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Melatonin exerts a variety of physiologic activities that are mainly relayed through the melatonin receptors MT1 and MT2 Low expressions of these receptors in tissues have led to widespread experimental use of the agonist 2-[(125)I]-iodomelatonin as a substitute for melatonin. We describe three iodinated ligands: 2-(2-[(2-iodo-4,5-dimethoxyphenyl)methyl]-4,5-dimethoxy phenyl) (DIV880) and (2-iodo-N-2-[5-methoxy-2-(naphthalen-1-yl)-1H-pyrrolo[3,2-b]pyridine-3-yl])acetamide (S70254), which are specific ligands at MT2 receptors, and N-[2-(5-methoxy-1H-indol-3-yl)ethyl]iodoacetamide (SD6), an analog of 2-[(125)I]-iodomelatonin with slightly different characteristics. Here, we further characterized these new ligands with regards to their molecular pharmacology. We performed binding experiments, saturation assays, association/dissociation rate measurements, and autoradiography using sheep and rat tissues and recombinant cell lines. Our results showed that [(125)I]-S70254 is receptor, and can be used with both cells and tissue. This radioligand can be used in autoradiography. Similarly, DIV880, a partial agonist [43% of melatonin on guanosine 5'-3-O-(thio)triphosphate binding assay], selective for MT2, can be used as a tool to selectively describe the pharmacology of this receptor in tissue samples. The molecular pharmacology of both human melatonin receptors MT1 and MT2, using a series of 24 ligands at these receptors and the new radioligands, did not lead to noticeable variations in the profiles. For the first time, we described radiolabeled tools that are specific for one of the melatonin receptors (MT2). These tools are amenable to binding experiments and to autoradiography using sheep or rat tissues. These specific tools will permit better understanding of the role and implication in physiopathologic processes of the melatonin receptors.

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 β-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.

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.

The accompanying multivariate analysis of the binding profiles of antiparkinson agents revealed contrasting patterns of affinities at diverse classes of monoaminergic receptor. Herein, we characterized efficacies at human (h)D(2SHORT(S)), hD(2LONG(L)), hD(3), and hD(4.4) receptors and at halpha(2A)-, halpha(2B)-, halpha(2C)-, and halpha(1A)-adrenoceptors (ARs). As determined by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding, no ligand displayed "full" efficacy relative to dopamine (100%) at all "D(2)-like" sites. However, at hD(2S) receptors quinpirole, pramipexole, ropinirole, quinerolane, pergolide, and cabergoline were as efficacious as dopamine (E(max)100%); TL99, talipexole, and apomorphine were highly efficacious (79-92%); piribedil, lisuride, bromocriptine, and terguride showed intermediate efficacy (40-55%); and roxindole displayed low efficacy (11%). For all drugs, efficacies were lower at hD(2L) receptors, with terguride and roxindole acting as antagonists. At hD(3) receptors, efficacies ranged from 33% (roxindole) to 94% (TL99), whereas, for hD(4) receptors, highest efficacies (approximately 70%) were seen for quinerolane, quinpirole, and TL99, whereas piribedil and terguride behaved as antagonists and bromocriptine was inactive. Although efficacies at hD(2S) versus hD(2L) sites were highly correlated (r = 0.79), they correlated only modestly to hD(3)/hD(4) sites (r = 0.44-0.59). In [(35)S]GTPgammaS studies of halpha(2A)-ARs, TL99 (108%), pramipexole (52%), talipexole (51%), pergolide (31%), apomorphine (16%), and quinerolane (11%) were agonists and ropinirole and roxindole were inactive, whereas piribedil and other agents were antagonists. Similar findings were obtained at halpha(2B)- and halpha(2C)-ARs. Using [(3)H]phosphatidylinositol depletion, roxindole, bromocriptine, lisuride, and terguride displayed potent antagonist properties at halpha(1A)-ARs. In conclusion, antiparkinson agents display diverse agonist and antagonist properties at multiple subtypes of D(2)-like receptor and alpha(1)/alpha(2)-AR, actions, which likely contribute to their contrasting functional profiles.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide, with a major role in stimulation of feeding behavior in mammals. MCH signals in the brain occur via two seven-transmembrane G protein-coupled receptors, namely MCH1 (SLC-1, MCH 1 , MCH-R1, or MCH-1R) and MCH2 (SLT, MCH 2 , MCH-R2, or MCH-2R). In this study, we demonstrate that the pro-MCH 131–165 peptide neuropeptide-glutamic acid-isoleucine (NEI)-MCH is more potent than MCH in stimulating feeding in the rat. Using rat MCH1-expressed human embryonic kidney 293 cells, we show that NEI-MCH exhibits 5-fold less affinity in a binding assay and 2-fold less potency in a cAMP assay than MCH. A similar 7- to 8-fold shift in potency was observed in a Ca 2+ i assay using rat MCH1 or human MCH2-transfected Chinese hamster ovary cell models. This demonstrates that NEI-MCH is not a better agonist than MCH at either of the MCH receptors. Then, we compared the proteolysis resistance of MCH and NEI-MCH to rat brain membrane homogenates and purified proteases. Kinetics of peptide degradation using brain extracts indicated a t 1/2 of 34.8 min for MCH and 78.5 min for NEI-MCH with a specific pattern of cleavage of MCH but not NEI-MCH by exo- and endo-proteases. Furthermore, MCH was found highly susceptible to degradation by aminopeptidase M and endopeptidase 24.11, whereas NEI-MCH was fully resistant to proteolysis by these enzymes. Therefore, our results strongly suggest that reduced susceptibility to proteases of NEI-MCH compared with MCH account for its enhanced activity in feeding behavior. NEI-MCH represents therefore the first MCH natural functional “superagonist” so far described.

