Abstract
The novel, potential antipsychotic, S33138 (N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenylacetamide), displayed ∼25-fold higher affinity at human (h) dopamine D3 versus hD2L (long isoform) and hD2S (short isoform) receptors (pKi values, 8.7, 7.1, and 7.3, respectively). Conversely, haloperidol, clozapine, olanzapine, and risperidone displayed similar affinities for hD3, hD2L, and hD2S sites. In guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]-GTPγS) filtration assays, S33138 showed potent, pure, and competitive antagonist properties at hD3 receptors, displaying pKB and pA2 values of 8.9 and 8.7, respectively. Higher concentrations were required to block hD2L and hD2S receptors. Preferential antagonist properties of S33138 at hD3 versus hD2L receptors were underpinned in antibody capture/scintillation proximity assays (SPAs) of Gαi3 recruitment and in measures of extracellular-regulated kinase phosphorylation. In addition, in cells cotransfected with hD3 and hD2L receptors that assemble into heterodimers, S33138 blocked (pKB, 8.5) the inhibitory influence of quinpirole upon forskolin-stimulated cAMP formation. S33138 had low affinity for hD4 receptors (<5.0) but revealed weak antagonist activity at hD1 receptors (Gαs/SPA, pKB, 6.3) and hD5 sites (adenylyl cyclase, 6.5). Modest antagonist properties were also seen at human serotonin (5-HT)2A receptors (Gαq/SPA, pKB, 6.8, and inositol formation, 6.9) and at 5-HT7 receptors (adenylyl cyclase, pKB, 7.1). In addition, S33138 antagonized hα2C adrenoceptors ([35S]GTPγS, 7.2; Gαi3/SPA, 6.9; Gαo/SPA, 7.3, and extracellular-regulated-kinase, 7.1) but not hα2A or hα2B adrenoceptors (<5.0). Finally, in contrast to haloperidol, clozapine, olanzapine, and risperidone, S33138 displayed negligible affinities for multiple subtypes of α1-adrenoceptor, muscarinic, and histamine receptor. In conclusion, S33138 possesses a distinctive receptor-binding profile and behaves, in contrast to clinically available antipsychotics, as a preferential antagonist at hD3 versus hD2 receptors.
Schizophrenia is a complex, progressive, and debilitating disorder of early onset that afflicts approximately 1% of the population. It is characterized by disorganized thought and a constellation of symptoms generally classified as positive (delusions and hallucinations), negative (social withdrawal, blunted affect, and mutism), and cognitive (deficits in attention, working and verbal memory, social cognition, and executive function) (Kapur and Mamo, 2003; Meltzer, 2004; Lieberman et al., 2005).
Conventional neuroleptics like haloperidol, which behaves principally as a dopaminergic antagonist, moderate positive symptoms. However, some 20 to 30% of patients are resistant, and efficacy against negative and cognitive symptoms is limited. Furthermore, there is only a modest therapeutic window to doses provoking extrapyramidal motor symptoms (EPSs), and long-term utilization may be associated with the development of irreversible tardive dyskinesia (Dean and Scarr, 2004; Lieberman et al., 2005; Margolese et al., 2005). The “atypical” antipsychotic, clozapine, which potently interacts with several classes of monoaminergic receptor, is active in certain neuroleptic-refractory patients, does not elicit EPS, and is more effective than haloperidol against negative symptoms (Dean and Scarr, 2004; Meltzer, 2004; Lieberman et al., 2005). However, its advantages are offset by the risk of agranulocytosis. Furthermore, clozapine elicits sedation, obesity, cardiovascular-autonomic, and metabolic side effects via actions at α1-adrenoceptors (ARs), histamine (H1) receptors, and muscarinic receptors (Cunningham-Owens, 1996; Millan et al., 2000a; Pacher and Kecskemeti, 2004). A number of other multireceptorial antipsychotics have been introduced with similar, although not identical, broad-based receptor-binding profiles: notably, olanzapine and risperidone (Millan et al., 2000a; Dean and Scarr, 2004; Lieberman et al., 2005; McCue et al., 2006). However, despite their undeniable utility, they do not fully reproduce the clinical benefits of clozapine, and therapeutic indexes to doses eliciting undesirable actions are limited; in particular, EPS for risperidone and obesity for olanzapine (Dean and Scarr, 2004; Lieberman et al., 2005; Margolese et al., 2005). Moreover, although the more recently launched partial dopaminergic agonist, aripiprazole, is well tolerated, its efficacy is not superior to that of other agents (Abi-Dargham and Laruelle, 2005; Burstein et al., 2005; McCue et al., 2006; Urban et al., 2007b). Clearly, there remains a need for innovative drugs possessing improved efficacy and favorable side-effect profiles.
It remains to be clinically demonstrated that antipsychotics that act independently of dopaminergic mechanisms are robustly and consistently effective, and several authorities have recently reasserted the importance of dopamine D2 receptors in the management of schizophrenia (Kapur and Mamo, 2003; McGowan et al., 2004; Abi-Dargham and Laruelle, 2005). Nonetheless, no clinically employed antipsychotic differentiates D2 from D3 receptors (Vanhauwe et al., 2000; Dean and Scarr, 2004; Burstein et al., 2005; Joyce and Millan, 2005), so their respective importance remains uncertain. Interestingly, several arguments support current interest in D3 receptors as a target for the potentially improved treatment of schizophrenia (Joyce and Millan, 2005; Boeckler and Gmeiner, 2006; Sokoloff et al., 2006).
First, although the density of D3 versus D2 receptors in rodent central nervous system is modest, they are better represented in primates and in man (Joyce, 2001; Sokoloff et al., 2006). In all species, in contrast to the enrichment of D2 sites in striatal regions, D3 receptors are concentrated in the nucleus accumbens and corticolimbic structures implicated in the control of mood, cognition, and the pathogenesis of schizophrenia (Joyce, 2001; Joyce and Millan, 2005; Sokoloff et al., 2006). Second, the density of D3, but not D2, receptors was elevated in (off-treatment) psychotic patients (Gurevich et al., 1997). Similar increases were seen in individuals chronically exposed to cocaine, which is known to aggravate and precipitate psychotic states (Richtand et al., 2001; Heidbreder et al., 2005). Third, the S9G polymorphism of D3 receptors [which more potently binds dopamine (DA)] has been correlated to a higher risk for schizophrenia. Although this association was not seen in all patient populations, studies of the interaction between D3 receptors and other genes support a relationship between this D3 receptor polymorphism and vulnerability to schizophrenia (Dubertret et al., 1998; Jonsson et al., 2003; Joyce and Millan, 2005). Fourth, long-term administration of D3 receptor antagonists reduces the spontaneous activity of mesolimbic but not nigrostriatal dopaminergic pathways (Ashby et al., 2000). Fifth, selective blockade of D3 versus D2 receptors improves cognitive performance and enhances frontocortical cholinergic transmission (Laszy et al., 2005; Millan et al., 2007a). Furthermore, consistent with an improvement in negative symptoms, pharmacological or genetic deletion of D3 receptors enhances social interaction in rodents (Millan, 2003). Finally, gene knockout and pharmacological studies in rodents and primates have shown that antagonism of D3 receptors favors motor function and counters its disruption by D2 receptor blockade (Millan et al., 2004b; Joyce and Millan, 2005; Sokoloff et al., 2006).
