Binding of methoxy-substituted N1-benzenesulfonylindole analogs at human 5-HT6 serotonin receptors

Comparison of several amine-substituted and methoxy-substituted analogs of N1-(4-aminobenzene)sulfonylindole suggests that these substituents might contribute to the 5-HT6 serotonin receptor affinity of these agents via their electronic effect on the indolic nucleus. Their 1,2,3,4-tetrahydrocarbazole counterparts behave differently.     

Binding of isotryptamines and indenes at h5-HT6 serotonin receptors

To determine if the indolic nitrogen atom is required for the binding of N(1)-benzyltryptamines at h5-HT(6) serotonin receptors, several isotryptamines and indene analogs were examined. The affinity of 3-benzyl-N(1)-(N,N-dimethylaminoethyl)indole (5, K(i)=32nM) and 1-benzyl-3-(N,N-dimethylaminoethyl)indene (11, K(i)=3nM) indicates that the indolic nitrogen atom is not essential for binding.     

Binding of beta-carbolines at 5-HT(2) serotonin receptors

A series of ring-substituted (i.e., methoxy and bromo) 3,4-dihydro- and 1,2,3,4-tetrahydro-β-carbolines was examined at 5-HT_2A and 5-HT_2C serotonin receptors. Whereas most of the methoxy-substituted derivatives typically displayed affinities similar to their unsubstituted parents, certain (particularly 8-substituted) bromo derivatives displayed enhanced affinity. A binding profile was obtained for selected β-carbolines.     

Interaction of N1-unsubstituted and N1-benzenesulfonyltryptamines at h5-HT6 receptors

Despite possessing a common tryptaminergic scaffold, examination of 28 (i.e., 14 pairs of) compounds suggests that N1-unsubstituted and N1-benzenesulfonyltryptamines likely bind at h5-HT6 receptors in a dissimilar manner (r2=0.201). Additionally, an examination of two rotationally constrained N1-benzenesulfonyltryptamine analogs indicates that a non-coplanar relationship between the two aryl groups might be preferred for interaction with the receptors.     

Binding thermodynamics of serotonin to rat-brain 5-HT1a, 5HT2a and 5-HT3 receptors

The thermodynamic parameters ΔG° , ΔH° and Δs° of the binding equilibrium of serotonin to 5-HT_1A, 5-HT_2A and 5-HT₃ rat-brain membrane receptors have been determined by means of affinity constant measurements at six temperatures in the range 0 –35 ° C and van't Hoff plots. At variance with 5-HT_1A and 5-HT₃, the binding at the 5-HT_2A receptors is strongly endothermic and entropy-driven. Comparison with the results obtained by other authors on 5-HT_2A receptors in rats and humans suggests that the observed differences can be explained by a single amino acid difference in the receptor sequence between these two species.     

gamma-Carbolines: binding at 5-HT5A serotonin receptors

Screening of various agents resulted in the identification of 5-methyl-1,2,3,4-tetrahydro-gamma-carboline (1; K(i)=5,300 nM) as a compound with modest affinity for mouse 5-HT(5A) receptors. Structure-affinity studies were conducted resulting in 5-methyl-2-[3-(4-fluorophenoxy)propyl]-1,2,3,4-tetrahydro-gamma-carboline (17; K(i)=13 nM). Although 17 also binds at 5-HT(2) receptors, it serves as a novel lead for the further development of 5-HT(5A) ligands.     

Mechanisms of agonism and inverse agonism at serotonin 5-HT1A receptors

Mechanisms of agonist and inverse agonist action at the serotonin 5-HT1A receptor have been studied using the modulation of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding in membranes of Chinese hamster ovary (CHO) cells expressing the receptor (CHO-5-HTA1A cells). A range of agonists increased [35S]GTPgammaS binding with different potencies and to different maximal extents, whereas two compounds, methiothepin and spiperone, inhibited both agonist-stimulated and basal [5S]GTPgammaS binding, thus exhibiting inverse agonism. Potencies of agonists to stimulate [35S]GTPgammaS binding in membranes from CHO-5-HT1A cells were reduced by adding increasing concentrations of GDP to assays, whereas changes in sodium ion concentration did not affect agonist potency. The maximal effect of the agonists was increased by increasing sodium ion concentrations. The affinities of agonists in ligand binding assays were unaffected by changes in sodium ion concentration. Increasing GDP in the assays of the inverse agonists increased potency for spiperone to inhibit [35S]GTPgammaS binding and had no effect for methiothepin, in agreement with the sensitivity of these compounds to guanine nucleotides in ligand binding assays. Potencies for these inverse agonists were unaffected by changes in sodium ion concentration. These data were simulated using the extended ternary complex model. These simulations showed that the data obtained with agonists were consistent with these compounds achieving agonism by stabilising the ternary complex. For inverse agonists, the simulations showed that the mechanism for spiperone may be to stabilise forms of the receptor uncoupled from G proteins. Methiothepin, however, probably does not alter the equilibrium distribution of different receptor species; rather, this inverse agonist may stabilise an inactive form of the receptor that can still couple to G protein.     

