S 14506: novel receptor coupling at 5-HT(1A) receptors

S 14506 is chemically related to the inverse agonist at 5-HT(1A) receptors, spiperone, but S 14506 behaves as one of the most potent agonists known at these receptors, both in vitro and in vivo. In hippocampal membranes, the specific binding of [(3)H]-S 14506 (K(d)=0.79+/-0.2 nM; B(max)=400+/-32 fmol/mg protein) to 5-HT(1A) receptors resembled that of an antagonist in that it was increased by GppNHp, whereas GppNHp reduced the binding of the classic agonist [(3)H]-8-OH-DPAT (K(d)=1.5+/-0.5 nM; B(max)=303+/-20 fmol/mg protein). Manganese, magnesium and calcium reduced the binding of [(3)H]-S 14506 to 5-HT(1A) receptors whereas the binding of [(3)H]-8-OH-DPAT was increased. Further, sodium markedly reduced the binding of [(3)H]-8-OH-DPAT, without affecting the binding of [(3)H]-S 14506. [(3)H]-S 14506 also bound with high affinity to h 5-HT(1A) receptors stably expressed in membranes of CHO cells (K(d)=0.13+/-0.05 nM; B(max)=2.99+/-0.60 pmol/mg protein): the B(max) was double that of [(3)H]-8-OH-DPAT. GppNHp strongly decreased [(3)H]-8-OH-DPAT binding but scarcely changed [(3)H]-S 14506 binding; calcium, magnesium and manganese had little effect on [(3)H]-S 14506 binding in CHO cells. Antagonists (WAY 100635, WAY 100135) and inverse agonists (spiperone and metitepine) displaced [(3)H]-S 14506 binding with high affinity and Hill slopes close to unity, whereas agonists (5-HT and 5-CT) displayed low affinity with low Hill slopes: partial agonists (buspirone, ipsapirone) showed intermediate properties. In fusion proteins of h 5-HT(1A) receptors with G(ialpha1) the compound potently increased high-affinity GTPase, with a steeper Hill slope than for 5-HT, which may indicate positive cooperativity. The maximum response for S 14506 in these assays was equivalent to 5-HT, indicating it to be a full agonist.In molecular modelling studies, using a three-site model of the 5-HT(1A) receptor, S 14506 spanned between the 5-HT recognition site and the "arginine switch" (DRY microdomain) postulated to activate the interaction of the receptor with the G protein. Thus it is possible to synthesise ligands at G-protein-coupled receptors which are highly potent agonists, but which are structurally related to inverse agonists and show some features of antagonist/inverse agonist binding.     

Efficacy of antipsychotic agents at human 5-HT(1A) receptors determined by [3H]WAY100,635 binding affinity ratios: relationship to efficacy for G-protein activation.

5-HT(1A) receptors are implicated in the aetiology of schizophrenia. Herein, the influence of 15 antipsychotics on the binding of the selective 'neutral' antagonist, [3H]WAY100,635 ([3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclo-hexanecarboxamide), was examined at human 5-HT(1A) receptors expressed in Chinese Hamster Ovary cells. In competition binding experiments, 5-HT displayed biphasic isotherms which were shifted to the right in the presence of the G-protein uncoupling agent, GTPgammaS (100 microM). In analogy, the isotherms of ziprasidone, quetiapine and S16924 (((R-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]-1-(4-fluoro-phenyl)-ethanone), were displaced to the right by GTPgammaS, consistent with agonist actions. Binding of several other antipsychotics, such as ocaperidone, olanzapine and risperidone, was little influenced by GTPgammaS. Isotherms of the neuroleptics, haloperidol, chlorpromazine and thioridazine were shifted to the left in the presence of GTPgammaS, suggesting inverse agonist properties. For most ligands, the magnitude of affinity changes induced by GTPgammaS (alteration in pK(i) values) correlated well with their previously determined efficacies in [35S]GTPgammaS binding studies [Eur. J. Pharmacol. 355 (1998) 245]. In contrast, the affinity of the 'atypical' antipsychotic agent, clozapine, which is a known partial agonist at 5-HT(1A) receptors, was less influenced by GTPgammaS. When the ratio of high-/low-affinity values was plotted against efficacy, hyperbolic isotherms were obtained, consistent with a modified ternary complex model which assumes that receptors can adopt active conformations in the absence of agonist. In conclusion, modulation of [3H]-WAY100,635 binding by GTPgammaS differentiated agonist vs. inverse agonist properties of antipsychotics at 5-HT(1A) receptors. These may contribute to differing profiles of antipsychotic activity.     

