Pharmacomodulation of a Sulfamide 5-HT6 Receptor Ligand

A series of N-ω-aminoalkyl- or N-ω-amidinoalkyl-2,4,6-triisopropyl benzenesulfonamides has been synthesized and their respective affinity indices on 5-HT₆ receptor determined. This evaluation clearly showed that the compounds possessing an arylpiperazine moiety or an amidine function exhibited good affinity for the model.     

N1-benzenesulfonylgramine and N1-benzenesulfonylskatole: novel 5-HT6 receptor ligand templates

1-Benzenesulfonyl-5-methoxy-N,N-dimethyltryptamine (3; K(i)=2.3 nM) is a 5-HT(6) receptor antagonist; removal of the 5-methoxy group (i.e., 6; K(i)=4.1 nM) has little impact on receptor affinity. In the present study, it is shown that the aminomethyl portion of 6 can be shortened to gramine analogue 10a (K(i)=3.1 nM); a related skatole derivative 11b (K(i)=12 nM) also binds with high affinity indicating that the aminoethyl portion of the tryptamines is not required for binding. Compounds 10a and 11b represent members of novel classes of 5-HT(6) antagonists.     

1,2,3,4-tetrahydrocarbazoles as 5-HT6 serotonin receptor ligands

An investigation of the structure-affinity relationships for the binding of 4-(N,N-dimethylaminomethyl)-N(9)-arylsulfonyl-9H-1,2,3,4-tetrahydrocarbazoles (conformationally-constrained analogues of the benzenesulfonyltryptamine 5-HT(6) antagonist MS-245) at human 5-HT(6) receptors revealed that various arylsulfonyl substituents are tolerated and that the 4-(N,N-dimethylaminomethyl) group is not required for binding. In particular, N(9)-(4-aminobenzenesulfonyl)-9H-1,2,3,4-tetrahydrocarbazole (20, K(i)=29 nM) was found to bind with high affinity and represents the first member of a new structural class of agents with 5-HT(6) antagonist properties (pA(2)=7.0; cAMP hydrolysis assay).     

The 5-HT3B subunit confers spontaneous channel opening and altered ligand properties of the 5-HT3 receptor

Current receptor theory suggests that there is an equilibrium between the inactive (R) and active (R*) conformations of ligand-gated ion channels and G protein-coupled receptors. The actions of ligands in both receptor types could be appropriately explained by this two-state model. Ligands such as agonists and antagonists affect receptor function by stabilizing one or both conformations. The 5-HT3 receptor is a member of the Cys-loop ligand-gated ion channel superfamily participating in synaptic transmission. Here we show that co-expression of the 5-HT3A and 5-HT3B receptor subunits in the human embryonic kidney (HEK) 293 cells results in a receptor that displays a low level of constitutive (or agonist-independent) activity. Furthermore, we also demonstrate that the properties of ligands can be modified by receptor composition. Whereas the 5-hydroxytryptamine (5-HT) analog 5-methoxyindole is a partial agonist at the 5-HT3A receptor, it becomes a "protean agonist" (functioning as an agonist and an inverse agonist at the same receptor) at the 5-HT3AB receptor (after the Greek god Proteus, who was able to change his shape and appearance at will). In addition, the 5-HT analog 5-hydroxyindole is a positive allosteric modulator for the liganded active (AR*) conformation of the 5-HT3A and 5-HT3AB receptors and a negative allosteric modulator for the spontaneously active (R*) conformation of the 5-HT3AB receptor, suggesting that the spontaneously active (R*) and liganded active (AR*) conformations are differentially modulated by 5-hydroxyindole. Thus, the incorporation of the 5-HT3B subunit leads to spontaneous channel opening and altered ligand properties.     

Discovery of 5-HT6 receptor ligands based on virtual HTS

Based on a pharmacophore alignment on a 5-HT(6) ligand applying 4SCan technology, a new lead series was identified and further structurally investigated. K(i)s down to 8 nM were achieved.     

Bicyclic heteroarylpiperazines as selective brain penetrant 5-HT6 receptor antagonists

Starting from the potent and selective but poorly brain penetrant 5-HT6 receptor antagonist SB-271046, a successful strategy for improving brain penetration was adopted involving conformational constraint with concomitant reduction in hydrogen bond count. This provided a series of bicyclic heteroarylpiperazines with high 5-HT6 receptor affinity. 5-Chloroindole 699929 combined high 5-HT6 receptor affinity with excellent brain penetration and also had good oral bioavailability in both rat and dog.     

