Multiple Serotonin Receptors: Opportunities for New Treatments for Obesity?

Recent progress in the molecular pharmacology of 5-HT receptors and the development of selective ligands for various 5-HT receptor subtypes has advanced our understanding of the role of 5-HT mechanisms in the control of food intake and body weight The most intensively investigated 5-HT receptor subtypes have been the 5-HT_1A receptor, the 5-HT_1B receptor andthe5-HT_2C receptor. The overall pattern of results to date suggests that selective 5-HT_2C agonists may be novel anorectic drugs and prove useful in the treatment of obesity. However, a number of issues remain unresolved, particularly regarding potential side-effects, as the 5-HT_2C receptor agonist mCPP has been reported to induce anxiety and nausea in humans, actions that would clearly limit its therapeutic utility. In addition, the possible role of recently cloned 5-HT receptor subtypes such as 5-ht₅,5-ht₆ and 5-ht₇ remains unexplored and the development of selective ligands for these sites has the potential to lead to new treatments for obesity .     

Design and synthesis of piperazinylpyrimidinones as novel selective 5-HT2C agonists

This Letter reports the design and synthesis of several novel series of piperazinyl pyrimidinones as 5-HT_2C agonists. Several of the compounds presented exhibit good in vitro potency and selectivity over the closely related 5-HT_2A and 5-HT_2B receptors. Compound 11 was active in in vivo models of stress urinary incontinence.     

Alpha-ethyltryptamines as dual dopamine–serotonin releasers

The dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter releasing activity and serotonin-2A (5-HT_2A) receptor agonist activity of a series of substituted tryptamines are reported. Three compounds, 7b, (+)-7d and 7f, were found to be potent dual DA/5-HT releasers and were >10-fold less potent as NE releasers. Additionally, these compounds had different activity profiles at the 5-HT_2A receptor. The unique combination of dual DA/5-HT releasing activity and 5-HT_2A receptor activity suggests that these compounds could represent a new class of neurotransmitter releasers with therapeutic potential.     

Discovery of a lead series of potent benzodiazepine 5-HT2C receptor agonists with high selectivity in functional and binding assays

A series of potential new 5-HT₂ receptor scaffolds based on a simplification of the clinically studied, 5-HT_2CR agonist vabicaserin, were designed. An in vivo feeding assay early in our screening process played an instrumental part in the lead identification process, leading us to focus on a 6,5,7-tricyclic scaffold. A subsequent early SAR investigation provided potent agonists of the 5-HT_2C receptor that were highly selective in both functional and binding assays, had good rat PK properties and that significantly reduced acute food intake in the rat.     

Novel N-biphenyl-2-ylmethyl 2-methoxyphenylpiperazinylalkanamides as 5-HT7R antagonists for the treatment of depression

5-HT₇ receptor (5-HT₇R) is a promising target for the treatment of depression and neuropathic pain. 5-HT₇R antagonists exhibited antidepressant effects, while the agonists produced strong anti-hyperalgesic effects. In our efforts to discover selective 5-HT₇R antagonists or agonists, N-biphenylylmethyl 2-methoxyphenylpiperazinylalkanamides 1 were designed, synthesized, and biologically evaluated against 5-HT₇R. Among the synthesized compounds, N-2′-chlorobiphenylylmethyl 2-methoxyphenylpiperazinylpentanamide 1–8 showed the best binding affinity with a K_i value of 8.69 nM and it was verified as a novel antagonist according to functional assays. The compound 1–8 was very selective over 5-HT_1DR, 5-HT_2AR, 5-HT₃R, 5-HT_5AR and 5-HT₆R and moderately selective over 5-HT_1AR, 5-HT_1BR and 5-HT_2CR. The novel 5-HT₇R antagonist 1–8 exhibited an antidepressant effect at a dose of 25 mg/kg in the forced swimming test in mice and showed a U-shaped dose–response curve which typically appears in 5-HT₇R antagonists such as SB-269970 and lurasidone.     