Clinical evidence that selective blockade of D3 receptors moderates the core symptoms of schizophrenia is awaited. Nonetheless, the above observations suggest that antipsychotics displaying a relative preference for D3 over D2 sites may conserve activity against positive symptoms, display strengthened efficacy against cognitive (and negative) symptoms and have a low EPS potential (Joyce and Millan, 2005). On this basis, we selected the benzopyranopyrrolidine derivative, S33138 (Fig. 1) (Dubuffet et al., 1999), for clinical evaluation. In the accompanying article (Millan et al., 2007), we characterize the actions of S33138 in a broad range of experimental models in vivo. The present article comprises a foundation for these studies in evaluating the interactions of S33138 with cloned, dopamine hD3, hD2, and hD2S receptors and with several other classes of G-protein-coupled receptor.
Materials and Methods
Drug Evaluation, Salts, and Sources. S33138 was fully characterized in all experimental procedures. In addition, its binding profile at hD3, hD2L, and hD2S receptors and at sites underlying metabolic, cardiovascular, and autonomic side effects was compared with those of the clinically established antipsychotics, haloperidol, clozapine, olanzapine, and risperidone. The majority of protocols employed have been extensively characterized in previous publications from this laboratory (see citations below). However, for those protocols that we have not, as yet, documented in detail, EC50s for endogenous agonists are given together with pKB values for prototypical antagonists evaluated in parallel as “internal” validators. In this regard, haloperidol was used as an internal reference agent for hD3, hD2L, and hD2S receptors. Efficacies of S33138 were determined at all subtypes of dopaminergic receptor, with the exception of D4 receptors, where its affinity (<5.0) was too low for study, and at all sites for which its affinities were within 2 “logs” (100-fold) of its affinity at hD3 receptors. In all studies, results are based on at least three independent determinations performed in triplicate. S33138 and MDL100,907 were synthesized by G. Lavielle (Servier); SB269,970, olanzapine base, and risperidone base were synthesized by J.-L. Péglion (Servier); clozapine base, RX821,002, dopamine HCl, SCH23390, (–)quinpirole diHCl, forskolin base, noradrenaline ditartrate, serotonin creatinine sulfate, and haloperidol base were purchased from Sigma-Aldrich (St. Quentin-Fallavier, France); [3H]spiperone and [125I]iodosulpride were purchased from GE Healthcare Europe (Orsay, France); and methiothepin maleate was purchased from Hoffman-LaRoche (Basel, Switzerland).
Chemical structure of S33138.
Evaluation of the Affinities of S33138 at Dopamine hD3,hD2L, and D2S Receptors and at Other Classes of Binding Site. All protocols used for determination of the affinities of S33138 at multiple classes of native and cloned receptor have been documented previously (Millan et al., 2000a,b, 2004a; Newman-Tancredi et al., 2002; for summaries, see tables). Isotherms were analyzed by non-linear regression using PRISM (GraphPad Software, San Diego, CA) to generate IC values. They were transformed into Ki values according to Cheng-Prussof: Ki = IC50/(1 + L/Kd), where L is the radioligand concentration, and Kd is the dissociation constant.
Antagonist Properties of S33138 at hD3, hD2L, and hD2S Receptors: Guanosine-5′-O-(3-[35S]thio)-Triphosphate Binding. The procedures used for determination of the functional actions of S33138 at Chinese hamster ovary (CHO) cell line-expressed hD3, hD2L, and hD2S receptors (15, 2.2, and 1.6 pmol/mg protein, respectively) by guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) (PerkinElmer Life and Analytical Sciences, Boston, MA) binding have been described in detail elsewhere (Newman-Tancredi et al., 1999; Millan et al., 2000b, 2004a). In brief, [35S]GTPγS was employed at a concentration of 1.0 nM for hD3 receptors and at a concentration of 0.1 nM for hD2L and hD2S receptors. In each case, the pH was 7.4, the temperature was 22°C, and the incubation period was 40 min. The buffer comprised HEPES (20 mM), NaCl (150 mM for hD3 receptors and l00 mM for hD2L and hD2S receptors), GDP (3 μM), and MgCl2 (3 mM for D3 receptors and 10 mM for hD2L and hD2S receptors). For evaluation of the actions of S33138 alone, membranes were incubated with incremental concentrations for 15 min before the addition of [35S]GTPγS. For evaluation of the antagonist properties of S33138, interaction studies were undertaken with a fixed concentration of DA (1 μM for hD3 sites, 10 μM for hD2L sites, and 3 μM for hD2S sites). In addition, the concentration-response curve for DA-induced [35S]GTPγS binding at hD3 receptors was examined in the presence of incremental, fixed concentrations of S33138 and the pA2 value derived by Schild analysis. Experiments were terminated by rapid filtration through Unifilter-96 GF/B filters using a filter mate harvester (PerkinElmer Life and Analytical Sciences). The radioactivity retained on the filters was quantified using a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences). KB values for inhibition of DA-stimulated [35S]GTPγS binding by S33138 were calculated according to Cheng-Prusoff: KB = IC50/(1 + (agonist/EC50)), where IC50 is the IC50 of S3318, agonist is the concentration of DA, and EC50 is the EC50 of DA alone.
Antagonist Properties of S33138 at hD3 and hD2L Receptors: Antibody-Capture/Scintillation Proximity Assay Studies of Coupling to Gαi3. The influence of S33138 upon DA-induced activation of Gαi3 subunits coupled to CHO-transfected hD3 and hD2L receptors was determined essentially as described previously (Millan et al., 2004a). In brief, [35S]GTPγS binding was performed, as outlined above for filtration protocols, in 96-well optiplates (PerkinElmer Life and Analytical Sciences). After incubation, 20 μl of Nonidet P-40 (Roche Diagnostics, Mannheim, Germany, 0.27% final concentration) was added to each well, and plates were incubated for 30 min. Then, 10 μl of mouse anti-Gαi3 monoclonal antibody (SA-281; Biomol, San Diego, CA) was added, and incubation was continued for 30 min. Scintillation proximity assay (SPA) beads coated with secondary, anti-mouse antibodies (GE Healthcare, Les Ulis, France) were added in a volume of 50 μl, and plates were incubated for 3 h with gentle agitation. They were then centrifuged (10 min, 1300g), and radioactivity was determined on a Top Count microplate scintillation counter (PerkinElmer Life and Analytical Sciences). Membranes were incubated with S33138 alone or with S33138 plus DA (1 μM for hD3 and 10 μM for hD2L receptors) for 15 min before addition of [35S]GTPγS. KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties of S33138 at hD3 and hD2L Receptors: Immunoblot Studies of Coupling to Extracellular-Regulated Kinase. Extracellular-regulated kinase [ERK; also known as mitogen-activated protein (MAP) kinase] phosphorylation was quantified as previously (Cussac et al., 1999; Millan et al., 2000b, 2004a). In brief, CHO cells expressing hD3 or hD2L receptors were grown in 24-well plates until 90% confluent and then starved overnight in serum-free medium. S33138 was diluted in the medium and added to each well at the desired concentration. For antagonist studies, cells were preincubated with S33138 for 20 min, then exposed to DA (0.1 μM for hD3 and hD2L receptors in each case) for 5 min. After incubation, 0.25 ml of Laemmli sample buffer containing 200 mM dithiothreitol was added. Whole-cell lysates were boiled for 3 min at 95°C, and 14 μl of cell extracts was loaded onto 15-well, 10% polyacrylamide gels. Phosphorylated forms of pp42MAP-KINASE (ERK 2) and pp44MAP-KINASE (ERK 1) were revealed using a monoclonal antibody (NanoTools, Denzlingen, Germany) followed by chemiluminescence detection with horseradish peroxidase coupled to the secondary antibody (GE Healthcare). Immunoblots shown are from representative experiments. Autoradiograms were analyzed, and phosphorylated ERK1/2 was quantified by computerized densitometry using AIS software (Imaging Research Inc., ON, Canada). Isotherms were analyzed by nonlinear regression with PRISM. KB values were calculated according to the Cheng-Prusoff equation (see above).