Bicyclic piperazinylbenzenesulphonamides are potent and selective 5-HT6 receptor antagonists

The synthesis of novel 3-(octahydropyrido[1,2-a]pyrazin-2-yl)- and 3-(hexahydropyrrolo[1,2-a]pyrazin-2-yl)phenyl-2-benzo[b]thiophene sulphonamide derivatives 3, (S)-4 and (R)-4 is described. The compounds show high affinity for the 5-HT6 receptor, excellent selectivity against a range of other receptors and good brain penetration.     

N-arylsulfonyl-2-vinyltryptamines as new 5-HT6 serotonin receptor ligands

Several new 2-vinyl-Nb,Nb-dimethyltryptamines were prepared using Fischer indole synthesis followed by simple functional group transformations and evaluated on 5-HT4, 5-HT5, 5-HT6 and 5-HT7 serotonin receptors. It was found that 2-vinyl substitution conferred a potent and selective 5-HT6 binding activity to these molecules which could be enhanced by Na-arylsulfonyl substituents.     

Neuropathic pain-alleviating effects of pyrazole-conjugated arylsulfonamides as 5-HT6 receptor antagonists

A novel series of arylsulfonylaminomethyl-3-(1-phenyl-5-isopropyl)pyrazoles was evaluated for serotonin receptor subtype 6 (5-HT₆R) antagonistic effects in vitro. We also investigated their neuropathic pain-alleviating effects in vivo using a rat spinal nerve ligation (SNL) model. Bicyclic aromatic sulfonamino groups, such as naphthalene and quinolin-substituted derivatives, showed good 5-HT₆ inhibitory activity in vitro. Among them, selected compounds, 12 and 13, having 8-quinoylsulfonamino groups, showed potent neuropathic pain-alleviating effects in the rat model.     

Conformationally constrained N1-arylsulfonyltryptamine derivatives as 5-HT6 receptor antagonists

Several series of conformationally constrained N1-arylsulfonyltryptamine derivatives were prepared and tested for 5-HT6 receptor binding affinity and ability to modulate cAMP production in a cyclase assay. The 3-piperidin-3-yl-, 3-(1-methylpyrrolidin-2-ylmethyl)-, and 3-pyrrolidin-3-yl-1H-indole arrays (8-13) appear to be able to adopt a conformation that allows high affinity 5-HT6 receptor binding, while the beta-carboline array 14 binds with a significantly weaker (10- to 100-fold) affinity. N1-Benzenesulfonyl-3-piperidin-3-yl-1H-indole 9a is a high affinity full agonist with EC50 = 24 nM. Several of the N1-arylsulfonyl-3-(1-methylpyrrolidin-2-ylmethyl)-1H-indole derivatives behave as very potent antagonists ((S)-11r, (S)-11t; IC50 = 0.8, 1.0 nM).     

Tricyclic azepine derivatives as selective brain penetrant 5-HT6 receptor antagonists

Starting from a benzazepine sulfonamide 5-HT(6) receptor antagonist lead with limited brain penetration, application of a strategy of conformational constraint and reduction of hydrogen bond donor count led to a novel series of tricyclic derivatives with high 5-HT(6) receptor affinity and excellent brain:blood ratios.     

Molecular cloning and functional characterization of a human 5-HT1B serotonin receptor: a homologue of the rat 5-HT1B receptor with 5-HT1D-like pharmacological specificity.

We describe a genomic clone encoding the human 5-HT1B receptor. This apparently intronless gene encodes a 390 amino acid polypeptide homologous to the rat 5-HT1B serotonin receptor, with which it shares 93% amino acid sequence identity. Remarkably, [3H]5-hydroxytryptamine binding studies with transfected HeLa cells show that the human 5-HT1B receptor has a pharmacological profile that is markedly different from that of the corresponding rat receptor. Instead, human 5-HT1B drug specificity is highly similar to that of the human 5-HT1D receptor, with which it shares 59% amino acid sequence identity. The human 5-HT1B receptor, like the 5-HT1D receptor, can couple to Gi proteins. The presence of the threonine355 in the human receptor rather than an asparagine, as found in the corresponding rat gene product, may explain much of the marked pharmacological difference between the human and rat 5-HT1B receptors.     