Characterisation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH-SY5Y: Comparative Stimulation by Hallucinogenic Drugs

Abstract: Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine_2A (5-HT_2A) or 5-HT_2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT_2C receptor (pEC₅₀ = 7.80 ± 0.06) compared with the 5-HT_2A receptor (pEC₅₀ = 7.30 ± 0.08). Activation of the 5-HT_2A receptor caused a transient fourfold increase in intracellular Ca²⁺ concentration. Whole-cell recordings of cells clamped at ?50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT_2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT_2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT_2C receptor, whereas activity at the 5-HT_2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.     

Tricyclic dihydroquinazolinones as novel 5-HT2C selective and orally efficacious anti-obesity agents

Agonists of the 5-HT_2C receptor have been shown to suppress appetite and reduce body weight in animal models as well as in humans. However, agonism of the related 5-HT_2B receptor has been associated with valvular heart disease. Synthesis and biological evaluation of a series of novel and highly selective dihydroquinazolinone-derived 5-HT_2C agonists with no detectable agonism of the 5-HT_2B receptor is described. Among these, compounds (+)-2a and (+)-3c were identified as potent and highly selective agonists which exhibited weight loss in a rat model upon oral dosing.     

Serotonin 5-HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus

Abstract: The serotonin 5-HT_2C receptor (formerly designated the 5-HT_1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5-HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five-fold basal level, with an EC₅₀ of 11 nM. This response was not inhibited by muscarinic or β-adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent K_i values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin-stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin-stimulated cGMP formation was calcium, phospholipase A₂, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A₂ or lipoxygenase inhibitors p-bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5-HT_2C receptors trigger cGMP formation in a calcium-sensitive manner.     

Discovery of TD-8954, a clinical stage 5-HT4 receptor agonist with gastrointestinal prokinetic properties

The discovery of a series of 5-HT₄ receptor agonists based on a novel 2-alkylbenzimidazole aromatic core is described. Optimization of the 2-substituent of the benzimidazole ring led to a series of agonists with subnanomolar binding affinity and moderate-to-high intrinsic activity relative to that of 5-HT. Consistent with our previously described multivalent design approach to this target, subsequent optimization of the linker and secondary binding group regions of the series afforded compound 18 (TD-8954), a potent and selective 5-HT₄ receptor agonist in vitro with demonstrated prokinetic activity in multiple species.     

SR 57227A is a partial agonist/partial antagonist of 5-HT3 receptor and inhibits subsequent 5-HT- or SR 57227A-induced 5-HT3 receptor current

The serotonin (5-hydroxytryptamine) type 3 (5-HT₃) receptors are transmembrane ligand-gated ion channels. Although several 5-HT₃ receptor agonists have been used as preclinical tools, SR 57227A is the most commonly used 5-HT₃ receptor agonist with the ability to cross the blood brain barrier. However, the precise pharmacological profile of SR 57227A remains unclear. Therefore, we examined the pharmacological profile of SR 57227A at the 5-HT₃A and 5-HT₃AB receptors. We microinjected Xenopus laevis oocytes with human 5-HT₃A complementary RNA (cRNA) or a combination of human 5-HT₃A and human 5-HT₃AB cRNA and performed two electrode voltage clamp recordings of 5-HT₃A and 5-HT₃AB receptor current in the presence of SR 57227A. Results showed that SR 57227A acts as partial agonist/partial antagonist at the 5-HT₃ receptor. Interestingly, SR 57227A specifically reduced subsequent current amplitudes induced by 5-HT or SR 57227A. Based on its 5-HT₃ receptor partial agonist/partial antagonist properties, we predict that SR 57227A functions as a serotonin stabilizer.     

