3D Pharmacophore,hierarchical methods,and 5-HT4 receptor binding data

5-Hydroxytryptamine subtype-4 (5-HT₄) receptors have stimulated considerable interest amongst scientists and clinicians owing to their importance in neurophysiology and potential as therapeutic targets. A comparative analysis of hierarchical methods applied to data from one thousand 5-HT₄ receptor–ligand binding interactions was carried out. The chemical structures were described as chemical and pharmacophore fingerprints. The definitions of indices, related to the quality of the hierarchies in being able to distinguish between active and inactive compounds, revealed two interesting hierarchies with the Unity (1 active cluster) and pharmacophore fingerprints (4 active clusters). The results of this study also showed the importance of correct choice of metrics as well as the effectiveness of a new alternative of the Ward clustering algorithm named Energy (Minimum E-Distance method). In parallel, the relationship between these classifications and a previously defined 3D 5-HT₄ antagonist pharmacophore was established.     

Synthesis and binding properties of novel selective 5-HT3 receptor ligands

This work reports on the synthesis and affinities for the 5-HT(3) versus the 5-HT(4) receptor of new piperazinyl-substituted thienopyrimidine derivatives 20-45 with a view to identify potent and selective ligands for the 5-HT(3) receptor. Some of the new compounds show good affinity for the 5-HT(3) receptor and, notably, do not display any affinity for the 5-HT(4) receptor. 4-(4-Methyl-1-piperazinyl)-2-methylthio-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine 31 exhibits the highest affinity for the 5-HT(3) receptor (Ki = 33 nM) and behaves as noncompetitive antagonist.     

Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.     

Pharmacophore modeling,comprehensive 3D-QSAR,and binding mode analysis of TGR5 agonists

Takeda G-protein-coupled receptor 5 (TGR5) is emerging as an important and promising target for the development of anti-diabetic drugs. Pharmacophore modeling and atom-based 3D-QSAR studies were carried out on a new series of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as highly potent agonists of TGR5. The generated best six featured pharmacophore model AAHHRR consists of two hydrogen bond acceptors (A): two hydrophobic groups (H) and two aromatic rings (R). The constructed 3D-QSAR model acquired excellent correlation coefficient value (R^2?=^?0.9018), exhibited good predictive power (Q^2?=^?0.8494) and high Fisher ratio (F?=?61.2). The pharmacophore model was validated through Guner–Henry (GH) scoring method. The GH value of 0.5743 indicated that the AAHHRR model was statistically valuable and reliable in the identification of TGR5 agonists. Furthermore, the combined approach of molecular docking and binding free energy calculations were carried out for the 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides to explore the binding mode and interaction pattern. The generated contour maps revealed the important structural insights for the activity of the compounds. The results obtained from this study could be helpful in the development of novel and more potent agonists of TGR5.     

The orphan receptor cDNA RDC4 encodes a 5-HT1D serotonin receptor.

The cDNA of RDC4, a putative receptor of the G protein-coupled receptor family, has been cloned by PCR methodology. The primary structure of this receptor showed homology with the serotonin 5-HT1A receptor. In this work, RDC4 mRNA has been injected in Y1 adrenal cells and Xenopus oocytes and RDC4 cDNA has been transfected transiently in cos-7 cells. In all these systems serotonin elicited a rise in cyclic AMP levels. Binding studies on membranes of the transfected cos-7 cells using [3H]-LSD showed a pattern of drug affinities consistent with the known properties of a 5-HT1D receptor. RDC4 therefore codes for a 5-HT1D receptor which in the studied systems is positively coupled to adenylate cyclase.     

