WAY-100635 has high selectivity for serotonin 5-HT1A versus dopamine D4 receptors
Jean-Claude Martel a,⁎, Nathalie Leduc a, Anne-Marie Ormière a, Valérie Faucillon a,
Nathalie Danty a, Charlène Culie a, Didier Cussac b, Adrian Newman-Tancredi a
a Division of Neurobiology 2 Centre de recherche Pierre Fabre, 17 avenue Jean Moulin, 81106 Castres cedex, France
b Cellular and Molecular Biology Department Centre de recherche Pierre Fabre, 17 avenue Jean Moulin, 81106 Castres cedex, France
Received 26 April 2007; received in revised form 18 June 2007; accepted 4 July 2007
Available online 13 July 2007
Abstract
The serotonin 5-HT1A receptor antagonist WAY-100635 was recently reported to have potent agonist properties at dopamine D4 receptors (Chemel et al., 2006, Psychopharmacology 188, 244–251.). Herein WAY-100635 (pKi at human (h) serotonin 5-HT1A receptors= 9.51; pKi at dopamine hD4.4 receptors= 7.42) stimulated [35S]GTPγS incorporation in membranes of Chinese Hamster Ovary cells expressing dopamine hD4.4 receptors with only moderate potency and modest efficacy (pEC50 = 6.63; Emax = 19% of dopamine). Moreover, in antagonism experiments, WAY-100635 had a much lower potency at dopamine hD4.4 receptors (pKB = 7.09), than at serotonin h5-HT1A receptors (pKB = 9.47). These data demonstrate that WAY-100635 has high selectivity for serotonin h5-HT1A versus dopamine hD4.4 receptors.
Keywords: WAY-100635; Serotonin 5-HT1A receptor; Dopamine D4 receptor
1. Introduction
Serotonin 5-HT1A receptors are key targets in the control of neuropsychiatric disorders including depression (Blier and Ward, 2003) and negative symptoms of schizophrenia (Bantick et al., 2001; Carli et al., 2006). Numerous ligands have been developed as tools to investigate the role of these receptors. WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride) is a selective seroto- nin 5-HT1A receptor antagonist that has been extensively pharma- cologically characterized (Fletcher et al., 1996; Forster et al., 1995). In receptor binding assays, WAY-100635 shows high selectivity for serotonin 5-HT1A receptors with 100 fold lower affinities for other serotoninergic, dopaminergic and adrenergic receptors (Fletcher et al., 1996). Over the past decade, WAY- 100635 has been extensively used to validate serotonin 5-HT1A receptors effects in both in vitro and in vivo models. The high selectivity of this compound for serotonin 5-HT1A receptors has also favoured the development of Positron Electron Tomography (PET) analogues of WAY-100635 for studying the role of serotonin 5-HT1A receptors in primates and humans (Cliffe, 2000). However, recent data have challenged the selectivity of WAY-100635 and suggest that this compound, and one of its main metabolite in primates (WAY-100634; descyclohexanecarbonyl- WAY-100635), may also be potent and efficacious dopamine D4 agonists (Chemel et al., 2006). Dopamine D4 receptors are mainly expressed in limbic brain structures such as prefrontal cortex, hippocampus and amygdala, and these receptors may have a key role in modulating cognitive processes (Browman et al., 2005; Van Tol et al., 1991).
In the present work, we evaluated the pharmacological prop- erties of WAY-100635 at dopamine hD4 receptors in comparison with those observed at serotonin h5-HT1A receptors. In contrast to the study of Chemel et al. (2006), the present data indicate that WAY-100635 has only modest agonist potency at dopamine hD4 receptors, and support the assertion that WAY-100635 is a highly selective serotonin 5-HT1A receptor antagonist.