S18616 ((S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4, 2'-(8'-chloro-1',2',3',4'-tetrahydronaphthalene)]) displayed high affinity at native rat alpha(2)-adrenoceptors (AR)s (pK(i), 9.8), native human (h)alpha(2A)-ARs (9.6), and cloned halpha(2A)- (9.5), halpha(2B)- (9.2), and halpha(2C)- (9.0) ARs. It showed 40-fold lower affinity for halpha(1A)-ARs (8.4) and/=100-fold lower affinity for rat alpha(1)-ARs (7.1), halpha(1B)-ARs (7.7), halpha(1D)-ARs (7.6), imidazoline(1) (7.4), and imidazoline(2) (7.4) sites and100-fold lower affinity for all other (50) sites. At halpha(2A)-ARs, in guanosine-5'-O-(3-[(35)S]thio)triphosphate binding studies, S18616 was a potent (partial) agonist: log effective concentration (pEC(50)), 9.3/maximal effect, 51%. This observation was corroborated employing a halpha(2A)-Gi1alpha fusion protein/GTPase assay (9.0/40%) in which the actions of S18616 were blocked by pertussis toxin. Employing guanosine-5'-O-(3-[(35)S]thio)triphosphate binding assays, S18616 was also a partial agonist at halpha(2C)-ARs (8.2/63%) but a full agonist (8.4/124%) at halpha(2B)-ARs. At halpha(2A)-, halpha(2B)-, and halpha(2C)-ARs, the selective alpha(2)-AR antagonist, atipamezole, abolished the actions of S18616: pK(b) values of 9.1, 9. 1, and 9.4, respectively. As determined by depletion of membrane-bound [(3)H]phosphatidyl inositols, S18616 behaved as a (less potent) agonist (7.8/79%) at halpha(1A)-ARs, an action abolished by prazosin (pK(b), 8.9). Reflecting alpha(2)-AR agonist properties, S18616 potently (/=1 microg/kg, s.c.) and dose dependently elicited hypothermia and antinociception (nine diverse models) in rodents. These actions were dose dependently inhibited by chemically diverse alpha(2)- versus alpha(1)-AR antagonists, atipamezole, idazoxan, RX821,002, and BRL44418 (a preferential alpha(2A)-AR ligand). In contrast, the actions of S18616 were unaffected by the alpha(1)-AR antagonists, ARC239 and prazosin (which preferentially block alpha(2B/2C)- versus alpha(2A)-ARs). Although the affinity of dexmedetomidine at alpha(2)-ARs was lower than S18616; it displayed a similar receptor and functional profile. Clonidine displayed lower efficacy than S18616, was substantially less potent, and had marked affinity for imidazoline(1) sites and alpha(1)-ARs. In conclusion, S18616 is a novel, selective, and highly potent agonist at alpha(2)-ARs.