Antagonist Actions of S33138 at hD3, hD2L, hD3/hD2L, and hD3i3(D2) Receptors: Adenylyl Cyclase Assays. The methods used have been detailed previously (Maggio et al., 2003; Novi et al., 2007). In brief, COS-7 cells, grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin, were seeded at a density of 5 × 105 per 100-mm dish. Twenty-four hours later, they were transiently cotransfected by the DEAE-dextran chloroquine method with plasmid DNA encoding adenylyl cyclase (AC)-V and hD3 receptors or with chimeric AC-V/VI plus hD3 receptors, hD2L receptors, hD3 and hD2L receptors, or chimeric hD3i3(D2) receptors. In this chimeric construct, kindly provided by Dr. Daniel Lévesque, the hD2 receptor sequence, corresponding to amino acids 327 to 338 (KTMSR-RKLSQQK) of the C-terminal portion of the “i3” loop, was introduced into the hD3 receptor, replacing amino acids 312 to 323. This substitution changes neither ligand recognition profile nor cell surface expression compared with wild-type hD3 receptors (Novi et al., 2007). Twenty-four hours after transfection, cells were trypsinized, recultured in 24-well plates, and, after an additional 24 h, the cells were assayed for AC activity. In brief, the cells were incubated for 2 h with 0.25 ml/well fresh growth medium containing 5 μCi/ml [3H]adenine. This medium was replaced with 0.5 ml/well Dulbecco's modified Eagle's medium containing 20 mM HEPES, pH 7.4, 0.1 mg of bovine serum albumin, and the phosphodiesterase inhibitors, 1-methyl-3-isobutylxanthine (0.5 mM) and RO 20-1724 (0.5 mM). AC activity was stimulated by addition of 1 μM forskolin in the presence or absence of S33138 and the D2/D3 receptor agonist, quinpirole, at concentrations indicated in the legend to Fig. 4. After 10 min of incubation at 30°C, the medium was removed, and the reaction was terminated by the addition of perchloric acid containing 0.1 mM unlabeled cAMP, followed by neutralization with KOH. The amount of [3H]cAMP formed was determined by a two-step column separation procedure, and disintegrations per minute values were expressed with respect to the protein content of each sample; the average value (mean ± S.E.M.) was 56.7 ± 4.2 μg. KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties of S33138 at hD1 Receptors: SPA Studies of Coupling to Gαs. Activation of hD1 receptor-coupled Gαs-protein was determined essentially as previously (Cussac et al., 2004). In brief, membranes of L cells expressing hD1 receptors (11 pmol/mg protein) were preincubated with S33138 or assay buffer containing 20 mM HEPES (pH 7.4, 1 μM GDP, 50 mM MgCl2, and 100 mM NaCl). The reaction was started by adding DA (1.0 μM) and [35S]GTPγS(∼0.3 nM) to 96-well optiplates (PerkinElmer Life and Analytical Sciences) for 60 min at room temperature. At the end of incubation, 20 μl of Nonidet P-40 (0.27% final concentration) was added to each well, and plates were incubated with gentle agitation for 30 min. Polyclonal antibodies against Gαs/olf (C18; Santa Cruz Biotechnology Inc., Santa Cruz, CA) were added to each well in a volume of 10 μl before 30 min of additional incubation. After addition of SPA beads coated with secondary antibodies (50 μl; GE Healthcare) and incubation for 3 h, plates were centrifuged (10 min, 1300g), and radioactivity was detected on a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences). KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties of S33138 at hD5 Receptors: Fluorescence Quantification of cAMP Production. GH4 cells stably expressing human D5 receptors (0.34 pmol/mg protein) were resuspended in Hanks' balanced salt solution buffer containing 20 mM HEPES/NaOH, pH 7.4, 138 mM NaCl, 5.3 mM KCl, 0.4 mM KH2PO4, 0.3 mM Na2HPO4, 1.25 mM CaCl2, 0.5 mM MgCl2, 0.4 mM MgSO4, and 0.1% glucose and preincubated for 30 min at 37°C with 0.5 mM 3-isobutyl-1-methylxanthine. Cells were distributed at a density of 1.104 cells/well in microplates and preincubated for 10 min at room temperature with S33138, the D5 receptor antagonist, SCH23390, or buffer (control). In antagonist studies, DA (30 nM) was added, and the mixture was incubated for 20 min at room temperature. The fluorescence acceptor (XL665-labeled cAMP) and the fluorescence donor (anti-cAMP antibody labeled with europium cryptate) were then added. After 60 min at room temperature, fluorescence transfer was measured at λex = 337 nm, λem = 620 nm, and λem = 665 nm using a “high time-resolved fluorescence” microplate reader (RUBY-star; BMG, Offenburg, Germany). The production of cAMP was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results were expressed as percentage inhibition of the level of cAMP induced by 30 nM DA. KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties of S33138 at hα2C-ARs: [35S]GTPγS Binding. [35S]GTPγS binding was determined as documented in detail elsewhere (Audinot et al., 2002; Newman-Tancredi et al., 2002). In brief, membranes of CHO cells expressing hα2C-AR (1.9 pmol/mg protein) were incubated for 60 min at 22°C with noradrenaline (NA) and [35S]GTPγS at a concentration of 0.1 nM in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 3 μM GDP, and 3 mM MgSO4. Antagonist properties of S33138 and the selective α2-AR antagonist, RX821,002, were evaluated against a fixed concentration of NA (10 μM). Incubations were terminated by rapid filtration through Whatman GF/B filters using a Filter Harvester (Perkin-Elmer Life and Analytical Sciences). Radioactivity retained on the filters was quantified by liquid scintillation counting. KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties of S33138 at hα2C-ARs: SPA Studies of Coupling to Gαi3 and Gαo. The coupling of hα2C-ARs to Gαi3 was evaluated using an SPA procedure essentially as described outlined above for hD3 receptors. The protocol used for Gαo was the same as for Gαi3. In brief, membranes were incubated on 96-well plates with NA (10 μM) and [35S]GTPγS (0.2 nM) for 1 h at 22°C following preincubation with S33138, RX821,002, or assay buffer for 30 min. The buffer contained 20 mM HEPES, pH 7.4, 0.3 μMGDP, 3 mM MgCl2, and 150 mM NaCl. The reaction was stopped by solubilizing membranes with detergent, Nonidet P-40 (0.27% final concentration). After gentle agitation for 30 min, 10 μl of mouse monoclonal anti-Gαi3 antibodies (see above) or anti-Gαo antibodies (SA-280; Biomol) were then added, and plates were incubated for a further 1 h. At the end of the incubation period, SPA beads coated with anti-mouse secondary antibodies (GE Healthcare) were added. They were incubated with gentle agitation overnight before counting radioactivity on a TopCount microplate scintillation counter. Nonspecific binding was defined with 10 μM GTPγS. KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties of S33138 at hα2C-ARs: ERK Phosphorylation. The procedure used was essentially that outlined above for hD3 receptors. CHO cells expressing hα2C-ARs were grown in six-well plates until confluent. Cells were then washed twice with serum-free medium and incubated overnight in this medium. Cells were preincubated for 20 min with S33138, RX821,002, or buffer and then stimulated with NA (1 μM) for 5 min. Phosphorylated ERK 1/2 was measured in cell extracts using a monoclonal antibody against phosphorylated pp42MAP-Kinase (ERK 2) and pp44MAP-Kinase (ERK 1) (NanoTools). Autoradiograms were analyzed, and KB values were calculated according to the Cheng-Prusoff equation (see above).