2. Materials and methods
2.1. Materials
All reagents used were from Sigma-Aldrich and were of the highest purity available. Binding and functional assays were performed in 96 well plates using membranes of Chinese Hamster Ovary (CHO) cells (Perkin-Elmer, Courtaboeuf, France) expressing high levels of human (h) serotonin 5-HT1A, or dopamine hD1, hD2S, or hD4.4 receptors as described previously (Newman-Tancredi et al., 2007) while CHO cells expressing dopamine hD3 receptors were from INSERM (U-573, Paris). Radioligands were all from Amersham ([3H]SCH23390: [3H](d)- 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H- 3-benzazepine; [3H]spiperone: 8-[4-(p-fluorophenyl)-4-oxol [2,3(n)-3H]butyl]-1-[4-3H]phenyl 1,3,8-triazaspiro[4,5]decan-4-one); [3H]8-OH-DPAT: (±)2-(N,N-Di[2,3(n)-3H]propyla-mino)-8-hydroxy-1,2,3,4-
tetrahydronaphthalene; [35S]GTPγS: Guanosine 5″-[γ-35S]thiotriphosphate; and [methoxy-3H] WAY-100635).
2.2. Receptor binding assays
Briefly, binding was performed by a 2 hour incubation with either 1 nM [3H]8-OH-DPAT (serotonin h5-HT1A receptors), 1 nM [3H]SCH23390 (dopamine hD1 receptors), or 0.6 nM [3H] spiperone (dopamine hD2S, hD3 or hD4.4 receptors) in 20 mM HEPES buffer containing salts (120 mM NaCl, 5 mM MgCl2 and KCl), 1 mM EDTA and WAY-100635 (11 concentrations) ranging from 10− 11 to 10− 5 M in semi-log intervals. Incubations on dopamine receptors were performed at 37 °C, while binding at serotonin h5-HT1A receptors was performed at room temper- ature, as these conditions allowed attainment of binding equilib- rium. Radioligand affinities (KD, in nM) and receptors densities (Bmax, in pmol/mg protein) were as follows (KD/Bmax): ([3H]8-receptors): 0.68/2.13; ([3H] 6.63 ± 0.08/100 ± 2% (3); WAY-100635: 6.83 ± 0.23/20 ± 1% (3) on dopamine hD4.4 receptors, WAY-100635 having a pKB of 7.09 ± 0.11 (3) when competing SCH23390; dopamine hD1 receptors):0.80/1.05; ([ H]spiper- one; dopamine hD2S receptors): 0.06/11.6 ([3H]spiperone; dopamine hD3 receptors): 0.09/12; ([3H]spiperone; dopamine hD4.4 receptors): 0.14/0.9. pKi values (=−Log[Ki]) were derived from competition binding isotherms using the Cheng–Prusoff equation (Lazareno and Birdsall, 1993): Ki = IC50 / (1 +[Ligand]/ KDLigand) where IC50 was the concentration of WAY-100635 necessary to displace 50% of the ligand specific binding, [Ligand] was the concentration of the radioligand and KDLigand was the affinity of the radioligand for the appropriate receptor.
Fig. 1. Effects of WAY-100635 (■), dopamine (●) or WAY-100635 together with 1 μM dopamine (○) on [35S]GTPγS binding to membranes of CHO cells expressing dopamine hD4.4 receptors (A), and of WAY-100635 (■), serotonin (5-HT; ) or WAY-100635 together with 30 nM 5-HT ( ) on [35S]GTPγS binding to membranes of CHO cells expressing serotonin h5-HT1A receptors (B). These data were obtained under standard [35S]GTPγS assay condition (HEPES buffer with 100 mM NaCl). Values derived from these experiments are (pEC50/Emax; mean±SEM of (N) independent determinations): (A) dopamine: OH-DPAT; serotonin h5-HT1A.
2.3. Functional assays
Functional assays were performed on CHO cell lines ex- pressing either serotonin h5-HT1A or dopamine hD4.4 receptors in 20 mM HEPES buffer containing either 30 or 100 mM NaCl, 3 mM MgCl2 and 3 mM GDP as previously described (Newman-Tancredi et al., 2007). Low sodium (30 mM) was used for antagonism experiments with L-745,870 as it improved detection of WAY-100635 partial agonism (higher relative efficacy compared with dopamine; compare Figs. 1A and 2A).