Novel tetrahydropyrido[3,2-c]pyrroles as 5-HT(7) antagonists

The synthesis and SAR for a novel series of tetrahydropyrido[3,2-c]pyrroles is described. Optimization of the pendant aryl ring lead to high binding affinity at the 5-HT₇ receptor when small lipophilic groups were placed in the para position. Modification of the N-benzyl group and secondary amine were not well tolerated. A representative set of compounds was shown to be functional antagonists of the 5-HT₇ receptor.     

Rapid Desensitization of Serotonin 5-HT2C Receptor-Stimulated Intracellular Calcium Mobilization in CHO Cells Transfected with Cloned Human 5-HT2C Receptors

Abstract: Serotonin 5-HT_2C receptor-mediated intracellular Ca²⁺ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5-HT_2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT_2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT_2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [³H]mesulergine binding (K_D = 0.49 nM). The addition of 5-HT mobilized intracellular Ca²⁺ in a dose-dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC₅₀ value for 5-HT of 0.029 µM. Exposure to 5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (a 5-HT_2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT_2C and 5-HT_2A agonist) resulted in increased intracellular Ca²⁺ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca²⁺ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myristate 13-acetate, protein kinase C activators, weakly inhibited the intracellular Ca²⁺ mobilization induced by 10 µM 5-HT. Furthermore, the protein kinase C inhibitor H-7 partially prevented the protein kinase C activator-induced inhibition of the 5-HT-mediated increase in intracellular Ca²⁺ concentration. The desensitization induced by pretreatment with 5-HT was blocked by W-7, added in conjunction with 5-HT, and partially inhibited by W-5, a nonselective inhibitor of protein kinases and weak analogue of W-7. Therefore, the 5-HT_2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5-HT_2C receptor activation mobilizes Ca²⁺ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase-mediated feedback.     

The serotonin 5-HT1D receptor: A progress review

Most of the known neurotransmitters interact with more than one type of receptor. Some of them even dispose of receptor subtypes to exert their actions. Serotonin, far from being an exception to that, possesses at least 3 classes of receptors, which have all been reported to be heterogeneous, although convincing data only exist for the 5-HT₁ class. This name has been proposed in 1979, two years before the introduction of A and B in the nomenclature to account for the observed heterogeneity of these cites. The 5-HT_1C receptor subtype was first described in 1984 and the last member of the family, named 5-HT_1D, was characterized in 1987. The pharmacological profiles, the signal transducing systems and the anatomical localizations, both at the regional and cellular levels, of all these subtypes have been investigated and possible functions have been proposed for each of them. Moreover, last and most definitive demonstration of the subtype individuality, the gene or complementary DNA coding for the 5-HT_1A and 5-HT_1C (and 5-HT₂) receptors have been cloned and sequenced. Such data are still missing for 5-HT_1D (and 5-HT_1B) receptors, but will certainly be provided in the next few years. However and waiting for this decisive clue, the characterization of the 5-HT_1D subtype leaves no doubt concerning its significance as a functional 5-HT receptor. This review will concentrate on the characteristics of this subtype of 5-HT receptor.Abbreviations 5-CT 5-carboxamidotryptamine - 5-MeOT 5-methoxy-tryptamine - 5-MeODMT N,N-dimethyl-5-methoxytryptamine - 8-OH-DPAT 8-hydroxy-2[di-n-propylamino]tetralin - CYP cyanopindolol - DHE dihydroergotamine - DOI 2,5-dimethoxy-4-iodophenylisopropylamine - DP-5-CT N,N-dipropyl 5-carboxamidotryptamine - ICPY 2-iodo-cyanopindolol - mCPP m-chloro-phenyl-piperazine - TFMPP m-trifluoro-methyl-phenyl-piperazine - E_MAX Maximal effect - EC₅₀ Half maximal effective concentration - K_D Dissociation constant - K_B Antagonist dissociation constant     

Model studies on a synthetically facile series of N-substituted phenyl-N′-pyridin-3-yl ureas leading to 1-(3-pyridylcarbamoyl) indolines that are potent and selective 5-HT2C/2B receptor antagonists

A model series of 5-HT_2C antagonists have been prepared by rapid parallel synthesis. These N-substituted phenyl-N′-pyridin-3-yl ureas were found to have a range of 5-HT_2C receptor affinities and selectivities over the closely related 5-HT_2A receptor. Extrapolation of simple SAR, derived from this set of compounds, to the more active but synthetically more complex 1-(3-pyridyl-carbamoyl)indoline series allowed us to target optimal substitution patterns and identify potent and selective 5-HT_2C/2B antagonists.     