S 14671: a novel naphthylpiperazine 5-HT1A agonist of high efficacy and exceptional in vivo potency.

The novel, naphthylpiperazine 5-HT1A agonist, S 14671 (4-[(thenoyl-2)aminoethyl]-1-(7-methoxynaphtylpiperazine], displayed very high affinity for 5-HT1A binding sites (pKi = 9.3) as compared to the serotonin (5-HT)1A agonists, 8-OH-DPAT (9.2) and (+)-flesinoxan (8.7) and the 5-HT1A partial agonists, buspirone (7.9) and BMY 7378 (8.8). In vivo, S 14671 induced the typical 5-HT1A agonist-induced responses of hypothermia and spontaneous tail-flicks at doses as low as greater than or equal to 5 micrograms/kg s.c. and greater than or equal to 40 micrograms/kg s.c., respectively. In each test, it was about 10-fold more potent than 8-OH-DPAT and 100-fold more potent than (+)-flesinoxan and buspirone. The actions of S 14671 could be blocked by BMY 7378 and the 5-HT1A receptor antagonist, (-)-alprenolol, but not by the 5-HT1C/2 receptor antagonist, ritanserin, nor the 5-HT3 receptor antagonist, ICS 205930. Thus, S 14671 is a novel 5-HT1A ligand of high efficacy and exceptional in vivo potency.     

Native rat hippocampal 5-HT1A receptors show constitutive activity

Previous studies have shown that human 5-hydroxytryptamine (5-HT)1A receptors stably expressed in transfected cell lines show constitutive G-protein activity, as revealed by the inhibitory effect of inverse agonists, such as spiperone, on basal guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding. In the present study, we evaluated the constitutive activity of native rat 5-HT1A receptors in hippocampal membranes. Using anti-Galphao-antibody capture coupled to scintillation proximity assay under low sodium (30 mM) conditions, we observed high basal [35S]GTPgammaS binding to Galphao subunits (defined as 100%). Under these conditions, 5-HT and the prototypic selective 5-HT1A agonist (+)8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] both stimulated [35S]GTPgammaS binding to Galphao to a similar extent, raising binding to approximately 130% of basal with pEC50 values of 7.91 and 7.87, respectively. The 5-HT1A-selective neutral antagonist [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) could block these effects in a competitive manner with pKb values (5-HT, 9.57; (+)-8-OH-DPAT, 9.52) that are consistent with its pKi value at r5-HT1A receptors (9.33). In this native receptor system, spiperone and methiothepin reduced basal [35S]GTPgammaS binding to Galphao in a concentration-dependent manner to 90% of basal with pIC50 values of 7.37 and 7.98, respectively. The inhibition of basal [35S]GTPgammaS binding induced by maximally effective concentrations of spiperone (10 microM) or methiothepin (1 microM) was antagonized by WAY100,635 in a concentration-dependent manner (pKb, 9.52 and 8.87, respectively), thus indicating that this inverse agonism was mediated by 5-HT1A receptors. These data provide the first demonstration that native rat serotonin 5-HT1A receptors can exhibit constitutive activity in vitro.     

Antibody capture assay reveals bell-shaped concentration-response isotherms for h5-HT(1A) receptor-mediated Galpha(i3) activation: conformational selection by high-efficacy agonists,and relationship to trafficking of receptor signaling

Although serotonin 5-HT(1A) receptors couple to several Gi/o G-protein subtypes, little is known concerning their differential activation patterns. In this study, in membranes of Chinese hamster ovary cells expressing h5-hydroxytryptamine(1A) receptors (CHO-h5-HT(1A)), isotherms of 5-HT-stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding were biphasic, suggesting coupling to multiple G-protein subtypes. The high potency component was abolished by preincubation with an antibody recognizing Galpha(i3) subunits and was resistant to induction of [(35)S]GTPgammaS dissociation by unlabeled GTPgammaS, thus yielding a bell-shaped concentration-response isotherm. To directly investigate Galpha(i3) activation, we adopted an antibody-capture/scintillation proximity assay. 5-HT and other high-efficacy agonists yielded bell-shaped [(35)S]GTPgammaS binding isotherms, with peaks at nanomolar concentrations. As drug concentrations increased, Galpha(i3) stimulation progressively returned to basal values. In contrast, the partial agonists (-)-pindolol and 4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) displayed sigmoidal stimulation isotherms, whereas spiperone and other inverse agonists sigmoidally inhibited [(35)S]GTPgammaS binding. Agonist-induced stimulation and inverse agonist-induced inhibition of Galpha(i3) activation were i) abolished by pretreatment of CHO-h5-HT(1A) cells with pertussis toxin; ii) reversed by the selective 5-HT(1A) antagonist (N-[2-[4-(2-methoxy-phenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclohexane-carboxamide) fumarate (WAY100,635), and iii) absent in nontransfected CHO cell membranes. 5-HT isotherms could be modified by altering sodium concentration; only stimulatory actions were observed at 300mM NaCl, whereas only inhibitory actions were seen at 10 mM NaCl. Furthermore, bell-shaped isotherms were not detected at short incubation times, suggesting time-dependent changes in receptor/Galpha(i3) coupling. Taken together, these data show that low but not high concentrations of high-efficacy 5-HT(1A) agonists direct receptor signaling to Galpha(i3). In contrast, partial agonists favor h5-HT(1A) receptor signaling to Galpha(i3) over a wide concentration range, whereas inverse agonists inhibit constitutive Galpha(i3) activation.     