Insights into binding modes of 5-HT2c receptor antagonists with ligand-based and receptor-based methods

5-hydroxytryptamine-2c (5-HT2c) receptor antagonists have clinical utility in the management of nervous system. In this work, ligand-based and receptor-based methods were used to investigate the binding mode of h5-HT2c receptor antagonists. First, the pharmacophore modeling of the h5-HT2c receptor antagonists was carried out by CATALYST. Then, the h5-HT2c antagonists were docked to the h5-HT2c receptor model. Subsequently, the comprehensive analysis of the pharmacophore and docking results revealed the structure-activity relationship of 5-HT2c receptor antagonists and the key residues involved in the interactions. For example, three hydrophobic points in the ligands corresponded to the region surrounded by Val135, Val208, Phe214, Ala222, Phe327, Phe328 and Val354 of the h5-HT2c receptor. The carbonyl group of compound 1 formed a hydrogen bond with Asn331. The nitrogen atom in the piperidine of compound 1 corresponding to the positive ionizable position of the best pharmacophore formed the electrostatic interactions with the carbonyl of Asp134, Asn331 and Val354, and with the hydroxyl group of Ser334. In addition, a predictive CoMFA model was developed based on the 24 compounds that were used as the training set in the pharmacophore modeling. Our results were not only useful to explore the detailed mechanism of the interactions between the h5-HT2c receptor and antagonists, but also provided suggestions in the discovery of novel 5-HT2c receptor antagonists.     

Exploring a potential palonosetron allosteric binding site in the 5-HT3 receptor

Palonosetron (Aloxi) is a potent second generation 5-HT₃ receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT₃ receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr⁷³, Phe¹³⁰, Ser¹⁶³, and Asp¹⁶⁵) and in the 5-HT3B receptor subunit (His⁷³, Phe¹³⁰, Glu¹⁷⁰, and Tyr¹⁴³) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT₃A) and heteromeric (5-HT₃AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT₃A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking.     

Characterization of a [3H]-5-hydroxytryptamine binding site in rabbit caudate nucleus that differs from the 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D subtypes

[3H]5-HT binding sites were analyzed in membranes prepared from the rabbit caudate nucleus (CN). [3H]5-HT labeled both 5-HT1A and 5-HT1C recognition sites, defined by nanomolar affinity for 8-OH-DPAT and mesulergine respectively; however, these represented only a fraction of total specific [3H]5-HT binding. Saturation experiments of [3H]5-HT binding in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine to block 5-HT1A and 5-HT1C sites revealed that non-5-HT1A/non-5-HT1C sites represented about 60% of the total 5-HT1 sites and that they exhibited saturable, high affinity, and homogeneous binding. The pharmacological profile of the non-5-HT1A/non-5-HT1C sites (designated 5-HT1R) also differed from that of 5-HT1B and 5-HT2 sites, but was similar to that of the 5-HT1D site. However, significant differences existed between the 5-HT1D and 5-HT1R sites for their Ki values for spiperone, spirilene (an analog of spiperone), metergoline, and methiothepin. The study of modulatory agents (calcium and GTP) also showed differences between the 5-HT1R and 5-HT1D sites. For example, the effects of GTP on agonist binding to the 5-HT1R sites were less than on the 5-HT1D sites in bovine caudate. In addition, calcium enhanced the effects of GTP on the 5-HT1R sites, whereas calcium inhibited the GTP effect on the 5-HT1D sites. The present findings demonstrate the presence of a high-affinity [3H]5-HT binding site in rabbit CN, designated 5-HT1R, that is different from previously defined 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT2 sites.     

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.     

New aza(nor)adamantanes are agonists at the newly identified serotonin 5-HT4 receptor and antagonists at the 5-HT3 receptor

New aza(nor)adamantanes , , and are described which exhibit properties of both 5-HT4 agonism and 5-HT3 antagonism. In particular, compound [SC-52491], an azanoradamantane, exhibits an EC₅₀ of 51 nM in a functional model of 5-HT₄ agonism and potent antagonism, Ki = 1.2 nM, at the 5-HT₃ receptor.     