Antagonists were preincubated for 30 min at room temper- ature before addition of agonist and 0.1 nM [35S]GTPγS, and incubation was continued for another 30 min at room temperature. pKB were derived from inhibition experiments using the Cheng–Prusoff equation: KB =IC50 / (1 + [Agonist]/ EC50 Agonist) where IC50 was the concentration of WAY- 100635 necessary to inhibit 50% of the response, [Agonist] was the concentration of the agonist used and EC50 Agonist was the potency of the agonist on the receptor system. pKB estimates from curve shift experiments using a fixed concentration of L-745,870 were calculated using the Gaddum equation (Lazareno and Birdsall, 1993): KB = [Antagonist] / ((EC50′ / EC50) − 1) where [Antagonist] is the concentration of antagonist, EC50′ and EC50 are respectively the agonist EC50 in the presence or absence of the antagonist. The agonism and antagonism assays were performed in parallel on the same membrane preparation.All assays (binding and functional) were stopped by rapid filtration, followed by 3 rapid rinses with cold (4 °C) buffer and radioactivity on the filter was measured by scintillation counting.
Fig. 2. Effects of WAY-100635 (■) or dopamine (●) on [35S]GTPγS binding to membranes of CHO cells expressing dopamine hD4.4 receptors in an assay condition favouring detection of partial agonism (HEPES with 30 mM NaCl) (A). Graph B shows the inhibition of a maximally effective concentration of WAY-100635 (10 μM; ▽) by increasing concentrations of the selective dopamine D4 antagonist L-745,870, this compound having no effect by itself (▼). In this assay, concentration–response curve to WAY-100635 (■) is shifted to the right in a parallel manner with a fixed concentration (100 nM) of L-745,870 (△) (C), this phenomenon being typical of competitive antagonism. Values derived from these experiments are (pEC50/Emax; mean±SEM of (N) independent determinations, unless stated otherwise): (A) dopamine: 7.04 ± 0.11/101 ± 2% (5); WAY-100635: 7.01 ± 0.20/33 ± 3% (6) on dopamine hD4.4 receptors, (B) L-745,870 had a pKB of 9.22 ± 0.28 (N = 3) when competing against 10 μM WAY-100635, (C) WAY- 100635 + 100 nM L-745,870: 5.17 ± 0.10/35 ± 2% (3), leading to an estimated pKB of 8.83 for L-745,870.
3. Results
In binding experiments, WAY-100635 showed high affinity for serotonin h5-HT1A receptors (pKi = 9.51 ± 0.06), while having low to moderate affinities at dopamine hD1, hD2s, hD3 and hD4.4 receptors with pKi of b 5, 6.67 ± 0.03, 6.98 ± 0.04 and 7.42 ± 0.08, respectively (N = 3 independent determination/ receptor). A Ki ratio was calculated, comparing WAY-100635 affinities at dopamine receptors with that at serotonin h5-HT1A receptors, this ratio being N 30,000 (dopamine hD1 receptors), 691 (dopamine hD2s receptors), 339 (dopamine hD3 receptors) and 123 (dopamine hD4.4 receptors).
In standard functional assay conditions with HEPES buffer containing 100 mM sodium, WAY-100635 showed weak partial agonism at dopamine hD4.4 receptors as revealed by its ability to increase [35S]GTPγS binding above basal (pEC50 = 6.63 and Emax = 20% of dopamine; Fig. 1A). In these assay conditions, WAY-100635 partially antagonised the effect of a submaxi- mally-effective concentration of dopamine (1 μM) down to an Emax of 23%, consistent with partial agonism at dopamine hD4.4 (Fig. 1A). The pKB (7.09) derived from antagonism of 1 μM dopamine by WAY-100635 in this system is consistent with the binding affinity at dopamine hD4.4 receptors reported above (pKi = 7.42). On the other hand, WAY-100635 showed neutral antagonism at serotonin h5-HT1A receptors (Fig. 1B) with a pKB of 9.47, this value being consistent with its binding affinity at serotonin h5-HT1A (pKi = 9.51). The KB ratio calculated from the potency of WAY-100635 for blockade of dopamine hD4.4 versus serotonin h5-HT1A receptors was superior by more than two orders of magnitude (240 fold) in favour of antagonism at serotonin h5-HT1A receptors.