Inactivation of 5-HT(1A) receptors in hippocampal and cortical homogenates

5-HT(1A) receptor function can be assessed in rat hippocampal and cortical membrane preparations as agonist-stimulated [35S]GTPgammaS binding. Membranes were preincubated in vitro with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). R(+)-8-hydroxy-2-(di-n-propylamino)tetralin [R(+)-8-OH-DPAT]-stimulated [35S]GTPgammaS binding and [3H]8-OH-DPAT binding assays were used to assess 5-HT(1A) receptor function and density, respectively. EEDQ decreased both R(+)-8-OH-DPAT-stimulated [35S]GTPgammaS and [3H]8-OH-DPAT binding in hippocampal and cortical membranes. The E(max) but not the EC(50) of R(+)-8-OH-DPAT to stimulate [35S]GTPgammaS binding was decreased by EEDQ in both preparations. Additionally, the IC(50) for EEDQ to reduce R(+)-8-OH-DPAT-stimulated [35S]GTPgammaS and [3H]8-OH-DPAT binding was the same for both brain regions in both assays. In contrast to EEDQ alone, agonist-stimulated [35S]GTPgammaS binding was not reduced in hippocampal membranes preincubated with EEDQ and the 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl- cyclohexanecarboxamide maleate (WAY 100,635), suggesting that EEDQ acts directly on the receptor. Due to parallel reductions in receptor density and maximal functional response, it is concluded that there is little or no reserve for 5-HT(1A) receptor coupling to G(alpha) in these preparations. In addition, the sensitivity of hippocampal and cortical 5-HT(1A) receptors to inactivation by EEDQ in vitro is the same.     

h5-HT(1B) receptor-mediated constitutive Galphai3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT

1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3) activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Galpha(i3).     

Inverse agonism and constitutive activity as functional correlates of serotonin h5-HT(1B) receptor/G-protein stoichiometry

This study evaluated the influence of receptor/G-protein (R:G) stoichiometry on constitutive activity and the efficacy of agonists, partial agonists, and inverse agonists at human (h) 5-hydroxytryphamine 1B (5-HT(1B)) receptors. Two Chinese hamster ovary cell lines were used; they expressed 8.5 versus 0.4 pmol h5-HT(1B) receptors/mg (determined by [(3)H]GR125,743 saturation analysis) and 3.0 versus 1.5 pmol receptor-activated G-proteins/mg [determined by guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) isotopic dilution], respectively. Thus, they displayed R:G ratios of approximately 3.0 (RGhigh) and approximately 0.3 (RGlow), respectively. In competition-binding experiments, the agonists, 5-HT and sumatriptan, displayed fewer high-affinity (HA)-binding sites and the partial agonists, BMS181, 101 and L775,606, displayed decreased affinity in RGhigh versus RGlow membranes. In contrast, the inverse agonists, SB224,289 and, to a lesser extent, methiothepin, showed increased affinity. In G-protein activation experiments, both basal and 5-HT-activated [(35)S]GTPgammaS binding were higher in RGhigh than in RGlow membranes. Constitutive activity (determined by inhibition of basal [(35)S]GTPgammaS binding with GTPgammaS in the absence of receptor ligands) was more pronounced in RGhigh versus RGlow membranes, as revealed by the >5-fold greater proportion of HA sites. Correspondingly, the negative efficacy of inverse agonists was strikingly augmented, inasmuch as they suppressed approximately two-thirds of HA [(35)S]GTPgammaS binding in RGhigh membranes, but only approximately one-third in RGlow membranes. Furthermore, the efficacy of partial agonists was greater at RGhigh versus RGlow membranes, as estimated by their ability to enhance [(35)S]GTPgammaS binding. In conclusion, an increase in R:G ratios at h5-HT(1B) receptors was associated with an increase in relative efficacy of partial agonists and, most notably, an increase in both constitutive G-protein activation and negative efficacy of inverse agonists.     