Evaluation of isotryptamine derivatives at 5-HT2 serotonin receptors

On the basis that meta-chlorophenylpiperazine (mCPP; 1) is a nonselective 5-HT_2C agonist, that benz-fused tryptamines (e.g., 5) display enhanced 5-HT₂ affinity, and that certain isotryptamines 3 reportedly bind with enhanced affinity and selectivity at 5-HT_2C receptors, we prepared and examined a series of isotryptamine-related analogues as potentially selective 5-HT_2C agonists. None of the compounds displayed selectivity for 5-HT_2C versus 5-HT_2A receptors. Detailed re-examination of a compound previously reported to display 100-fold 5-HT_2C selectivity [i.e., S(+)-5,6-difluoro-α-methylisotryptamine] revealed that its selectivity versus 5-HT_2A receptors was, at best, only 10-fold.     

Further studies on the binding of N1-substituted tryptamines at h5-HT6 receptors

N(1)-Arylsulfonyl-substituted analogs of N,N-dimethyltryptamine bind at 5-HT(6) receptors. Replacement of the aryl moiety with similarly hydrophobic alkyl substituents results in decreased affinity, as does replacement of a benzenesulfonyl moiety with a benzyl group. Current findings indicate that an aryl (or substituted aryl) sulfonyl (rather than alkylsulfonyl or benzyl) moiety is optimal for high-affinity binding, and further suggest that the N(1)-benzenesulfonyl- and their corresponding N(1)-benzyltryptamine counterparts bind in a different fashion.     

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.     

Binding of tryptamine analogs at h5-HT1E receptors: a structure-affinity investigation

Structure-affinity requirements for the binding of serotonin (5-HT) analogs at human 5-HT1E receptors were investigated by examining the affinities of >40 tryptamine-related compounds. No tryptamine analog was found to bind with substantially higher affinity than 5-HT. The results indicate that hydrogen bonding plays a key role in the 5-HT1E/receptor interaction. This finding was supported using quantitative structure-activity analysis (QSAR) techniques such as comparative molecular field analysis (CoMFA) and the program QsarIS.     

Target size analysis of serotonin 5-HT1 and 5-HT2 receptors in bovine brain membranes

Freeze-dried crude synaptic membranes prepared from bovine cerebral cortex and striatum were exposed to high energy gamma ray from the source of 60Co. The size of serotonin 5-HT1 receptors labeled by [3H]serotonin and that of 5-HT2 receptors labeled by [3H]spiperone or [3H]ketanserin was determined by target size analyses. The values were 57,000 daltons, 145,000 daltons and 152,000 daltons for the cerebral cortex and 56,000 daltons, 141,000 daltons and 150,000 daltons for the striatum, respectively. The estimated sizes were deduced by reference to enzyme standards with known molecular masses and which were irradiated in parallel. Our results demonstrate that the molecular entities in situ for 5-HT1 receptors are distinct from those for 5-HT2 receptors, thus supporting data on the existence of two distinct populations of serotonin receptors, hitherto evidenced physiopharmacologically.     

5-HT1B serotonin receptors and antidepressant effects of selective serotonin reuptake inhibitors ]

We used knockout mice and receptor antagonist strategies to investigate the contribution of the serotonin (5-hydroxytryptamine, 5-HT) 5-HT1B receptor subtype in mediating the effects of selective serotonin reuptake inhibitors (SSRIs). Using in vivo intracerebral microdialysis in awake mice, we show that a single systemic administration of paroxetine (1 or 5 mg/kg, i.p.) increased extracellular serotonin levels [5-HT]ext in the ventral hippocampus and frontal cortex of wild-type and mutant mice. However, in the ventral hippocampus, paroxetine at the two doses studied induced a larger increase in [5-HT]ext in knockout than in wild-type mice. In the frontal cortex, the effect of paroxetine was larger in mutants than in wild-type mice at the 1 mg/kg dose but not at 5 mg/kg. In addition, either the absence of the 5-HT1B receptor or its blockade with the mixed 5-HT1B/1D receptor antagonist, GR 127935, potentiates the effect of a single administration of paroxetine on [5-HT]ext more in the ventral hippocampus than in the frontal cortex. Furthermore, we demonstrate that SSRIs decrease immobility in the forced swimming test; this effect is absent in 5-HT1B knockout mice and blocked by GR 127935 in wild-type suggesting therefore that activation of 5-HT1B receptors mediate the antidepressant-like effects of SSRIs. Taken together these data demonstrate that 5-HT1B autoreceptors appear to limit the effects of SSRI on dialysate 5-HT levels particularly in the hippocampus while presynaptic 5-HT1B heteroreceptors are likely to be required for the antidepressant activity of SSRIs.     