Identification of the binding sites and selectivity of sarpogrelate,a novel 5-HT2 antagonist,to human 5-HT2A, 5-HT2B and 5-HT2C receptor subtypes by molecular modeling

The aim of the present study was to investigate the binding sites interactions and the selectivity of sarpogrelate to human 5-HT(2) receptor family (5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor subtypes) using molecular modeling. Rhodopsin (RH) crystal structures were used as template to build structural models of the human serotonin-2A and -2C receptors (5-HT(2A)R, 5-HT(2C)R), whereas for 5-HT(2B)R, we used our previously published three-dimensional (3D) models based on bacteriorhodopsin (BR). Sarpogrelate, a novel 5-HT(2)R antagonist, was docked to the receptors. Molecular dynamics (MD) simulations produced the strongest interaction for 5-HT(2A)R/sarpogrelate complex. Upon binding, sarpogrelate constraints aromatic residues network (Trp(3.28), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2A)R; Phe(3.35), Phe(6.51), Trp(7.40) in 5-HT(2B)R; Trp(3.28), Phe(3.35), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2C)R) in a stacked configuration, preventing activation of the receptor. The models suggest that the structural origin of the selectivity of sarpogrelate to 5-HT(2A)R vs both 5-HT(2B)R and 5-HT(2C)R comes from the following results: (1) The tight interaction between the antagonist and the transmembrane domain (TMD) 3. Asp(3.32) neutralizes the cationic head and interacts simultaneously with carboxylic group hydrogen of the antagonist molecule. (2) Due to steric hindrance, Ser(5.46) (vs Ala(5.46) in 5HT(2B) and 5HT(2C)) prevents sarpogrelate to enter deeply inside the hydrophobic core of the helix bundle and to interact with Pro(5.50). (3) The side chain of Ile(4.56) (vs Ile(4.56) in 5HT(2B)R and Val(4.56) in 5HT(2C)R) constraints sarpogrelate to adjust its position by translating toward the strongly attractive Asp(3.32). These results are in good agreement with binding affinities (pKi) of sarpogrelate for 5-HT(2) receptor family expressed in transfected cell.     

1-Aryl-4-(4-succinimidobutyl)piperazines and their conformationally constrained analogues: synthesis, binding to serotonin (5-HT1A, 5-HT2A, 5-HT7), alpha1-adrenergic, and dopaminergic D2 receptors, and in vivo 5-HT1A functional characteristics

Starting with the structure of potent 5-HT(1A) ligands, that is, MM77 [1-(2-methoxyphenyl)-4-(4-succinimidobutyl)piperazine, 4] and its constrained version 5 (MP349), previously obtained in our laboratory, a series of their direct analogues with differently substituted aromatic ring (R=H, m-Cl, m-CF(3), m-OCH(3), p-OCH(3)) were synthesized. The flexible and the corresponding 1e,4e-disubstituted cyclohexane derivatives were designed in order to investigate the influence of rigidification on 5-HT(1A) affinity, selectivity for 5-HT(2A), 5-HT(7), D(1), and D(2) binding sites and functional profile at pre- and postsynaptic 5-HT(1A) receptors. The new compounds 19-25 were found to be highly active 5-HT(1A) receptor ligands (K(i)=4-44 nM) whereas their affinity for other receptors was: either significantly decreased after rigidification (5-HT(7)), or controlled by substituents in the aromatic ring (alpha(1)), or influenced by both those structural modifications (5-HT(2A)), or very low (D(2), K(i)=5.3-31 microM). Since a distinct disfavor towards rigid compounds was observed for 5-HT(7) receptors only, it seems that the bioactive conformation of chain derivatives at those sites should differ from the extended one. Several in vivo models were used to asses functional activity of 19-25 at pre- (hypothermia in mice) and postsynaptic 5-HT(1A) receptors (lower lip retraction in rats and serotonin syndrome in reserpinized rats). Unlike the parent antagonists 4 and 5, all the new derivatives tested were classified as partial agonists with different potency, however, similar effects were observed within pairs (flexible and rigid) of the analogues. The obtained results indicated that substitution in the aromatic ring, but not spacer rigidification, controls the 5-HT(1A) functional activity of the investigated compounds. Moreover, an o-methoxy substituent in the structure of 5 seems to be necessary for its full antagonistic properties. Of all the new compounds studied, trans-4-(4-succinimidocyclohexyl)-1-(3-trifluoromethylphenyl)piperazine 24 was the most potent 5-HT(1A) receptor ligand in vitro (K(i)=4 nM) and in vivo, with at least 100-fold selectivity for the other receptors tested.     

