Tetrahydrothienopyridylbutyl-tetrahydrobenzindoles: new selective ligands of the 5-HT(7) receptor

The synthesis and the affinity for the 5-HT(7) receptor and other receptors of a novel series of fused-ring tetrahydropyridine derivatives are described. Some of the compounds showed high affinity for the 5-HT(7) receptor. Tetrahydrothienopyridylbutyl-tetrahydrobenzindoles and are potent ligands for the 5-HT(7) receptor, with high selectivity over the 5-HT(2) receptor and other receptors. These compounds should be useful tools for clarifying the biological role of the 5-HT(7) receptor.     

Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT1A serotonin receptor ligands

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2-adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents.     

Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.

Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.     

Pyrazino[1,2-a]indoles as novel high-affinity and selective imidazoline I(2) receptor ligands

1,2,3,4-Tetrahydropyrazino[1,2-a]indoles are described as a novel class of I(2) imidazoline receptor ligands. In particular, 8-methoxy-1,2,3,4-tetrahydropyrazino[1,2-a]indole (8-OMe THPI; 3c) binds with high affinity at I(2) imidazoline receptors (K(i)=6.2 nM) and with exceptional (> or =1000-fold) selectivity relative to its affinity for I(1) imidazoline receptors, alpha(2)adrenergic receptors, and 5-HT(2A) and 5-HT(2C) serotonin receptors.     

Phenylpyrroles, a new chemolibrary virtual screening class of 5-HT7 receptor ligands

Virtual screening studies have identified a series of phenylpyrroles as novel 5-HT7 receptor ligands. The synthesis and the affinity for the 5-HT7 receptor of these phenylpyrroles are described. Some of these compounds exhibited high affinity for the 5-HT7 receptors.     

The 5-HT3 antagonist tropisetron (ICS 205-930) is a potent and selective alpha7 nicotinic receptor partial agonist

The 5-HT3 receptor antagonist tropisetron (ICS 205-930) was found to be a potent and selective partial agonist at alpha7 nicotinic receptors. Two other 5-HT3 receptor antagonists, ondansetron and LY-278,584, were found to lack high affinity at the alpha7 nicotinic receptor. Quinuclidine analogues (1 and 2) of tropisetron were also found to be potent and selective partial agonists at alpha7 nicotinic receptors.     

Structure-intrinsic activity relationship studies in the group of 1-imido/amido substituted 4-(4-arylpiperazin-1-yl)cyclohexane derivatives; new, potent 5-HT1A receptor agents with anxiolytic- like activity

Introduction of 1,4-disubstituted cyclohexane ring in the structure of flexible long chain arylpiperazines resulted in linearly constrained, potent serotonin (5-HT)(1A) ligands. In order to trace structure-intrinsic activity relationships in this group, a new series of 1-substituted 4-(4-arylpiperazin-1-yl)cyclohexane derivatives with different cyclic imide/amide termini, and their flexible, tetramethylene analogues were synthesized and pharmacologically evaluated for 5-HT(1A) receptors. In vitro binding experiments revealed that all the compounds were potent 5-HT(1A) receptor agents (K(i) = 1.9-74 nM). Some derivatives tested additionally showed also high affinity for alpha(1)-adrenergic receptors (K(i) = 2.9-101 nM) and for 5-HT(7) receptors. Functional in vivo examination revealed that rigid ligands with o-OCH(3) group in the aryl moiety and cyclic imide system in the opposite terminal behaved like postsynaptic 5-HT(1A) receptor antagonists. On the other hand, unsubstituted, m-Cl, or m-CF(3) substituted derivatives as well as those with cyclic amide group in the terminal fragment exhibited agonistic or partial agonistic activity. Three out of four derivatives tested, that is, postsynaptic 5-HT(1A) antagonists 9 and 10, and partial agonist 16, showed anxiolytic-like activity in the conflict drinking (Vogel) test in rats.     