5-HT(1A) receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

1. It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT(1A) receptors. 2. Saturation studies using [(3)H]-8-OH-DPAT and [(3)H]-MPPF revealed a single, high affinity site (K(D)approximately 1 nM) in HEK293 cells expressing human 5-HT(1A) receptors and rat cortex. In recombinant cells, [(3)H]-MPPF labelled 3 - 4 fold more sites than [(3)H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [(3)H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT(1A) receptors but did not bind to native tissue 5-HT(1A) receptors. These data suggest that, in transfected HEK293 cells, human 5-HT(1A) receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. 3. Receptor agonists inhibited [(3)H]-MPPF binding from recombinant 5-HT(1A) receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [(3)H]-8-OH-DPAT and [(3)H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [(3)H]-MPPF and [(3)H]-8-OH-DPAT in a monophasic manner. 4. Functional evaluation of compounds, using [(35)S]-GTPgammaS binding, produced a range of intrinsic activities from full agonism, displayed by 5-HT and 5-CT to inverse agonism displayed by spiperone. 5. [(3)H]-8-OH-DPAT : [(3)H]-MPPF pK(i) difference correlated well with functional intrinsic activity (r=0.86) as did [(3)H]-8-OH-DPAT : [(3)H]-spiperone pK(i) difference with functional intrinsic activity (r=0.96). 6. Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5-HT(1A) receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5-HT(1A) receptors in which only the high agonist affinity state was detectable.     

Regulation of 5-HT(1A) receptor-stimulated [35S]-GtpgammaS binding as measured by quantitative autoradiography following chronic agonist administration

1. Because changes 5-HT(1A) receptor number do not occur following repeated agonist treatment, we hypothesized that the basis for 5-HT(1A) receptor desensitization involves changes in receptor-G protein coupling. We measured the effect of repeated agonist administration on 5-HT(1A) receptor-stimulated [(35)S]-GTPgammaS binding in forebrain areas, (i.e. anterior cingulate cortex, lateral septum, hippocampus, entorhinal cortex), and serotonergic cell body areas, the dorsal and median raphe nuclei. 2. Following treatment of rats with (+/-)8-OH-DPAT (1 mg kg(-1), s.c.) for 7 or 14 days, 5-HT(1A) receptor-stimulated [(35)S]-GTPgammaS binding was significantly attenuated in both the dorsal and median raphe nuclei. 3. 5-HT(1A) receptor-stimulated [(35)S]-GTPgammaS binding was significantly attenuated in the CA(1) region of the hippocampus after 7, but not 14 days of 8-OH-DPAT administration. 5-HT(1A) receptor-stimulated [(35)S]-GTPgammaS binding was not altered in other forebrain areas examined. 4. The binding of [(3)H]-MPPF to 5-HT(1A) receptor sites was not altered in any brain region examined following repeated agonist administration, suggesting that the observed changes in (+/-)8-OH-DPAT-stimulated [(35)S]-GTPgammaS binding were not due to changes in 5-HT(1A) receptor number. 5. Our data indicate that in serotonergic cell body areas the regulation of presynaptic 5-HT(1A) receptor function following repeated agonist administration occurs at the level of receptor-G protein interaction. In forebrain areas, however, the regulation of postsynaptic 5-HT(1A) receptor sensitivity appears not to be at the level of receptor-G protein coupling.     

The 5HT(1A) receptor ligand, S15535, antagonises G-protein activation: a [35S]GTPgammaS and [3H]S15535 autoradiography study

4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) is a highly selective ligand at 5-HT(1A) receptors. The present study compared its autoradiographic labelling of rat brain sections with its functional actions, visualised by guanylyl-5'-[gamma-thio]-triphosphate ([35S]GTPgammaS) autoradiography, which affords a measure of G-protein activation. [3H]S15535 binding was highest in hippocampus, frontal cortex, entorhinal cortex, lateral septum, interpeduncular nucleus and dorsal raphe, consistent with specific labelling of 5-HT(1A) receptors. In functional studies, S15535 (10 microM) did not markedly stimulate G-protein activation in any brain region, but abolished the activation induced by the selective 5-HT(1A) agonist, (+)-8-hydroxy-dipropyl-aminotetralin ((+)-8-OH-DPAT, 1 microM), in structures enriched in [3H]S15535 labelling. S15535 did not block 5-HT-stimulated activation in caudate nucleus or substantia nigra, regions where (+)-8-OH-DPAT was ineffective and [3H]S15535 binding was absent. Interestingly, S15535 attenuated (+)-8-OH-DPAT and 5-HT-stimulated G-protein activation in dorsal raphe, a region in which S15535 is known to exhibit agonist properties in vivo [Lejeune, F., Millan, M.J., 1998. Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)(1A) receptors: WAY100,635-reversible actions of the highly selective ligands, flesinoxan and S15535. Synapse 30, 172-180.].The present data show that (i) [3H]S15535 labels pre- and post-synaptic populations of 5-HT(1A) sites in rat brain sections, (ii) S15535 exhibits antagonist properties at post-synaptic 5-HT(1A) receptors in corticolimbic regions, and (iii) S15535 also attenuates agonist-stimulated G-protein activation at raphe-localised 5-HT(1A) receptors.     