Antagonist Properties of S33138 at h5-HT1D Receptors: [35S]GTPγS Binding. Membranes of CHO cells expressing h5HT1D receptors (1.6 pmol/mg protein) were incubated for 30 min at 22°C with 5-HT and [35S]GTPγS at a concentration of 0.1 nM in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 3 μM GDP, and 3 mM MgCl2. Incubations were terminated by rapid filtration through Whatman GF/B filters using a Filter Harvester (PerkinElmer Life and Analytical Sciences). Radioactivity retained on the filters was quantified by liquid scintillation counting. Antagonist properties of S33138 and the 5-HT1D receptor antagonist, methiothepin, were evaluated against a fixed concentration of 5-HT (10 nM). KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties at h5-HT2A Receptors: SPA Studies of Coupling to Gαq. Efficacy at h5-HT2A receptors was determined by Gαq activation in CHO cells expressing h5-HT2A receptors (4.4 pmol/mg protein) employing an SPA protocol previously used for study of Gαq-coupled h5-HT2C receptors (Cussac et al., 2002). Cells were preincubated with S33138, the selective 5-HT2A receptor antagonist, MDL100,987, or buffer for 30 min at 22°C. They were then incubated for 1 h with 5-HT (at a concentration of 100 nM in antagonist studies) and [35S]GTPγS (0.3 μM). KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties at h5-HT2A Receptors: Studies of Inositol Phosphate Production. Determination of the accumulation of inositol phosphate (IP) in CHO cells expressing h5-HT2A receptors was performed in 96-well plates (0.25 × 106 cells/well) after overnight labeling with [3H]myoinositol (0.5 μCi/well). Stimulation by 5-HT (0.3 μM) was conducted for 30 min in a medium containing 10 mM LiCl and S33138, MDL100,907, or buffer. The reaction was stopped by adding formic acid (0.1 M). Supernatants were recovered, and IPs were purified by ion-exchange chromatography using DOWEX AG1-X8 resin (Bio-Rad, Hercules, CA) in 96-well filter plates (Millipore, Bedford, MA). The total radioactivity remaining in the membrane fraction was counted after treatment of the cells by a mixture of 10% Triton X-100 and 0.1 N NaOH. Radioactivity was quantified using a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences). Data are expressed (percentage) as (amount of total IP produced/amount of radioactivity remaining in membranes) × 100. KB values were calculated according to the Cheng-Prussoff equation (see above).
Antagonist Properties of S33138 at h5-HT7 Receptors: Fluorescence Quantification of cAMP Production. Actions of S33138 were determined in CHO cells stably expressing human 5-HT7 receptors (0.25 pmol/mg protein) (1.104 cells/well in microplates) as described above for hD5 receptors. Cells were preincubated for 10 min at room temperature with S33138, the selective 5-HT7 receptor antagonist, SB269,970, or buffer. In antagonist studies, 5-HT (100 nM) was added, and incubations were performed for 20 min at room temperature. Fluorescence was quantified as described above for hD5 receptors. The results are expressed as percent inhibition of the level of cAMP induced by 5-HT. KB values were calculated according to the Cheng-Prussoff equation (see above).
Results
Binding Profile of S33138 at Multiple Subtypes of Dopamine Receptor (Table 1;Fig. 2). S33138 concentration-dependently and potently displaced the binding of [3H]spiperone to hD3 receptors (Fig. 2A). S33138 similarly competed with [3H]spiperone at hD2L receptors (long isoform) and hD2S receptors (short isoform), but its affinity was markedly (approximately 25-fold) lower compared with hD3 receptors (Fig. 2A). A similar pattern of data was acquired with [125I]iodosulpride (used at 0.2 nM for hD3 receptors and 0.1 nM for hD2L receptors); this radioligand yielded affinities (pKi values) of S33138 of 8.66 ± 0.09 and 7.43 ± 0.02 at hD3 and hD2L receptors, respectively. The affinity of S33138 for native, rat striatal D2 receptors labeled by [3H]spiperone was close to that seen at recombinant hD2 receptors (Table 1). In contrast to hD3 sites, the affinity of S33138 for cloned hD4 receptors was low, although it displayed modest affinity for hD1 and hD5 receptors. S33138 displayed low affinity for native, rat, and cloned, human DA reuptake sites (pKi values, <5.0 and 4.91 ± 0.03, respectively).
Affinities of S33138 at multiple classes of dopamine receptor, 5-HT receptor, and AR
For procedural details, see Millan et al. (2000a,b, 2004a) and Newman-Tancredi et al. (2002). Data are means ± S.E.M. of pKi values, based on three to four determinations, each performed in triplicate. S33138 displayed negligible (pKi < 5.0) affinities for the following sites: histamine H2; opiate μ, δ, and κ; adenosine (A1, A2 adenosine); angiotensin I; benzodiazepine; bradykinin B2; calcitonin gene-related peptide; cannabinoid (CB1, CB); cholecystokinin (A, B); choline uptake; endothelin (A, B); GABA (A, B); glutamate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, N-methyl-d-aspartate); imidazoline (I2); melatonin (MT1, MT2); neurokinin (NK1, NK2); neuropeptide Y (Y1, Y5); and nicotinic, prostanoid (thromboxane A2/PGH2), σ (1, 2), estrogen, progesterone, and testosterone receptors. It also showed low affinity (pKi < 5.0) for Ca2+ channels (diltiazem site), Na+ channels (batrachotoxin site), and K+ channels (ATP, Ca2+ and voltage dependent) and for a variety of enzymes, including acetylcholinesterase, adenyl cyclase, cyclooxygenase 1, guanylyl cyclase, 5-lipoxygenase, monoamine oxydase A and B, nitric-oxide synthase, phospholipase A2, phospholipase C, phosphodiesterase I and III, protein kinase C, and sodium-potassium ATPase.