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.     

Unaltered D1, D2, D4, and D5 dopamine receptor mRNA expression and distribution in the spinal cord of the D3 receptor knockout mouse

Dopamine (DA) acts through five receptor subtypes (D1–D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.     

Quinoline- and isoquinoline-sulfonamide derivatives of LCAP as potent CNS multi-receptor-5-HT1A/5-HT2A/5-HT7 and D2/D3/D4-agents: the synthesis and pharmacological evaluation

Two series of arylpiperazinyl-alkyl quinoline-, isoquinoline-, naphthalene-sulfonamides with flexible (13-26) and semi-rigid (33-36) alkylene spacer were synthesized and evaluated for 5-HT(1A), 5-HT(2A), 5-HT(6), 5-HT(7) and selected compounds for D(2), D(3), D(4) receptors. The compounds with a mixed 5-HT and D receptors profile 16 (N-{4-[4-(3-chlorophenyl)-piperazin-1-yl]-butyl}-3-quinolinesulfonamide) and 36 (4-(4-{2-[4-(4-chloro-phenyl)-piperazin-1-yl]-ethyl}-piperidine-1-sulfonyl)-isoquinoline), displaying antagonistic activity at 5-HT(7), 5-HT(2A), D(2) postsynaptic sites, produced antidepressant-like effects in the forced swim test in mice and showed significant anxiolytic activity in the plus-maze test in rats. The lead compound 36, a multi-receptor 5-HT(2A)/5-HT(7)/D(2)/D(3)/D(4) agent, also displayed significant antipsychotic properties in the MK-801-induced hyperlocomotor activity in mice.     

5-Alkyltryptamine derivatives as highly selective and potent 5-HT1D receptor agonists

A series of 5-alkyltryptamines (6) and the corresponding conformationally constrained analogues (8) have been synthesized. The structure activity relationships (SAR) at the 5-position of the indole skeleton and the ethylamine side chain have been studied. Functional activities were assessed using isolated rabbit saphenous vein. Potent, selective ligands were found (6e, Ki 2.5 nM, 5-HT1B/5-HT1D 125-fold) that have potential for treating acute migraine.     

Enantio- and diastereocontrolled dopamine D1, D2, D3 and D4 receptor binding of N-(3-pyrrolidinylmethyl)benzamides synthesized from aspartic acid

Subreceptor selectivity tuning of N-(3-pyrrolidinyl)benzamides leading to the selective dopamine D3 ligand ent1h and the derivatives 1g and 1e/ent1e which preferably recognize human D2 or D4 receptors, respectively, is described. Binding profiles were controlled by both, absolute and relative configuration. The enantiopure target compounds were synthesized from aspartic acid.     

Novel, flexible, and conformationally defined analogs of gepirone: synthesis and 5-HT1A, 5-HT2A, and D2 receptor activity

Novel, flexible arylpiperazine gepirone analogs (1a-3a) with a mixed 5-HT1A/5-HT2A receptor profile, low D2 receptor affinity, and agonistic (2a) or partial agonistic (1a, 3a) activity toward 5-HT1A receptor sites were synthesized. Their conformationally restricted counterparts (1b-3b) were selective 5-HT1A ligands (over 5-HT2A and D2 receptors), which turned out to be agonists (2b, 3b), or partial agonist (1b) of 5-HT1A receptors.     

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.