In order to further examine receptor agonist properties of WAY-100635 on [35S]GTPγS binding to dopamine hD4.4-ex- pressing CHO cell membranes, sodium concentrations were reduced to 30 mM (Fig. 2A), a modification that favours de- tection of partial agonism (Newman-Tancredi et al., 2007). Under these conditions, WAY-100635 stimulated [35S]GTPγS incorporation with a potency of 7.01 and an Emax of 33% of dopamine. The effect of a maximally effective concentration of WAY-100635 (10 mM) could be reversed with the selective dopamine D4 receptor antagonist L-745,870 in a concentration- dependent manner (Fig. 2B). The calculated pKB for this inhibition is 9.22 ± 0.28 (N = 3), a value consistent with the binding affinity of L-745,870 at dopamine hD4.4 receptors (pKi = 9.15 ± 0.03, N = 3). Furthermore, a fixed concentration of L-745,870 (100 nM) shifted the concentration–response curve of WAY-100635 to the right in a parallel manner with an estimated pKB of 8.83 (Fig. 2C).
4. Discussion
WAY-100635 has been extensively used as a selective serotonin 5-HT1A receptor antagonist in both in vitro and in vivo experiments. The recent finding by Chemel et al. suggests that WAY-100635 is also a potent dopamine D4 receptor agonist, and has raised the question of the selectivity of this drug for serotonin 5-HT1A receptors (Chemel et al., 2006). We compared affinities and potencies of WAY-100635 in a set of CHO cell lines expressing human dopamine receptor subtypes (hD1, hD2S, hD3 or hD4.4) to data obtained with serotonin h5-HT1A receptors. WAY-100635 acts as a low efficacy partial receptor agonist at dopamine hD4.4 receptors while being a highly potent antagonist at serotonin h5-HT1A receptors. The present comparison of the pharmacological properties of WAY-100635 at dopamine hD4.4 and serotonin h5-HT1A receptors suggest that the properties of this drug at dopamine hD4.4 receptors are likely to be of minor significance for two reasons: 1) the receptor binding assays confirmed that WAY-100635 has a much lower affinity at dopamine hD4.4 (pKi = 7.42) than at serotonin h5-HT1A receptors (pKi = 9.51), and 2) in agreement with binding data, the relative potencies of WAY-100635 at antagonising endogenous receptor agonist effects on [35S]GTPγS binding at dopamine hD4.4 and serotonin h5-HT1A receptors demonstrated a greater than 200 fold selectivity for antagonism of serotonin h5-HT1A receptors. Thus, these pharmacological data confirm the high selectivity of WAY-100635 for serotonin h5-HT1A receptors.
One major argument of the study of Chemel et al. (2006) was that WAY-100635 exhibited only a 10 fold selectivity for serotonin h5-HT1A versus dopamine hD4 receptors. The present study did not confirm this finding and is more consistent with the affinity data reported in the literature (Fletcher et al., 1996; Forster et al., 1995; Khawaja et al., 1997; Newman-Tancredi et al., 1996). Indeed there are some inconsistencies in the study of Chemel et al. (2006) between the Ki obtained for WAY-100635 in competition binding experiments performed on serotonin h5-HT1A expressing CHO cell membranes (2.2 nM corresponding to a pKi of 8.7) and the affinity obtained on the same cell line using [3H]WAY-100635 saturation binding experiment (KD = 0.28 nM corresponding to a pKD of 9.6). This latter value is much closer to the values reported herein and in the literature, and is equivalent to the value that we obtained in saturation binding assays performed with [Methoxy-3H]WAY- 100635 on CHO cell membranes expressing serotonin h5-HT1A receptors (KD = 0.14 ± 0.02 nM; N = 4). On the other hand, unlike Chemel et al. (2006), concentrations of [3H]WAY-100635 from 0.4 to 25 nM did not yield specific binding to membranes of CHO cells expressing dopamine hD4.4 receptors, consistent with low affinity of WAY-100635 for this receptor. In that regard, it should be noted that Chemel et al. (2006) used a different cell line: human embryonic kidney (HEK) cells for expressing high levels of dopamine hD4.2 receptors. One possibility is that the CHO and HEK have different basal levels of G-protein coupling to dopamine hD4 receptors. Indeed, the inhibition data on Fig. 4 of Chemel et al. (2006) suggest that HEK cells expressing dopamine hD4.2 receptors have strong G-protein coupling to these receptors, as revealed by increase in forskolin-induced cAMP production above basal when dopamine D4 antagonists were present (suggesting the presence of constitutive activity). Furthermore, the CHO cells used here expressed approximately ten fold lower levels of dopamine hD4.4 receptors, and exhibited no evidence of constitutive activity. Thus, binding experiments of WAY-100635 on HEK cell membranes expressing high levels of tightly coupled dopamine hD4 receptors may accentuate the apparent affinity of this compound at dopamine D4 receptors.