Binding Profile of S33138 at Nondopaminergic Receptors (Table 1). S33138 displayed significant affinity for h5-HT7 receptors as well as for h5-HT2A receptors, although its affinity for native, rat 5-HT2A receptors was slightly lower than for h5-HT2A sites. Modest affinity of S33138 also was seen at h5-HT1D and, less markedly, at h5-HT1A, h5-HT1B, h5-HT2B, and h5-HT2C receptors. The affinity of S33138 was, however, negligible at h5-HT3, h5-HT4, h5-HT5A, and h5-HT6 receptors. S33138 also displayed low affinity for cloned, human 5-HT reuptake sites ([lt]5.0). The affinity of S33138 for multiple subtypes of hα1-ARs was modest, and it manifested negligible affinity for hα2A- and hα2B-ARs. Conversely, S33138 showed greater affinity for hα2C-ARs. The affinity of S33138 for hβ1 and hβ2-ARs was negligible, and it also showed low affinity for cloned, human NA reuptake sites (pKi, 5.34 ± 0.14). S33138 showed negligible (<5.0) affinity for numerous other sites listed in the legend to Table 1.
Antagonism by S33138 of hD3, hD2L, and hD2S receptor-coupled G-protein stimulation as determined by “total” [35S]GTPγS binding. A, competition isotherms for binding of S33138 to hD3 receptors compared with other dopamine receptor subtypes. B to D, concentration-dependent blockade by S33138 of the actions of DA (1, 10, and 3 μM, respectively) at hD3, hD2L, and hD2S receptors. E, dextral displacement of the concentration-response to DA at hD3 receptors in the presence of incremental (fixed) concentrations of S33138. F, Schild transformation of data in E. Data are representative of three to four independent experiments, each of which was performed in triplicate.
Low Affinity of S33138 as Compared with Prototypical Antipsychotics for Receptors Underlying Metabolic, Cardiovascular, and Autonomic Side Effects (Table 2). Compared with its high affinity for hD3 receptors, S33138 displayed only low affinities for h5-HT2C receptors, hα1A-ARs, hα1B-ARs, hα1C-ARs, and hH1 receptors. It displayed negligible affinity for hM1, hM2, hM3, and hM4 receptors and weak affinity (Ki, 525 ± 11, n = 3) for hM5 receptors. The affinity of haloperidol for h5-HT2C compared with hD3 receptors was low, although it showed marked affinities at α1A-ARs, hα1B-ARs, and hα1C-ARs. It only weakly recognized hH1 sites and multiple subtypes of muscarinic receptor. Compared with hD3 receptors, clozapine manifested high affinity for h5-HT2C receptors, for each subtype of hα1-AR, and for hH1 receptors. Its affinities at hM1, hM3, and hM4 receptors were also high, and it displayed modest affinity for hM2 receptors. Olanzapine revealed high affinities for h5-HT2C receptors, for each subtype of hα1-AR, for hH1 receptors, and for all subtypes of muscarinic receptor, except hM3, for which its affinity was modest. The affinities of risperidone for h5-HT2C receptors, hα1-AR subtypes, and hH1 receptors were high, but, in contrast to clozapine and olanzapine, it only weakly interacted with muscarinic receptors.
Interaction of S33138 and other antipsychotics at hD3, hD2L, and hD2S receptors compared with sites underlying metabolic, cardiovascular, and autonomic side effects
All studies were performed in CHO cell lines. Drug affinities at hD3, hD2L, and hD2S sites were determined as summarized in Table 1. Radioligands (concentrations) for the other sites were as follows: h5-HT2C receptors ([3H]mesulergine, 0.3 nM); hα1A-, hα1B-, and hα1C-ARs ([3H]prazosin, 0.3 nM); hH1 receptors ([3H]pyrilamine, 2.0 nM); hM1 receptors ([3H]pirenzepine, 2.0 nM); hM2 receptors ([3H]AF-DX384, 2.0 nM); and hM3 and hM4 receptors ([3H]4-diphenylacetoxy-N-methylpiperidine, 0.2 nM). Data for S33138 at hα1-AR subtypes and h5-HT2C receptors are transformed from pKi values in Table 1 and are included for the sake of completeness. Data are means ± S.E.M. based on three to four determinations, each performed in triplicate.
Antagonist Properties of S33138 at hD3 versus hD2L and hD2S Receptors: Filtration Assays of [35S]GTPγS Binding (Fig. 2;Table 3). In line with previous studies (Newman-Tancredi et al., 1999; Millan et al., 2000b, 2004a), DA concentration-dependently (approximately 2-fold) stimulated [35S]GTPγS binding at hD3, hD2L, and hD2S receptors, actions blocked by haloperidol with pKB values of 8.51 ± 0.06, 9.49 ± 0.16, and 8.90 ± 0.16, respectively. S33138 did not modify [35S]GTPγS binding when applied alone. Indeed, it potently suppressed the stimulation of [35S]GTPγS binding at hD3 receptors by DA with a pKB of 8.85 ± 0.10; this value corresponds well to its affinity (8.68) at these sites. Furthermore, in the presence of incremental concentrations of S33138 (3–100 nM), the concentration-response curve for DA-induced [35S]GTPγS binding at hD3 receptors was shifted in parallel to the right with no loss of maximal effect (Fig. 2E). Schild analysis (Fig. 2F) yielded a slope not significantly different to unity (1.18, r = 0.88) and a pA2 (8.69) very close to its pKB and pKi values at hD3 sites. These observations indicate that S33138 behaves as a competitive and reversible antagonist at hD3 receptors. S33138 likewise suppressed DA-induced [35S]GTPγS binding at hD2L and hD2S receptors, indicating that it is also an antagonist at these sites (Fig. 2, C and D). However, pKB values of 7.82 ± 0.03 and 7.79 ± 0.06 at hD2L and hD2S receptors, respectively, were lower than for hD3 sites.
Summary of antagonist properties of S33138 at hD3, hD2L, and hD2S receptors as determined by blockade of DA-stimulated [35S]GTPγS binding, Gαi3 recruitment, and extracellular-regulated-kinase (ERK) phosphorylation
Data (pKi and pKB values) are means ± S.E.M. of three to four independent experiments.
Antagonist Properties of S33138 at hD3 versus hD2L Receptors: SPA Studies of Coupling to Gαi3 (Table 3). At hD3 and hD2L receptors, in line with previous work (Millan et al., 2004a), DA enhanced [35S]GTPγS binding to Gαi3 by approximately 2-fold; its actions were blocked by haloperidol, with pKB values of 8.25 ± 0.01 and 9.18 ± 0.26, respectively. In contrast, S33138 alone was without activity. At hD3 receptors, S33138 concentration-dependently suppressed DA-induced stimulation of Gαi3 with a pKB of 8.24 ± 0.07. S33138 also behaved as an antagonist at hD2L receptor-coupled Gαi3 in blocking its activation by DA with a pKB of 7.76 ± 0.14.
Antagonist Properties of S33138 at hD3 versus hD2LReceptors: Immunoblot Studies of ERK Phosphorylation (Fig. 3;Table 3). By analogy to previous studies (Cussac et al., 1999; Millan et al., 2004a), in CHO cells transfected with hD3 or hD2L receptors, DA triggered a transient yet marked activation (phosphorylation) of ERK 1/2 (MAP kinase). In distinction, S33138 did not increase levels of the phosphorylated forms of ERK. Furthermore, S33138 concentration-dependently abolished induction of ERK 1/2 at hD3 receptors with a pKB of 9.08 ± 0.02 (Fig. 3A). S33138 similarly behaved as an antagonist of DA-stimulated ERK 1/2 phosphorylation at hD2L receptors with a pKB of 8.03 ± 0.02 (Fig. 3B).