Promiscuous Dimerization of the Growth Hormone Secretagogue Receptor (GHS-R1a) Attenuates Ghrelin-mediated Signaling

G protein-coupled receptors (GPCRs), such as the ghrelin receptor (GHS-R1a), the melanocortin 3 receptor (MC₃), and the serotonin 2C receptor (5-HT_2C), are well known for their key role in the homeostatic control of food intake and energy balance. Ghrelin is the only known gut peptide exerting an orexigenic effect and has thus received much attention as an anti-obesity drug target. In addition, recent data have revealed a critical role for ghrelin in dopaminergic mesolimbic circuits involved in food reward signaling. This study investigates the downstream signaling consequences and ligand-mediated co-internalization following heterodimerization of the GHS-R1a receptor with the dopamine 1 receptor, as well as that of the GHS-R1a-MC₃ heterodimer. In addition, a novel heterodimer between the GHS-R1a receptor and the 5-HT_2C receptor was identified. Interestingly, dimerization of the GHS-R1a receptor with the unedited 5-HT_2C-INI receptor, but not with the partially edited 5-HT_2C-VSV isoform, significantly reduced GHS-R1a agonist-mediated calcium influx, which was completely restored following pharmacological blockade of the 5-HT_2C receptor. These results combined suggest a potential novel mechanism for fine-tuning GHS-R1a receptor-mediated activity via promiscuous dimerization of the GHS-R1a receptor with other G protein-coupled receptors involved in appetite regulation and food reward. These findings may uncover novel mechanisms of significant relevance for the future pharmacological targeting of the GHS-R1a receptor in the homeostatic regulation of energy balance and in hedonic appetite signaling, both of which play a significant role in the development of obesity.     

The Pre-nervous Serotonergic System of Developing Sea Urchin Embryos and Larvae: Pharmacologic and Immunocytochemical Evidence

Forty serotonin-related neurochemicals were tested on embryos and larvae of Lytechinus variegatus and other sea urchin species. Some of these substances (agonists of 5-HT₁ receptors, antagonists of 5-HT₂, 5-HT₃ or 5-HT₄ receptors, and inhibitors of the serotonin transporter, SERT) perturbed post-blastulation development, eliciting changes in embryonic/larval phenotypes typical for each class of receptor ligand. These developmental malformations were prevented completely or partially by serotonin (5-HT) or 5-HT analogs (5-HTQ, AA-5-HT), providing evidence for the putative localization of cellular targets. Immunoreactive 5-HT, 5-HT receptors and SERT were found in pre-nervous embryos and larvae of both L. variegatus and Strongylocentrotus droebachiensis. During gastrulation, these components of the serotonergic system were localized to the archenteron (primary gut), mesenchyme-like cells, and often the apical ectoderm. These results provide evidence that pre-nervous 5-HT may regulate early events of sea urchin embryogenesis, mediated by 5-HT receptors or the 5-HT transporter.     