Cloning of the human serotonin 5-HT2 and 5-HT1C receptor subtypes.

We report the cloning and the deduced amino acid sequence of cDNAs encoding both the human serotonin 5-HT2 and 5-HT1C receptors. The human 5-HT2 and 5-HT1C receptors shared 87% and 90% amino acid homology, respectively, with their rat counterparts. The most divergent regions of the 5-HT2 receptor between human and rat were the N-terminal extracellular domain (75% homology) and the C-terminal intracellular domain (67% homology between amino acids 426-474). The greatest variability between the human and rat 5-HT1C receptors were at the N-terminal extracellular domain (78% homology) and the third cytoplasmic loop (71% homology). The availability of the cloned human 5-HT2 and 5-HT1C receptors will help facilitate the further understanding of the molecular pharmacology and physiology of these receptors.     

Possible differences in modes of agonist and antagonist binding at human 5-HT6 receptors

A graphics model of the human 5-HT6 receptor was constructed and automated docking studies were performed. The model suggests that 5-HT6 antagonist arylsulfonyltryptamines might bind differently than that of the agonist serotonin. Furthermore, the model explains many of the empirical results from our previous structure-affinity studies.     

Molecular dynamics of buspirone analogues interacting with the 5-HT1A and 5-HT2A serotonin receptors

Three-dimensional (3-D) models of the human serotonin 5-HT1A and 5-HT2A receptors were constructed, energy refined, and used to study the interactions with a series of buspirone analogues. For both receptors, the calculations showed that the main interactions of the ligand imide moieties were with amino acids in transmembrane helix (TMH) 2 and 7, while the main interactions of the ligand aromatic moieties were with amino acids in TMH5, 6 and 7. Differences in binding site architecture in the region of highly conserved serine and tyrosine residues in TMH7 gave slightly different binding modes of the buspirone analogues at the 5-HT1A and 5-HT2A receptors. Molecular dynamics simulations of receptor-ligand interactions indicated that the buspirone analogues did not alter the interhelical hydrogen bonding patterns upon binding to the 5-HT2A receptor, while interhelical hydrogen bonds were broken and others were formed upon ligand binding to the 5-HT1A receptor. The ligand-induced changes in interhelical hydrogen bonding patterns of the 5-HT1A receptor were followed by rigid body movements of TMH2, 4 and 6 relative to each other and to the other TMHs, which may reflect the structural conversion into an active receptor structure.     

Caveolin-1 affects serotonin binding and cell surface levels of human 5-HT7(a) receptors

Studies using HeLa cells expressing 5-HT7 receptors showed that detergent resistant membranous lipid rafts purified by sucrose gradient centrifugation contained 5-HT7 receptors and caveolin-1. Caveolin-1 siRNA-mediated knockdown or serotonin (5-HT) treatment caused significant reductions of maximum [3H]5-HT binding to 5-HT7 receptors and total immunoreactivity of membranous 5-HT7 receptors in intact cells. Co-treatment with 5-HT, caveolin-1 siRNA and methyl-beta-cyclodextrin had no additive effects on reducing maximum binding of [3H]5-HT to 5-HT7 receptors. 5-HT-mediated reduction of [3H]5-HT binding to 5-HT7 receptors was counteracted by genistein, but not by sucrose. Caveolin-1, specifically localized in cholesterol-enriched lipid rafts, appears to regulate constitutive and agonist-stimulated cell surface levels of 5-HT7 receptors via a clathrin-independent mechanism.     

Arylguanidine and arylbiguanide binding at 5-HT3 serotonin receptors: a QSAR study

For a series of monosubstituted arylguanidines, 5-HT3 receptor affinity was found generally related to the electron withdrawing nature of the substituent at the aryl 3-position and the lipophilicity of the 4-position substituent. A broader examination of 35 arylguanidines and arylbiguanides revealed that affinity could be described by molecular polarizability, a Chi index term (8chiP), and the sum of all (-Cl) E-State values (SsCl) in the molecule.     

Stimulation- and palmitoylation-dependent changes in oligomeric conformation of serotonin 5-HT1A receptors

In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP- and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.     

Interaction of chiral MS-245 analogs at h5-HT6 receptors

Optically active pyrrolidinylmethylindole analogs related in structure to the benzenesulfonyltryptamine 5-HT(6) receptor antagonist MS-245 were evaluated and their R-isomers were found to bind with affinity higher than their S-enantiomers.