The marked differences in efficacy measures detected on dopamine hD4 receptors between the present study and that of Chemel et al. (2006) may relate to the discrepancy between the levels of dopamine hD4 receptors expression as men- tioned above. Detection of partial agonism is highly dependent on receptor expression levels, the presence of high receptor to G-protein stoichiometry leading to increase efficacy of partial agonists (Newman-Tancredi et al., 2000). Furthermore, efficacy differences may also relate to the assay system used, as mea- surement of cAMP production is less suitable for different- iating partial from full agonists, most likely due to intracellular signal amplification (Niedernberg et al., 2003). On the other hand, [35S]GTPγS binding assay represents a measure closer to receptor agonist interaction and is less subject to signal ampli- fication, rendering it better able to discriminate partial recep- tor agonist properties (Harrison and Traynor, 2003; Milligan, 2003). Finally, partial agonism may be affected by the expres- sion system, each cell line having a particular G-protein make- up (Newman-Tancredi, 2003). Thus, the use of a different dopamine hD4 receptors-expressing cell line between the present study (CHO) and that of Chemel et al. (2006) (HEK), and different functional assay may have contributed to the reported efficacy differences.
Another issue discussed by Chemel et al. (2006) concerns WAY-100634, the main metabolite of WAY-100635 in primates. This metabolite was reported to be more potent than WAY- 100635 for inhibition of forskolin-induced cAMP formation in HEK cells expressing dopamine hD4.4 receptors, albeit with a lower efficacy. While this observation may be of importance to interpret clinical or pre-clinical data performed in human or primates with pharmacological doses of WAY-100635, this should not concern most pre-clinical studies performed in rodents as this metabolite was not detected in the plasma of rats 30 min following acute administration of [O-methyl-11C]WAY- 100635 (Pike et al., 1994). At that time more than 80% of radioactivities present in plasma were other metabolites of [O- methyl-11C]WAY-100635, this contrasting with cerebrum where 80% of radioactivity was still the original ligand (Pike et al., 1994). Thus, peripheral metabolism of WAY-100635 does not lead to WAY-100634 in rat plasma and the compound is poorly metabolised in rat brain, both observations suggesting that a central effect of WAY-100634 in rat is unlikely. In primate plasma, [O-methyl-11C]WAY-100634 is rapidly detected fol- lowing administration of [O-methyl-11C]WAY-100635, and this metabolite readily accesses the brain (Osman et al., 1996). As discussed by Chemel et al. (2006), this issue was addressed in PET studies by the development of [Carbonyl 11C]WAY- 100635 which does not lead to radiolabelled WAY-100634 (Osman et al., 1998).
In summary, the present data show clear pharmacological differences between the high potency of WAY-100635 for blocking serotonin 5-HT1A receptors and a much lower potency for interacting with dopamine D4 receptors. Our data also show that WAY-100635 has modest potency and efficacy for activating dopamine D4 receptors. The high selectivity of WAY-100635 for serotonin 5-HT1A receptors suggests that this compound should block serotonin 5-HT1A receptor mediated responses at much lower doses or concentrations than those at which it would have an influence on dopamine D4 recep- tors. Thus, at appropriate doses or concentrations, WAY-100635 shows a sufficient degree of selectivity to specifically anta- gonise serotonin 5-HT1A receptors.
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