A robust and high-capacity [(35)S]GTPgammaS binding assay for determining antagonist and inverse agonist pharmacological parameters of histamine H(3) receptor ligands

Guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays were established and utilized as a reliable and high-capacity functional assay for determining antagonist and inverse agonist pharmacological parameters of novel histamine H(3) ligands, at the recombinant human H(3) receptor. [(35)S]GTPgammaS binding assays were performed with membranes prepared from human embryonic kidney 293 cells stably expressing the full-length (445 amino acids) human H(3) receptor isoform, at approximately 1 pmol/mg of protein. Utilizing robotic liquid handling, assay filtration, and scintillation counting in a 96-well format, concentration-response curves were determined for up to 40 compounds per assay. The imidazole-containing H(3) receptor antagonist ciproxifan and the non-imidazole antagonist ABT-239 inhibited (R)-alpha-methylhistamine (RAMH)-stimulated [(35)S]GTPgammaS binding in a competitive manner, and negative logarithm of the dissociation equilibrium constant (pK(b)) values determined for nearly 200 structurally diverse H(3) antagonists were very similar to the respective negative logarithm of the equilibrium inhibition constant values from N-alpha-[(3)H]methylhistamine competition binding assays. H(3) antagonists also concentration-dependently decreased basal [(35)S]GTPgammaS binding, thereby displaying inverse agonism at the constitutively active H(3) receptor. At maximally effective concentrations, non-imidazole H(3) antagonists inhibited basal [(35)S]GTPgammaS binding by approximately 20%. For over 100 of these antagonists, negative logarithm of the 50% effective concentration values for inverse agonism were very similar to the respective pK(b) values. Both H(3) receptor agonist-dependent and -independent (constitutive) [(35)S]GTPgammaS binding were sensitive to changes in assay concentrations of sodium, magnesium, and the guanine nucleotide GDP; however, the potency of ABT-239 for inhibition of RAMH-stimulated [(35)S]GTPgammaS binding was not significantly affected. These robust and reliable [(35)S]GTPgammaS binding assays have become one of the important tools in our pharmacological analysis and development of novel histamine H(3) receptor antagonists/inverse agonists.     

Synthesis and molecular modeling of new 1-aryl-3-[4-arylpiperazin-1-yl]-1-propane derivatives with high affinity at the serotonin transporter and at 5-HT(1A) receptors

It has been proposed that 5-HT(1A) receptor antagonists augment the antidepressant efficacy of selective serotonin (5-HT) reuptake inhibitors. In a search toward new and efficient antidepressants, 1-(aryl)-3-[4-arylpiperazin-1-yl]-1-propane molecular hybrids were designed, synthesized, and evaluated for 5-HT reuptake inhibition and 5-HT(1A) receptor affinity. The design was based in coupling structural moieties related to inhibition of serotonin reuptake, such as benzo[b]thiophene derivatives to arylpiperazines, typical 5-HT(1A) receptor ligands. In binding studies, several compounds showed affinity at the 5-HT transporter and at 5-HT(1A) receptors. Molecular modeling studies predicted the pharmacophore elements required for high affinity binding and the features that enable to discriminate between agonist, partial agonist, or antagonist action at 5-HT(1A) receptors and 5-HT transporter inhibition. Solvent interactions in desolvation prior to the binding step along with enthalpy and enthropy compensations might be responsible to explain agonist, partial agonist, and antagonist character. Hydrogen-bonding capability seems to be important to break hydrogen interhelical hydrogen bonds or alternatively to form other bonds upon ligand binding. Partial agonists and antagonists are unable to do this as the full agonist, which interacts closely by long-range forces or directly. The compounds showing the higher affinity at both the 5-HT transporter (K(i) < 50 nM) and the 5-HT(1A) receptors (K(i) < 20 nM) were further explored for their ability to stimulate [(35)S]GTPgammaS binding or to antagonize 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT)-stimulated [(35)]GTPgammaS binding to rat hippocampal membranes, an index of agonist/antagonist action at 5-HT(1A) receptors, respectively. Compound 8g exhibited agonist activity (EC(50) = 30 nM) in this assay, whereas compounds 7g and 8h,i behaved as weak partial agonists and 7h-j and 8j,l antagonized the R(+)-8-OH-DPAT-stimulated GTPgammaS binding. Functional characterization was performed by measuring the antagonism to 8-OH-DPAT-induced hypothermia in mice.     