7-Arylpiperazinylalkyl and 7-tetrahydroisoquinolinylalkyl derivatives of 8-alkoxy-purine-2,6-dione and some of their purine-2,6,8-trione analogs as 5-HT(1A), 5-HT(2A), and 5-HT(7) serotonin receptor ligands

On the basis of our earlier studies with the serotonin receptor ligands in the group of 1,3-dimethyl-3,7-dihydropurine-2,6-dione derivatives, a series of new arylpiperazinylalkyl and tetrahydroisoquinolinylalkyl analogs of 8-alkoxy-1,3-dimethyl-3,7-dihydropurine-2,6-dione (10-25) and 1,3-dimethyl-7,9-dihydro-3H-purine-2,6,8-trione (26-30) were synthesized and their 5-HT(1A), 5-HT(2A), and 5-HT(7) receptor affinities were determined. The new compounds 17, 18, 20, and 21 were found to be highly active 5-HT(1A) receptor ligands (K(i)=11-19nM) with diversified affinity for 5-HT(2A) receptors (K(i)=15-253nM). Compounds 12, 13, 15, and 19 were moderately potent 5-HT(2A) ligands (K(i)=23-57nM), whereas 17, 18, 24, and 25 showed distinct affinity for 5-HT(7) receptors (K(i)=51-83nM). Purine-2,6,8-triones showed weak affinities for 5-HT(1A) and 5-HT(7) receptors; among them, 27 and 29 were classified as 5-HT(2A) receptor ligands. The selected compounds 17 and 21 were pharmacologically evaluated to determine their functional activities at pre-(hypothermia in mice) and post-(lower lip retraction in rats) synaptic 5-HT(1A) receptors. Compound 17 showed features of a potential agonist of pre- and post-synaptic 5-HT(1A) receptors, whereas 21 was classified as a potential, weak partial agonist of postsynaptic sites. Last of all, the most interesting compound 17 tested in behavioral models showed potential anxiolytic and antidepressant activities.     

Bivalent ligand approach on 4-[2-(3-methoxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine: synthesis and binding affinities for 5-HT(7) and 5-HT(1A) receptors

We here report on the synthesis and binding properties at 5-HT(7) and 5-HT(1A) receptors of ligands 3-12, that were designed according to the 'bivalent ligand' approach. Two moieties of the 5-HT(7)/5-HT(1A) ligand 4-[2-(3-methoxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine (1) were linked through their 3-methoxy substituent by polymethylene chains of variable length, with the aim to increase the affinity for 5-HT(7) receptor and the selectivity over 5-HT(1A) receptors. In the best cases, the dimers showed affinities for 5-HT(7) receptors as high as the monomer with no improvement in selectivity. Some dimers displayed 5-HT(1A) receptor affinities slightly higher than monomer 1.     

Synthesis and structure-affinity relationship investigations of 5-aminomethyl and 5-carbamoyl analogues of the antipsychotic sertindole. A new class of selective alpha1 adrenoceptor antagonists

A new class of selective alpha(1) adrenoceptor antagonists derived from the antipsychotic drug sertindole is described. The most potent and selective compound 1-(2-(4-[5-aminomethyl-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl)ethyl)-2-imidazolidinone (11) binds with 0.50 nM affinity for alpha(1) adrenergic receptors and with more than 44 times lower affinity for dopamine D(2),D(3), D(4) and serotonin 5-HT(1A), 5-HT(1B), 5-HT(2A) and 5-HT(2C) receptors. The molecular features providing high affinity for adrenergic alpha(1) receptors and high selectivity towards dopamine D(2) and serotonin 5-HT(2A) and 5-HT(2C) receptors are discussed.     

A single mutation in the 5-HT4 receptor (5-HT4-R D100(3.32)A) generates a Gs-coupled receptor activated exclusively by synthetic ligands (RASSL)

To better understand G-protein-coupled receptor (GPCRs) signaling, cellular and animal physiology, as well as gene therapy, a new tool has recently been proposed. It consists of GPCR mutants that are insensitive to endogenous ligands but sensitive to synthetic ligands. These GPCRs are called receptor activated solely by synthetic ligands (RASSL). Only two examples of such engineered receptors have been described so far: one G(i)-coupled (opioid receptors) and one G(s)-coupled (beta(2)-adrenergic receptors). Here, we describe the first RASSL related to serotonin receptors (D100(3.32)A G(s)-coupled 5-HT(4) receptor or 5-HT(4)-RASSL). 5-HT(4)-RASSL is generated by a single mutation, is totally insensitive to serotonin (5-HT), and still responds to synthetic ligands. These ligands have affinities in the range of nanomolar concentrations for the mutant receptor and exhibit full efficacy. More interestingly, two synthetic ligands behave as antagonists on the wild type but as agonists on the 5-HT(4)-RASSL.     