Antagonism by S33138 of hD3 and hD2L receptor-coupled extracellular-regulated-kinase phosphorylation as determined by immunoblot assays. Concentration-dependent antagonist properties of S33138 at hD3 receptors (A) and at hD2L receptors (B) against DA (0.1 μM). Immunoblots are shown to the right of the S33138 concentration-response curves. Data are representative of three to four independent experiments, each of which was performed in triplicate.
Antagonist Properties of S33138 at hD3 Receptors, hD2L Receptors, Cotransfected hD3 and hD2L Receptors, and hD3i3(D2) Receptors: Enzymatic Studies of AC Activity (Fig. 4). In COS-7 cells transfected with a chimeric AC-V/VI, forskolin elicited a marked (approximately 6-fold) increase in cAMP accumulation. This AC isoform is insensitive to D3 receptors, and, confirming previous work (Maggio et al., 2003; Novi et al., 2007), quinpirole did not modify the influence of forskolin in cells cotransfected with hD3 receptors. On the other hand, quinpirole (EC50, 1.2 nM) robustly and potently suppressed forskolin-induced cAMP accumulation in cells cotransfected with AC-V/VI and hD2L receptors. With similar potency, it reduced the activity of AC-V/VI in cells cotransfected with both hD2L and hD3 receptors (EC50, 0.97 nM). S33138 alone did not modify the effects of forskolin in hD3, hD2L, or hD3/hD2L cotransfected cells. However, it concentration-dependently abolished the inhibitory influence of quinpirole both in cells expressing hD2L receptors (pKB, 7.57 ± 0.12) and, more potently, in cells cotransfected with hD3 and hD2L receptors (pKB, 8.69 ± 0.14) (Fig. 4A). In COS-7 cells transfected with AC-V/VI and chimeric hD3i3(D2) receptors, where a 12-amino acid sequence of the third intracellular loop was substituted by the corresponding sequence of hD2L receptors to enhance coupling efficacy to AC-V/VI, quinpirole (EC50, 1.01 nM) markedly suppressed cAMP formation. Its action was blocked by S33138 with pKB of 8.71 ± 0.16 (Fig. 4B). In contrast to AC-V/VI, the AC-V isoform is responsive to D3 receptors, although effects of agonists are less robust than at hD2L receptors (Robinson and Caron, 1997; Maggio et al., 2003). Quinpirole significantly reduced the influence of forskolin upon cAMP accumulation in cells transfected with hD3 receptors and AC-V; this effect was reversed by S33138, which was inactive alone (Fig. 4C).
Antagonist Properties of S33138 at hD1 and hD5 Receptors. In L cells transfected with hD1 receptors, DA (EC50, 11.5 nM) elicited an approximately 2-fold increase in [35S]GTPγS binding to Gαs as quantified using an SPA protocol. In contrast, S33138 was ineffective and concentration-dependently eliminated the action of DA with a pKB of 6.21 ± 0.11. In CHO cells transfected with hD5 receptors, DA elevated cAMP accumulation with an EC50 of 41.7 nM. This action of DA was blocked by the D5 receptor antagonist, SCH23390, with a pKB of 8.82 ± 0.08. It was also concentration-dependently abrogated by S33138 (pKB, 6.46 ± 0.16), which was inactive alone. S33138 then behaves as an antagonist of hD1 and hD5 receptors with modest potency.
Antagonist Properties of S33138 at hα2C-ARs (Fig. 5). Corroborating previous studies at CHO-transfected hα2C-ARs (Audinot et al., 2002; Newman-Tancredi et al., 2002), NA elicited an approximately 2-fold enhancement in [35S]GTPγS binding, whereas S33138 was without effect. The action of NA (10 μM) was abolished by S33138 with a pKB of 7.23 ± 0.15. In SPA assays, NA robustly (approximately 1.5-fold in each case) enhanced the binding of [35S]-GTPγStoGαi3 (EC50, 513 nM) and Gαo (EC50, 145 nM), actions blocked by the prototypical α2-AR antagonist, RX821,002, with pKB values of 9.04 ± 0.09 and 8.57 ± 0.05, respectively. S33138, which was inactive alone, also abolished the effects of NA with pKB values of 6.85 ± 0.07 and 7.29 ± 0.14 (Fig. 5, A and B), respectively. Likewise, in CHO cells transfected with hα2C-ARs, NA (EC50, 27.7 nM) elicited a robust increase in ERK 1/2 phosphorylation. This effect of NA (1 μM) was blocked by RX821,002 (pKB, 8.32 ± 0.10) and dose-dependently abolished by S33138 (pKB, 7.11 ± 0.15) (Fig. 5C). In line with its weak affinity for hα2A- and hα2B-ARs, S33138 showed no antagonist properties (up to 10 μM) in [35S]GTPγS binding studies of these sites (data not shown).
Antagonism by S33138 of quinpirole-induced inhibition of adenylyl cyclase activity in CHO cells transfected with hD2L and/or hD3 receptors or with hD3i3(D2) receptors. A, concentration-dependent blockade by S33138 of the inhibitory influence of the D3/D2 receptor agonist, quinpirole (10 nM), upon forskolin-stimulated cAMP formation in CHO cells transfected either with hD2L receptors or with hD2L receptors and hD3 receptors. In both cases, cells were also transfected with a chimeric adenylate cyclase (V/VI) resistant to hD3 receptors. B, concentration-dependent blockade by S33138 of the inhibitory influence of quinpirole (1 nM) upon forskolin-stimulated cAMP formation in CHO cells transfected with chimeric hD3i3(D2) receptors that couple more robustly than their wild-type counterparts to AC-V/VI. C, blockade by a fixed concentration of S33138 (1 μM) of the inhibitory influence of quinpirole (1 μM) upon forskolin (1 μM)-stimulated cAMP accumulation in CHO cells transfected with hD3 receptors together with adenylate cyclase-V. Data are representative of three to four independent experiments, each of which was performed in triplicate. *, P < 0.01 versus forskolin alone.
Antagonist Properties of S33138 at 5-HT Receptor Subtypes (Fig. 5). Serotonin (EC50, 1.3 nm) evoked an approximately 1.3-fold increase in [35S]GTPγS binding at h5-HT1D receptors. The antagonist, methiothepin, antagonized the action of 5-HT with a pKB of 7.55 ± 0.07. S33138 also blocked the influence of 5-HT upon [35S]GTPγS binding at h5-HT1D receptors with a pKB of 6.79 ± 0.18. It was inactive alone at these sites. At h5-HT2A receptors, an SPA assay revealed activation by 5-HT (EC50, 27.8 nM) of [35S]GTPγS binding to Gαq with a maximal 2-fold increase versus basal values. The effect of 5-HT (0.3 μM) was abolished by the prototypical 5-HT2A antagonist, MDL100,907, with a pKB of 9.26 ± 0.20. Activation of Gαq coupled to h5-HT2A receptors was also abolished by S33138 with a pKB of 6.86 ± 0.02 (Fig. 5D). S33138 did not influence Gαq alone. Serotonin (EC50, 31.9 nM) also activated phospholipase C coupled to h5-HT2A receptors as reflected in a marked increase in IP formation. This action of 5-HT (0.3 μM) was abolished by MDL100,907 with a pKB of 8.9 ± 0.1. It was also blocked by S33138 with a pKB of 6.79 ± 0.07 (Fig. 5E). At h5-HT7 receptors, 5-HT enhanced the activity of AC with an EC50 of 30.0 nM. The 5-HT7 antagonist, SB269,970, blocked the action of 5-HT with a pKB of 8.85 ± 0.12. S33138 was inactive alone and blocked this action of 5-HT with a pKB of 7.06 ± 0.25 (Fig. 5F).