Mechanisms of G protein activation via the D2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation

BACKGROUND AND PURPOSE: The aim of this report is to study mechanisms of G protein activation by agonists. EXPERIMENTAL APPROACH: The association and dissociation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D(2short) receptor was studied in the presence of a range of agonists. KEY RESULTS: Binding of [(35)S]GTPgammaS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D(2) receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [(35)S]GTPgammaS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [(35)S]GTPgammaS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [(35)S]GTPgammaS binding was reduced with longer association times and increased [(35)S]GTPgammaS concentrations. CONCLUSIONS AND IMPLICATIONS: These data are consistent with [(35)S]GTPgammaS binding being initially to receptor-linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [(35)S]GTPgammaS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.     

Constitutive activity at serotonin 5-HT(1D) receptors: detection by homologous GTPgammaS versus [(35)S]-GTPgammaS binding isotherms

Although many G-protein-coupled receptors (GPCRs) may display constitutive activity, their detection has, to date, depended on the use of inverse agonists. The present study exploited a novel procedure to investigate constitutive activity at recombinant human (h) serotonin (5-HT) 5-HT(1D) receptors stably expressed in Chinese hamster ovary (CHO) cells. 5-HT modestly stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]-GTPgammaS) binding to CHO-h5-HT(1D) membranes whereas methiothepin and the 5-HT(1B/1D)-selective ligand, SB224,289, exerted robust inhibition of basal [(35)S]-GTPgammaS binding (inverse agonism). These actions were specific inasmuch as they were reversed by the novel, selective 5-HT(1B/1D) ligand, S18127. Constitutive activity was investigated by homologous inhibition of [(35)S]-GTPgammaS binding to CHO-h5-HT(1D) membranes with unlabelled GTPgammaS. Under 'basal' conditions (absence of receptor ligand), biphasic isotherms were observed. Most (80%) [(35)S]-GTPgammaS binding sites were in the high affinity (HA) versus low affinity (LA) component of the isotherms. HA binding was augmented by 5-HT (to 155%; relative to basal values=100%), but decreased by methiothepin (to 23%) and by SB224,289 (to 67%). In contrast, LA binding was not altered. Further, membranes of untransfected CHO cells exhibited only LA binding sites, indicating that the latter are not related to h5-HT(1D) receptor-G-protein coupling. Thus, at 5-HT(1D) receptors expressed in this CHO cell line, HA binding detected in homologous inhibition experiments (GTPgammaS versus [(35)S]-GTPgammaS) under basal conditions provides a measure of constitutive G-protein activation. Thus, it is suggested that for h5-HT(1D) receptors and, possibly, other GPCRs, inverse agonists will be detectable by [(35)S]-GTPgammaS binding if a HA component is present under basal conditions.     

The naturally occurring Arg219Leu variant of the human 5-HT1A receptor: impairment of signal transduction

The present study in transfected HEK293 cells aimed to investigate whether the pharmacological and/or transductional properties of the naturally occurring Arg219Leu variant (VAR) in the third intracellular loop of the h5-HT1A receptor differ from those of the wild-type receptor. Binding of [3H]8-hydroxy-2-(di-n-propylamino)tetraline ([H]8-OH-DPAT) and of [35S]GTPgammaS to membranes, as well as inhibition of forskolin-stimulated [3H]cAMP formation by 5-HT receptor agonists in whole cells, were estimated. The VAR and wild-type h5-HT1A receptors were found to be expressed at virtually identical densities. The VAR and wild-type receptors did also not differ with respect to the potencies of 5-HT receptor agonists and antagonists in inhibiting [3H]8-OH-DPAT binding. The ability of 5-HT to stimulate [35S]GTPgammaS binding (a measure of G protein coupling) to the VAR receptor and of the agonists 5-HT, buspirone and urapidil to inhibit forskolin-stimulated cAMP accumulation in HEK293 cells expressing the VAR receptor was decreased by 60-90%. In conclusion, the Arg219Leu variation of the human 5-HT1A receptor does not change the binding properties, but is associated with a drastic impairment of signal transduction. In patients carrying this variation, disturbances of 5-HT1A receptor-mediated functions and diminished responses to drugs acting via this receptor may occur.     