Influence of exercise on serotonergic neuromodulation in the brain

Summary. Implications of exercise on serotonergic neuromodulation in the brain have been investigated in two studies. Acute paroxetine (selective serotonin (5-HT) reuptake inhibitor) administration to endurance athletes, who performed a cycle ergometer test to exhaustion at moderate intensity, reduced time to exhaustion and post exercise cognitive performance in comparison to trials with placebo or BCAA administration. Furthermore, during a 3-week moderate endurance training of sedentary males basaline values of B_max of 5-HT transporters (5-HTT) and 5-HT_2A receptors (5-HT_2AR) on isolated platelet membranes increased while plasma prolactin (PRL) concentrations decreased as well as mood and physical efficiency improved. In contrast, after an excessive training program over four weeks, well-trained endurance athletes showed no change of B_max of 5-HTT, but a decline of 5-HT_2AR density and an increase in basal plasma PRL concentration. Mood was impaired and central fatigue increased. Thus, the impact of exercise on 5-HT neurotransmission may depend on training state of athletes and extent of exertion. The theoretical background of the implication of exercise and the effect of long lasting exhaustive exercise in athletes on mental and physical efficiency or central fatigue are evaluated. The significance of the primary disturbance of central neuromodulation and dysfunction of 5-HTT, 5-HT receptor subtypes and the phosphoinositol signal transduction as well as the limited modulation capacity of the 5-HT system in overstrain are also addressed. Received December 1, 1999 / Accepted February 1, 2000     

Sequence and Functional Analysis of Cloned Guinea Pig and Rat Serotonin 5-HT1D Receptors: Common Pharmacological Features Within the 5-HT1D Receptor Subfamily

Abstract: This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)_1D receptor sites. Guinea pig, rat, and mouse 5-HT_1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT_1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13–27%) in the N-terminal extracellular region of these 5-HT_1D receptors. Guinea pig and rat 5-HT_1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT_1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT_1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT_1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT_1D receptor-mediated antagonist potency for methiothepin and the 5-HT₂ receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT_1D receptor subtype seems, unlike the 5-HT_1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

Design and synthesis of pyridazinone-based 5-HT2C agonists

The SAR of a series of pyridazinone derived 5-HT_2C agonists has been explored and resulted in identification of a compound with excellent levels of 5-HT_2C functional agonism and selectivity over 5-HT_2A and 5-HT_2B. This compound displayed good in vivo efficacy in pre-clinical models of stress urinary incontinence, despite having physiochemical properties commensurate with impaired CNS penetration.     

Identification of neurons that express 5-hydroxytryptamine4 receptors in intestine

5-Hydroxytryptamine (5-HT) is an endogenous stimulant of intestinal propulsive reflexes. It exerts its effects partly through 5-HT₄ receptors; 5-HT₄ receptor agonists that are stimulants of intestinal transit are in clinical use. Both pharmacological and recent immunohistochemical studies indicate that 5-HT₄ receptors are present on enteric neurons but the specific neurons that express the receptors have not been determined. In the present work, we describe the characterization of an anti-5-HT₄ receptor antiserum that reveals immunoreactivity for enteric neurons and other cell types in the gastrointestinal tract. With this antiserum, 5-HT₄ receptor immunoreactivity has been found in the muscularis mucosae of the rat oesophagus, a standard assay tissue for 5-HT₄ receptors. It is also present in the muscularis mucosae of the guinea-pig and mouse oesophagus. In guinea-pig small intestine and rat and mouse colon, 5-HT₄ receptor immunoreactivity occurs in subpopulations of enteric neurons, including prominent large neurons. Double-staining has shown that these large neurons in the guinea-pig small intestine are also immunoreactive for two markers of intrinsic primary afferent neurons, cytoplasmic NeuN and calbindin. Some muscle motor neurons in the myenteric ganglia are immunoreactive for this receptor, whereas it is rarely expressed by secretomotor neurons. Immunoreactivity also occurs in the interstitial cells of Cajal but is faint in the external muscle. Expression of the protein and mRNA has been confirmed in extracts containing enteric neurons. The observations suggest that one site of action of 5-HT₄ receptor agonists is the intrinsic primary afferent neurons.This work was supported by the National Health and Medical Research Council of Australia and Pfizer Pharmaceuticals, Japan.