Structural basis of ligand recognition in 5-HT3 receptors

The 5‐HT₃ receptor is a pentameric serotonin‐gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti‐emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5‐HT₃ receptor. In the serotonin‐bound structure, we observe hydrophilic interactions with loop E‐binding site residues, which might enable transitions to channel opening. In the granisetron‐bound structure, we observe a critical cation–π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5‐HT₃ receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high‐affinity ligand binding in the human 5‐HT₃ receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti‐emetics in the 5‐HT₃ receptor.     

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.     

Design and synthesis of dual 5-HT1A and 5-HT7 receptor ligands

5-HT_1A and 5-HT₇ receptors have been at the center of discussions recently due in part to their major role in the etiology of major central nervous system diseases such as depression, sleep disorders, and schizophrenia. As part of our search to identify dual targeting ligands for these receptors, we have carried out a systematic modification of a selective 5HT₇ receptor ligand culminating in the identification of several dual 5-HT_1A and 5-HT₇ receptor ligands. Compound 16, a butyrophenone derivative of tetrahydroisoquinoline (THIQ), was identified as the most potent agent with low nanomolar binding affinities to both receptors. Interestingly, compound 16 also displayed moderate affinity to other clinically relevant dopamine receptors. Thus, it is anticipated that compound 16 may serve as a lead for further exploitation in our quest to identify new ligands with the potential to treat diseases of CNS origin.     

Novel 7-phenylsulfanyl-1,2,3,4,10,10a-hexahydro-pyrazino[1,2-a]indoles as dual serotonin 5-HT2C and 5-HT6 receptor ligands

Novel 7-phenylsulfanyl-1,2,3,4,10,10a-hexahydro-pyrazino[1,2-a]indoles are synthesized using a six-step protocol. Notably, the synthesis route make use of a new and improved ring-closing methodology for the assembly of the hexahydro-pyrazino[1,2-a]indole scaffold, which is based on intramolecular C–H insertion of a carbene. The compounds act as dual serotonin 5-HT_2C- and 5-HT₆-ligands.     

Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant

Several variants of the serotonin 5-HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT4(hb). This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT4(a) and 5-HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT4 ligands, no significant differences were detected. However, the broadly used 5-HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT4(a) and 5-HT4(b) variant but showed partial agonistic activity on the 5-HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT4 ligands in different model systems.     

Synthesis and pharmacological evaluation of aryl aminosulfonamide derivatives as potent 5-HT6 receptor antagonists

A series of novel aryl aminosulfonamides was designed and synthesized as 5-HT₆ receptor ligands. Many compounds screened in a functional reporter gene based assay displayed potent antagonistic activity with Kb values in the range of 0.02–10 nM. The lead compound 11m exemplified in this series showed good ADME surrogate properties, acceptable pharmacokinetic profile and is active in animal models of cognition like novel object recognition test and Morris water maze. The compound was selected for detailed profiling.     

The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn

The human 5-HT(6) receptor (5-HT(6)R) is one of the latest cloned receptors among the known 5-HT receptors. Its abundant distribution in the limbic region, which participates in the control of mood and emotion and is involved in nervous system diseases such as depression and Alzheimer disease, has caused it to generate much interest. However, the cellular mechanisms of 5-HT(6)R are poorly understood. In the present study we found, using a yeast two-hybrid assay, that the carboxyl-terminal region of 5-HT(6)R interacts with the Fyn-tyrosine kinase. We also determined using a glutathione S-transferase pulldown assay that this interaction was mediated through the SH3 domain of Fyn and confirmed this by co-immunoprecipitation assays in two different transfected cell lines as well as in adult rat brains. Immunocyto(histo)chemistry also showed prominent co-localization between 5-HT(6)R and Fyn in transfected cells and a similar distribution between 5-HT(6)R and Fyn in the rat brain. Based on this interaction, we further examined the modulation of 5-HT(6)R by Fyn and vice versa. In addition, we demonstrated that the activation of 5-HT(6)R activated the extracellular signal-regulated kinase1/2 via an Fyn-dependent pathway. These findings suggest that Fyn may play an important role in 5-HT(6)R- mediated signaling pathways in the central nervous system.