Labelling of recombinant human and native rat serotonin 5-HT1A receptors by a novel, selective radioligand, [3H]-S 15535: Definition of its binding profile using agonists, antagonists and inverse agonists

The novel benzodioxopiperazine, 5-HT_1A receptor weak partial agonist, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) bound with high affinity and selectivity to membranes of Chinese Hamster Ovary cells stably expressing the human (h) 5-HT_1A receptor (K_i = 0.6 nM versus [³H]-8-hydroxy-dipropylamino-tetralin, [³H]-8-OH-DPAT): its affinity at h5-HT_1A receptors was more than 70-fold higher than its affinity at > 50 other binding sites. S 15535 was tritiated to high specific activity (50 Ci/mmol) and its binding profile characterised. At 22° C, [³H]-S 15535 associated and dissociated from h5-HT_1A receptors with half-times of 2.9 and 5.0 min, respectively, yielding a K_d estimate of 3.6 nM. In saturation binding experiments, [³H]-S 15535 displayed a B_max value for h5-HT_1A receptors (1630 fmol/mg), higher than that obtained with the agonist [³H]-8-OH-DPAT (1023 pmol/mg). Guanylyl imidodiphosphate (GppNHp, 100 μM) reduced the binding of [³H]-S 15535 by only 25% compared with 79% for [³H]-8-OH-DPAT at h5-HT_1A receptors. [³H]-S 15535 also showed high affinity, saturable binding to rat hippocampal membranes (B_max = 820 fmol/mg versus 647 fmol/mg for [³H]-8-OH-DPAT). For both h5-HT_1A and rat 5-HT_1A receptors, the K_i values for competition binding of 15 serotonergic ligands with [³H]-S 15535 was highly correlated with that of [³H]-8-OH-DPAT. However, important differences were also observed. The agonist, 5-hydroxytryptamine (5-HT), displayed biphasic competition curves with [³H]-S 15535 but not with [³H]-8-OH-DPAT at h5-HT_1A receptors. Similarly, the ‘antagonists’, spiperone, methiothepin and (+)butaclamol, showed biphasic competition isotherms versus [³H]-S 15535 but not [³H]-8-OH-DPAT. When [³H]-S 15535 competition binding experiments were carried out in the presence of GppNHp (100 μM) the 5-HT and 8-OH-DPAT competition curves shifted to the right, whereas the spiperone and methiothepin competition curves shifted to the left. In contrast, in the presence of GppNHp, the competition isotherms for N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100,635) were not altered. Taken together, these data show that (i) [³H]-S 15535 is a highly selective 5-HT_1A receptor ligand which labels both G-protein-coupled and uncoupled 5-HT_1A receptors, (ii) antagonists, such as WAY 100,635, which yield monophasic isotherms in competition with both [³H]-agonists and [³H]-antagonists, are not sensitive to the G-protein coupling state of the receptor, but (iii) spiperone and methiothepin behaved as inverse agonists, their competition isotherms with [³H]-S 15535 being modulated in an opposite manner to those of agonists. Received: 12 September 1997 / Accepted: 1 December 1997     

Modulation of 5-HT(1A) receptor activation by its interaction with wild-type and mutant g(alphai3) proteins

Constitutive and agonist-dependent activation of the recombinant human 5-HT(1A) receptor (RC: 2.1.5HT.01A) was investigated by co-expression with a rat G(alphai3) protein in Cos-7 cells. The interaction between the 5-HT(1A) receptor and rat G(alphai3) protein was modulated by substitution of the G(alphai3) protein site for pertussis toxin-catalysed ADP-ribosylation (cysteine(351)) by each of the natural amino acids. Enhanced basal [(35)S]GTPgammaS binding responses (+24 to +189%) were observed with the mutant G(alphai3) proteins containing at position 351 either a histidine, glutamine, serine, tyrosine or a nonpolar amino acid with the exception of a proline. With each of these mutant G(alphai3) proteins, spiperone (10 microM), but not WAY 100635 (10 microM), reduced (-22 to -60%, p<0.05) the enhanced basal [(35)S]GTPgammaS binding response. 5-HT (10 microM)-mediated [(35)S]GTPgammaS binding responses attained for some of the mutant G(alphai3)Cys(351) proteins (Phe, Met, Val and Ala) more than 300% of that obtained with the wt G(alphai3) protein. Similar results were also obtained with the prototypical 5-HT(1A) agonist 8-OH-DPAT and the partial agonist (-)-pindolol. Fusion proteins assembled from the 5-HT(1A) receptor and either the wt G(alphai3)Cys(351), mutant G(alphai3)Cys(351)Gly or G(alphai3)Cys(351)Ile protein displayed similar observations for these ligands as obtained by co-expression of the 5-HT(1A) receptor with each of these G(alphai3) proteins. Both the degree of 5-HT(1A) receptor activation by 8-OH-DPAT and (-)-pindolol, and its inhibition by spiperone, strongly correlate (r(2): 0.78-0.81) with the octanol/water partition coefficients of the mutated amino acid at position 351 of the G(alphai3) protein. The present data also suggest the wt G(alphai3) protein does not result in maximal activation of the 5-HT(1A) receptor by the agonists being investigated.     