Novel aminoethylbiphenyls as 5-HT7 receptor ligands

The synthesis of a series of aminoethylbiphenyls as novel 5-HT(7) receptor ligands is described. The novel derivatives exhibit high affinity for the 5-HT(7) receptor with selectivity toward 5-HT(1A) receptor.     

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon

Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine samples tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.     

Dual role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors

The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of alpha7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of alpha4beta2 nicotinic and 5-HT3 serotonergic receptors. Both the N-terminal region of hRIC-3, which contains two transmembrane segments, and the C-terminal region are needed for these differential effects. hRIC-3 inhibits receptor expression by hindering export of mature receptors to the cell membrane. By using chimeric proteins made of alpha7 and 5-HT3 receptors, we have shown that the presence of an extracellular isoleucine close to the first transmembrane receptor fragment is responsible for the transport arrest induced by hRIC-3. Enhancement of alpha7 receptor expression occurs, at least, at two levels: by increasing the number of mature receptors and facilitating its transport to the membrane. Certain amino acids of a putative amphipathic helix present at the large cytoplasmic region of the alpha7 subunit are required for these actions. Therefore, hRIC-3 can act as a specific regulator of receptor expression at different levels.     

In vivo regulation of the serotonin-2 receptor in rat brain

Serotonin-2 (5-HT-2) receptors in brain were measured using [3H]ketanserin. We examined the effects of amitriptyline, an antidepressant drug, of electroconvulsive shock (ECS) and of drug-induced alterations in presynaptic 5-HT function on [3H]ketanserin binding to 5-HT-2 receptors in rat brain. The importance of intact 5-HT axons to the up-regulation of 5-HT-2 receptors by ECS was also investigated, and an attempt was made to relate the ECS-induced increase in this receptor to changes in 5-HT presynaptic mechanisms. Twelve days of ECS increased the number of 5-HT-2 receptors in frontal cortex. Neither the IC50 nor the Hill coefficient of 5-HT in competing for [3H]ketanserin binding sites was altered by ECS. Repeated injections of amitriptyline reduced the number of 5-HT-2 receptors in frontal cortex. Reserpine, administered daily for 12 days, caused a significant increase in 5-HT-2 receptors, but neither daily injections of p-chlorophenylalanine (PCPA) nor lesions of 5-HT axons with 5,7-dihydroxytryptamine (5,7-DHT) affected 5-HT-2 receptors. However, regulation of 5-HT-2 receptors by ECS was dependent on intact 5-HT axons since ECS could not increase the number of 5-HT-2 receptors in rats previously lesioned with 5,7-DHT. Repeated ECS, however, does not appear to affect either the high-affinity uptake of [3H]5-HT or [3H]imipramine binding, two presynaptic markers of 5-HT neuronal function. 5-HT-2 receptors appear to be under complex control. ECS or drug treatments such as reserpine or amitriptyline, which affect several monoamine neurotransmission systems including 5-HT, can alter 5-HT-2 receptors. While depleting 5-HT alone (5,7-DHT or PCPA) does not alter [3H]ketanserin binding to 5-HT-2 receptors, intact 5-HT axons are necessary for the adaptive up-regulation of the receptor following ECS.     