Discussion
Preferential Antagonism by S33138 of hD3 versus hD2L and hD2S Receptors. The higher affinity of S33138 for hD3 versus hD2L and hD2S receptors distinguishes it from haloperidol, clozapine, olanzapine, risperidone, and other clinically available antipsychotics displaying similar affinities for hD3, hD2L, and hD2S sites (Table 2) (Millan et al., 2000a; Vanhauwe et al., 2000; Burstein et al., 2005). Furthermore, the marked D3 versus D2 receptor preference of S33138 likewise differentiates it from aripiprazole and bifeprunox, which behave as partial agonists at hD3 and hD2 receptors (Shapiro et al., 2003; Burstein et al., 2005; Novi et al., 2007; Urban et al., 2007b). Although the preference of S33138 for hD3 versus hD2 sites is less pronounced than the 100-fold selectivity of antagonists like S33084, SB414,796, and A437,203 (Millan et al., 2000b; Reavill et al., 2000; Joyce and Millan, 2005; Boeckler and Gmeiner, 2006), it was selected for clinical development precisely on this basis. That is, rather than “absolute” selectivity, preferential D3 versus D2 receptor blockade should allow for antipsychotic activity against positive and cognitive-negative symptoms in the relative absence of extrapyramidal dysfunction (Joyce and Millan, 2005). This preferential interaction of S33138 with D3 versus D2 receptors is supported by in vivo findings reported in the accompanying article (Millan et al., 2007b).
hD3 receptors couple via pertussis-sensitive G-proteins to AC and ERK 1/2 (MAP kinases) (Ahlgren-Beckendorf and Levant, 2004; Beom et al., 2004; Neve et al., 2004; Sokoloff et al., 2006). Activation of hD3 receptors by DA led to enhanced [35S]GTPγS binding (Newman-Tancredi et al., 1999, 2002; Vanhauwe et al., 2000), an effect potently and reversibly blocked by S33138, indicating competitive antagonist properties. Moreover, using an SPA procedure coupled to a highly selective antibody (Millan et al., 2004a; Neve et al., 2004), the activation of Gαi3 by DA was also shown to be antagonized by S33138. Coupling of hD3 receptors to AC is variable and isoform-dependent (Griffon et al., 1997; Robinson and Caron, 1997; Hall and Strange, 1999; Neve et al., 2004). Nonetheless, in COS-7 cells transfected with hD3 receptors, quinpirole inhibited forskolin-stimulated AC-V activity, and this action was abolished by S33138. Furthermore, S33138 blocked the inhibition of cAMP formation by quinpirole in cells transfected with a chimeric hD3i3(D2) receptor displaying enhanced coupling efficacy to AC-V/VI (Novi et al., 2007), yet identical ligand recognition profiles and cell surface expression levels versus wild-type hD3 receptors (Novi et al., 2007). D3 receptors recruit the ERK pathway by pertussis toxin-sensitive Gαi (including Gαi3) proteins and an array of intracellular cascades involving both α and β subunits, a “phosphoinoside-3 kinase,” and an atypical protein kinase C (Cussac et al., 1999; Newman-Tancredi et al., 1999; Beom et al., 2004; Neve et al., 2004). Correspondingly, in CHO cells bearing hD3 receptors, DA stimulated ERK 1/2 phosphorylation, an effect abolished by S33138. The lack of effect of S33138 alone is important because ERK 1/2 activation is highly sensitive even to weak partial agonist actions; for example, aripiprazole and bifeprunox (M. J. Millan and C. Mannoury la Cour, unpublished observation; Cussac et al., 1999; Shapiro et al., 2003; Burstein et al., 2005; Urban et al., 2007b). In certain cell lines, D3 receptors couple to Go, Gq/11, Gz, and (via pertussis-insensitive pathways) phospholipase D (Everett and Senogles, 2004; Neve et al., 2004). Thus, it would be of interest to extend the present work to other intracellular cascades.
Antagonist properties of S33138 at hα2C-adrenoceptors, h5-HT2A receptors, and h5-HT7 receptors. Concentration-dependent blockade by S33138 of hα2C-adrenoceptors as measured by NA-induced activation of Gαi3 (A), Gαo (B), and ERK phosphorylation (C). D, concentration-dependent blockade by S33138 of the stimulation of h5-HT2A receptor-coupled Gαq by 5-HT (0.3 μM). E, concentration-dependent blockade by S33138 of the stimulation of h5-HT2A receptor-coupled IP production by 5-HT (0.3 μM). F, concentration-dependent blockade by S33138 of the stimulation of h5-HT7 receptor-coupled adenylyl cyclase by 5-HT (0.1 μM). Data are representative of three to four independent experiments, each of which was performed in triplicate.
D2 receptors show subtle differences to hD3 sites as concerns their interactions with signaling pathways (Hall and Strange, 1999; Vanhauwe et al., 1999'02; Ahlgren-Beckendorf and Levant, 2004; Beom et al., 2004; Neve et al., 2004). Nonetheless, like their D3 counterparts, both hD2S and hD2L receptors (which are principally localized presynaptically and postsynaptically to dopaminergic pathways, respectively) couple via Go/i to AC and ERK 1/2. Across a broad range of measures, [35S]GTPγS binding to hD2L and hD2S receptors (Vanhauwe et al., 2000; Newman-Tancredi et al., 2002), as well as hD2L receptor coupling to Gαi3 (Millan et al., 2004a; Lane et al., 2007), AC (O'Hara et al., 1996; Hall and Strange, 1999), and ERK 1/2 (Beom et al., 2004; Neve et al., 2004), S33138 behaved as a pure antagonist at both hD2L and hD2S receptors with lower potency than at hD3 receptors. Recent work on “agonist-directed” trafficking and “protean” agonism at hD2L sites emphasizes that drug efficacy can vary as a function of the intracellular pathway (Neve et al., 2004; Lane et al., 2007; Urban et al., 2007a). Thus, it would be interesting to examine the influence of S33138 upon other D2 receptor-coupled transduction cascades.
Antagonism by S33138 of Cotransfected hD3 and hD2L Receptors. D3 and D2 receptors are partially colocalized in dopaminergic pathways and postsynaptic neurones (Surmeier et al., 1992; Joyce and Millan, 2005), and they assemble into functional D3/D2L heterodimers in vitro (Maggio et al., 2003; Novi et al., 2007). Heterodimers display functional characteristics distinct from monomers, so it is of significance that S33138 abolished the inhibitory influence of quinpirole upon AC in cells expressing both hD3 and hD2L receptors. Furthermore, S33138 was more potent in cells expressing hD3 and hD2 sites than in cells expressing hD2 receptors alone. This finding resembles the altered potencies of other drugs at D3/D2 heterodimers compared with constituent monomers (Lee et al., 2003; Maggio et al., 2003; Novi et al., 2007). The underlying reasons remain to be defined. Nonetheless, proof that functional heterodimers form in the brain is awaited. Thus, relevance of the actions of S33138 (and other antipsychotics) at D3/D2 heterodimers to the control of psychotic states remains to be clarified (Lee et al., 2003; Maggio et al., 2003).
