)-, and halpha(2C)-AR.

Design and synthesis of naphthalenic dimers as selective MT1 melatoninergic ligands.

We report the synthesis and binding properties at MT(1) and MT(2) receptors of the first example of agomelatine (N-[2-(7-methoxynaphth-1-yl)ethyl]acetamide) dimers in which two agomelatine moieties are linked together through their methoxy substituent by a polymethylene side chain according to the "bivalent ligand" approach. Some of these compounds behave as MT(1)-selective ligands. The most selective one (5) behaves as an antagonist.

Synthesis and structure-affinity-activity relationships of novel benzofuran derivatives as MT(2) melatonin receptor selective ligands.

A series of N-(2-phenylbenzofuran-3-yl) ethyl amide and N-(2-arylalkylbenzofuran-3-yl) ethyl amide derivatives were synthesized and evaluated as melatonin receptor ligands. The affinity of each compound for the two MT(1) and MT(2) melatonin receptor subtypes was determined by binding studies using 2-[(125)I]iodomelatonin on human embryonic kidney cell line HEK293 membrane homogenates. The intrinsic activity of the most interesting compounds was evaluated on the [(35)S]GTPgammaS binding assay. Introduction of a 2-phenyl substituent in the C-2 benzofuran position leads to an agonist compound, 10q, which binds more strongly than melatonin itself to both MT(1) and MT(2) subtypes. On the other hand, a 2-benzyl group in the same position allows MT(2) antagonist selective ligands to be obtained. The MT(2) selectivity and antagonist potency can be modulated with suitable modifications on the N-acyl and benzyl substituents, and the most selective compounds 10c and 19 show affinity ratios of 123 and 192, respectively, and bind to the MT(2) subtype similarly to melatonin itself (0.1 nM). Nevertheless, 10c acts as an MT(1) and MT(2) antagonist, whereas 19 is a partial agonist.

Molecular pharmacology of the ovine melatonin receptor: comparison with recombinant human MT1 and MT2 receptors.

The variations of the pharmacological properties of melatonin receptors between different mammalian species in transfected cell lines have been poorly investigated. In the present study, melatonin analogues have been used to characterize the pharmacology of the recombinant ovine melatonin receptor (oMT1) expressed in CHO cell lines and the native oMT1 from the pars tuberalis (PT). Studies with selective ligands on native and transfected oMT1 showed similar properties for binding affinities [r2(PT/CHO) = 0.85]. The affinities and the functional activities of these ligands were compared with the human receptors (hMT1 or hMT2) expressed in CHO cells as well. The oMT1 and hMT1 receptors had similar pharmacological profiles (r2=0.82). Nevertheless, some of the selective compounds at the human receptor presented a reduced affinity at the ovine receptor. Furthermore, some compounds showed marked different functional activities at oMT1 vs. hMT1 receptors. Our findings demonstrated differences in the pharmacological properties of melatonin receptors in ovine and human species.

New selective ligands of human cloned melatonin MT1 and MT2 receptors.

Melatonin has a key role in the circadian rhythm relay to periphery organs. Melatonin exerts its multiple roles mainly through two seven transmembrane domain, G-coupled receptors, namely MT1 or MT2 receptors. A pharmacological characterization of these human cloned melatonin hMT1 and hMT2 receptors stably expressed in HEK-293 or CHO cells is presented using a 2-[125I]-iodo-melatonin binding assay and a [35S]-GTPgammaS functional assay. Both reference compounds and new chemically diverse ligands were evaluated. Binding affinities at each receptor were found to be comparable on either HEK-293 or CHO cell membranes. Novel non-selective or selective hMT1 and hMT2 ligands are described. The [35S]-GTPgammaS functional assay was used to define the functional activity of these compounds which included partial, full agonist and/or antagonist activity. None of the compounds acted as an inverse agonist. We report new types of selective antagonists, such as S 25567 and S 26131 for MT1 and S 24601 for MT2. These studies brought other new molecular tools such as the selective MT1 agonist, S 24268, as well as the non-selective antagonist, S 22153. Finally, we also discovered S 25150, the most potent melatonin receptor agonist, so far reported in the literature.

Preparation of 4-azaindole and 7-azaindole dimers with a bisalkoxyalkyl spacer in order to preferentially target melatonin MT1 receptors over melatonin MT2 receptors.

Several 4-azaindole and 7-azaindole dimer analogues of melatonin with a bisalkoxyalkyl spacer between the position 5 of each heterocycle were synthetized. Our aim was to investigate the influence of the spacers length on the selectivity of such compounds for the MT(1) receptors over the MT(2) receptors. Our results suggest the distance between indole ring seems to be an important parameter in determining the potency of binding with melatonin receptor site.

Peripheral injections of melanin-concentrating hormone receptor 1 antagonist S38151 decrease food intake and body weight in rodent obesity models.

The compound S38151 is a nanomolar antagonist that acts at the melanin-concentrating hormone receptor 1 (MCH(1)). S38151 is more stable than its purely peptide counterpart, essentially because of the blockade of its N-terminus. Therefore, its action on various models of obesity was studied. Acute intra-cerebroventricular (i.c.v.) administration of S38151 in wild-type rats counteracted the effect of the stable precursor of melanin-concentrating hormone (MCH), NEI-MCH, in a dose-dependent manner (from 0.5 to 50 nmol/kg). In genetically obese Zucker fa/fa rats, daily i.c.v. administration of S38151 induced dose-dependent (5, 10, and 20 nmol/kg) inhibition of food intake, water intake, and body weight gain, as well as increased motility (maximal effect observed at 20 nmol/kg). In Zucker fa/fa rats, intraperitoneal injection of S38151 (30 mg/kg) induced complete inhibition of food consumption within 1 h. Daily intraperitoneal injection of S38151 (10 and 30 mg/kg) into genetically obese ob/ob mice or diet-induced obese mice is able to limit body weight gain. Furthermore, S38151 administration (10 and 30 mg/kg) does not affect food intake, water intake, or body weight gain in MCHR1-deleted mice, demonstrating that its effects are linked to its interaction with MCH(1). These results validate MCH(1) as a target of interest in obesity. S38151 cannot progress to the clinical phase because it is still too poorly stable in vivo.

S38151 [p-guanidinobenzoyl-[Des-Gly(10)]-MCH(7-17)] is a potent and selective antagonist at the MCH(1) receptor and has anti-feeding properties in vivo.

Structure-activity relationships studies have established the minimal sequence of melanin-concentrating hormone (MCH) that retains full agonist potency at the MCH(1), to be the dodecapeptide MCH(6-17). The alpha-amino function is not required for activity since arginine(6) can be replaced by p-guanidinobenzoyl, further improving activity. We report that the deletion of glycine in this short potent agonist (EC(50) 3.4nM) turns it into a potent and new MCH(1) antagonist (S38151, K(B) 4.3nM in the [(35)S]-GTPgammaS binding assay), which is selective versus MCH(2). A compared Ala-scan of the agonist and antagonist sequences reveals major differences in the residues that are mandatory for affinity, including arginine(11) and tyrosine(13) for the agonist and leucine(9) for the antagonist, whereas methionine(8) was necessary for both agonist and antagonist activities. A complete molecular study of the antagonist behavior is described in the present report, with a particular focus on the description of several analogues, attempting to find structure-activity relationships. Finally, S38151 antagonizes food intake when injected intra-cerebroventricularly in the rat. This is in agreement with the in vitro data and with our previous demonstration of a good correlation between in vitro and in vivo data on MCH(1) agonists.