Regulation of serotonin receptor function in the nervous system by lipid rafts and adaptor proteins

5-HT is a phylogenetically conserved monoaminergic neurotransmitter which is crucial for a number of physiological processes and is dysregulated in several disease states including depression, anxiety and schizophrenia. 5-HT neurons in the central nervous system are localized in the raphe nuclei and project to a wide range of target areas. 5-HT exerts its functions through 14 subtypes of 5-HT receptors. The tertiary structures of seven transmembrane 5-HT receptors contain several important features, including cholesterol consensus motifs, prominent intracellular loops and free C-termini. Alterations of cholesterol levels affect binding of ligands to 5-HT receptors and cholesterol-enriched microdomains in the cell membrane, termed lipid rafts, regulate 5-HT receptor internalization and signaling. The intracellular loops and the C-termini of 5-HT receptors provide binding sites for interacting adaptor proteins. Adaptor proteins affect internalization, desensitization as well as G-protein dependent and independent signaling via 5-HT receptors. We will here briefly review recent progress on the role of lipid rafts and adaptor proteins in the regulation of localization, trafficking, signaling and ligand bias of 5-HT receptors.     

High affinity binding of epibatidine to serotonin type 3 receptors

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of (125)I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT(3)Rs) block the remaining 25%. (125)I-Epibatidine binds with a high affinity to native 5-HT(3)Rs of N1E-115 cells and to receptors composed of only 5-HT(3A) subunits expressed in HEK cells. In these cells, serotonin, the 5-HT(3)R-specific antagonist MDL72222, and the 5-HT(3)R agonist chlorophenylbiguanide readily competed with (125)I-epibatidine binding to 5-HT(3)Rs. Nicotine was a poor competitor for (125)I-epibatidine binding to 5-HT(3)Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of (125)I-epibatidine binding to 5-HT(3)Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT(3)Rs in neuroblastoma cell lines and 5-HT(3A)Rs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT(3)Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT(3)R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT(3)Rs.     

Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor

Homomeric complexes of 5-HT(3A) receptor subunits form a ligand-gated ion channel. This assembly does not fully reproduce the biophysical and pharmacological properties of native 5-HT(3) receptors which might contain the recently cloned 5-HT(3B) receptor subunit. In the present study, heteromeric assemblies containing human 5-HT(3A) and 5-HT(3B) subunits were expressed in HEK 293 cells to detail the functional diversity of 5-HT(3) receptors. We designed patch-clamp experiments with homomeric (5-HT(3A)) and heteromeric (5-HT(3AB)) receptors to emphasize the kinetics of channel activation and desensitization. Co-expression of the 5-HT(3B) receptor subunit reduced the sensitivity for 5-HT (5-HT(3A) receptor: EC(50) 3 micro M, Hill coefficient 1.8; 5-HT(3AB) receptor: EC(50) 25 micro M, Hill coefficient 0.9) and markedly altered receptor desensitization. Kinetic modeling suggested that homomeric receptors, but not heteromeric receptors, desensitize via an agonist-induced open-channel block. Furthermore, heteromeric 5-HT(3AB) receptor assemblies recovered much faster from desensitization than homomeric 5-HT(3A) receptor assemblies. Unexpectedly, the specific 5-HT(3) receptor agonist mCPBG induced an open-channel block at both homomeric and heteromeric receptors. Because receptor desensitization and resensitization massively affect amplitude, duration, and frequency of synaptic signaling, these findings are evidence in favor of a pivotal role of subunit composition of 5-HT(3) receptors in serotonergic transmission.     

The mouse 5HT5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family.

Serotonin (5-HT) is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with G proteins, we have isolated a cDNA encoding a new serotonin receptor from a mouse brain library. Amino acid sequence comparisons revealed that this receptor was a distant relative of all previously identified 5-HT receptors; we therefore named it 5HT5. When expressed in Cos-7 cells and NIH-3T3 cells, the 5HT5 receptor displayed a high affinity for the serotonergic radioligand [125I]LSD. Surprisingly, its pharmacological profile resembled that of the 5HT1D receptor, which is a 5-HT receptor subtype which has been shown to inhibit adenylate cyclase and which is predominantly expressed in basal ganglia. However, unlike 5HT1D receptors, the 5HT5 receptor did not inhibit adenylate cyclase and its mRNA was not found in basal ganglia. On the contrary, in situ hybridization experiments revealed that the 5HT5 mRNA was expressed predominantly in cerebral cortex, hippocampus, habenula, olfactory bulb and granular layer of the cerebellum. Our results therefore demonstrate that the 5HT1D receptors constitute a heterogeneous family of receptors with distinct intracellular signalling properties and expression patterns.