Antagonism by S33138 of hD1 and hD5 Receptors. Dopamine D1 and (closely related) D5 receptors couple positively to AC via Gs (Cai et al., 1999; Neve et al., 2004). As quantified by SPA coupled to a specific anti-Gαs antibody (Cussac et al., 2004), recruitment of Gs by hD1 receptors was blocked by S33138, indicating mild antagonist properties at these sites. Blockade of mesolimbic D1 receptors may participate in the antipsychotic properties of certain antipsychotics, and equilibrated antagonism of striatal D1 and D2 receptors contributes to the low EPS potential of clozapine (Josselyn et al., 1997; Tauscher et al., 2004). However, in vivo studies suggest that blockade of D1 sites is unlikely to play a major role in the therapeutic actions of S33138 (Millan et al., 2007). Antagonist properties of S33138 at hD5 receptors were also expressed at concentrations well below those blocking D3 receptors. Furthermore, although clozapine likewise antagonizes D5 receptors (Neve et al., 2004), any potential advantage in the management of schizophrenia remains conjectural.
Antagonist Actions of S33138 at hα2C-ARs. An intriguing finding differentiating S33138 from other antipsychotics was its interaction with hα2C-ARs despite low affinities for their hα2A and hα2B counterparts. All hα2-AR subtypes couple via Gi/Go to downstream pathways converging on ERK 1/2 (Hieble et al., 1995), and, by analogy to other α2-AR antagonists, S33138 abolished activation of hα2C-ARs in procedures of G-protein activation, recruitment of Gαi3 and Gαo, and ERK 1/2 phosphorylation (Alblas et al., 1993; Jasper et al., 1998; Audinot et al., 2002). Few ligands possessing a marked preference for α2C-versus α2A/α2B-ARs have been described previously (Hieble et al., 1995), so S33138 may serve as a useful template for construction of chemically novel selective α2C-AR antagonists. Exploration of the significance of α2C-AR blockade to the functional profile of S33138 would be of interest because activation of α2C-ARs in frontal cortex and hippocampus may compromise cognitive performance (Marcus et al., 2005; Soto-Moyano et al., 2005). Moreover, antagonism of corticolimbic and striatal α2C-ARs may contribute to the atypical profile of clozapine, including its low EPS potential (Millan et al., 2001'02; Kalkman and Loetscher, 2003; Svensson, 2003).
Antagonist Actions of S33138 at h5-HT2A and h5-HT7 Receptors. S33138 behaved as a pure antagonist at h5-HT2A receptors in protocols quantifying the activation of Gαq and the turnover of IP (Cussac et al., 2002; Kurrasch-Orbaugh et al., 2003). This observation is of potential importance because the more marked affinity of atypical antipsychotics for 5-HT2A versus D2 receptors has been related to improved control of negative symptoms and a low propensity to elicit EPS (Meltzer et al., 2003; Werkman et al., 2006). Activation of 5-HT7 receptors engages AC via recruitment of Gs (Thomas and Hagan, 2004), and modest antagonist actions of S33138 at h5-HT7 sites were seen in a protocol of cAMP formation. Blockade of 5-HT7 receptors by S33138 mimics clozapine and several other antipsychotics (Roth et al., 1994; Thomas and Hagan, 2004). 5-HT7 receptors are enriched in the cortex, hippocampus, striatum, and suprachiasmatic nucleus, and their blockade may favorably influence mood, sleep, and circadian rhythms, which are perturbed in schizophrenic patients (Thomas and Hagan, 2004; Monti and Monti, 2005).
Low Affinity of S33138 for hH1, Human Muscarinic, and hα1-AR Receptors. The weak affinity of S33138 for H1 receptors suggests a low sedative potential and a low risk of obesity compared with antipsychotics that potently block these sites (Table 2) (Cunningham-Owens, 1996; Kroeze et al., 2003; Kim et al., 2007). A low risk of sedation is consistent with the weak affinity of S33138 for α1-ARs, which also suggests a low risk of orthostatic hypotension and cardiovascular perturbation (Pacher and Kecskemeti, 2004; Lieberman et al., 2005). Moreover, S33138 did not interact with hM1-hM4 muscarinic receptors, suggesting that it should not provoke autonomic side effects such as troubled vision and gastrointestinal discomfort (Cunningham-Owens, 1996). Importantly, moreover, S33138 should not compromise cognitive function by blocking postsynaptic populations of muscarinic and H1 receptors (Lieberman et al., 2005).
Conclusions
The novel benzopyranopyrrolidine derivative, S33138, behaves as a preferential antagonist of hD3 versus hD2L and hD2S receptors. It also displays modest antagonist properties at hα2C-ARs, h5-HT2A receptors, and h5-HT7 receptors, actions likewise of potential relevance to the treatment of schizophrenia. This distinctive receptor-binding profile of S33138 was corroborated by in vivo studies in rodents and primates (Millan et al., 2007), underscoring its innovative profile for the improved management of psychotic states. Conceivably, low doses may suffice for maintenance therapy, whereas during acute exacerbation of psychosis, doses can be increased to more markedly antagonize D2 receptors. This remains to be seen. In any case, ongoing (phase II) clinical studies should soon shed light on the long-standing question of how antipsychotics that preferentially block D3 versus D2 receptors compare with clinically established agents acting with similar affinity at these sites.
Acknowledgments
We thank L. Verrièle, C. Chaput, and M. Touzard for technical assistance and M. Soubeyran for secretarial support.
Footnotes
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.107.126706.
-
ABBREVIATIONS: EPS, extrapyramidal motor symptom; AR, adrenoceptor; H1, histamine; DA, dopamine; S33138 N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenylacetamide; h, human; MDL100,907, R(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol; SB269,970, (R)-1-{2-[1-(3-hydroxy benzensulfonyl) pyrrolidin-2-yl] ethyl}-4-methylpiperidine; RX821,002, 2-(2,3-dihydro-2-methoxy-1,4-benzodioxan-2-yl)-4,5-dihydro-1H-imidazoline; SCH23390, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; CHO, chinese hamster ovary; [35S]GTPγS, guanosine-5′-O-(3-[35S]thio)-triphosphate; SPA, scintillation proximity assay; ERK, extracellular-regulated kinase; MAP, mitogen-activated protein; AC, adenylyl cyclase; Ro 20-1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; NA, noradrenaline; 5-HT, serotonin; IP, inositol phosphate; CGP-12177, 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one; SB414,796, trans-3-(2-(4-((3-(3-(5-methyl-1,2,4-oxadiazolyl))-phenyl)carboxamido)cyclohexyl)ethyl)-7-methylsulfonyl-2,3,4,5-tetrahydro-1H-3-benzazepine; A437,203, 2-{3-4-(2-tert-butyl-6-trifluoromethyl-pyrimidin-4-yl)-piperazin-1-yl]propyl-sulfanyl}-3H-pyrimidin-4-one.
- Received June 4, 2007.
- Accepted November 14, 2007.
- The American Society for Pharmacology and Experimental Therapeutics