Reciprocal regulation of agonist and inverse agonist signaling efficacy upon short-term treatment of the human delta-opioid receptor with an inverse agonist

Rapid regulation of receptor signaling by agonist ligands is widely accepted, whereas short-term adaptation to inverse agonists has been little documented. In the present study, guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding and cAMP accumulation assays were used to assess the consequences of 30-min exposure to the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174864) (1 microM) on delta-opioid receptor signaling efficacy. ICI174864 pretreatment increased maximal effect (E(max)) for the partial agonist Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP) at the two levels of the signaling cascade, whereas E(max) values for more efficacious agonists like (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) and bremazocine were increased in [(35)S]GTPgammaS binding but not in cAMP accumulation assays. Pre-exposure to ICI174864 also induced a shift to the left in dose-response curves for bremazocine and TIPP. On the other hand, E(max) for the inverse agonist H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH was reduced in both assays, but no changes in potency were observed. For the weaker inverse agonist naloxone, E(max) in [(35)S]GTPgammaS binding was drastically modified because the drug turned from inverse agonist to agonist after ICI174864 pretreatment. Likewise, ICI174864 turned from inverse agonist to agonist when tested in cAMP accumulation assays. In both cases, inversion of efficacy was concomitant with marked increase in potency for agonist effects. Together with functional changes, short-term treatment with ICI174864 reduced basal receptor phosphorylation and increased immunoreactivity for Galpha(i3) in membrane preparations. Functional consequences of ICI174864 pretreatment were simulated in the cubic ternary complex model by increasing receptor/G protein coupling or G protein amount available for interaction with the receptor. Taken together, these data show that inverse agonists may induce rapid regulation in receptor signaling efficacy.     

S 14506: A novel,potent, high-efficacy 5-HT1A agonist and potential anxiolytic agent

The studies characterized the newly discovered 7-methoxy naphtylpiperazine S 14506 and examined the possible relationship between the apparent in vivo efficacy of 5-HT_1A ligands and the magnitude of their anxiolytic potential. In vitro radioligand binding studies indicated S 14506 to bind with high affinity and in an apparently competitive manner to 5-HT_1A sites, while having much less affinity for other 5-HT binding sites. S 14506 demonstrated exceptional anxiolytic potential in the pigeon conflict procedure; the magnitude of its effects exceeded that of the other 5-HT_1A ligands 8-hydroxy-2-(n-propylamino) tetralin (8-OH-DPAT), (+)-flesinoxan, buspirone, and BMY 7378. The anticonflict action of S 14506 was antagonized by the 5-HT_1A antagonist (–)-alprenolol. Like the prototypical 5-HT_1A agonist 8-OH-DPAT, S 14506 induced forepaw treading, hypothermia and spontaneous tail flicks, antagonized morphine-induced analgesia, and produced stimulus generalization in rats discriminating 8-OH-DPAT from saline. The apparent in vivo efficacy of the 5-HT_1A ligands in stimulating corticosterone release varied as follows: S 14506 > 8-OH-DPAT > (+)-flesinoxan > buspirone > BMY 7378. This rank order corresponded with the rank order of magnitude of the anxiolytic action which the same compounds produced in the pigeon conflict procedure. The rank order of in vivo potency of these compounds in producing 5-HT_1A agonist actions in rodents. (i.e., S 14506) > 8-OH-DPAT > (+)-flesinoxan > bus-pirone > BMY 7378) also corresponded with their rank order of potency in producing anticonflict effects. The data characterize S 14506 as an extremely potent, orally active, high-efficacy agonist at 5-HTl_1A receptors that demonstrates exceptional anxiolytic potential in the pigeon conflict procedure. The results provide strong evidence that the activation of central nervous system (CNS) 5-HT_1A receptors yields marked anticonflict effects and that the anxiolytic potential of 5-HT_1A ligands is proportional to their efficacy at 5-HT_1A re-ceptors. © 1992 Wiley-Liss, Inc.