Introduction

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In the past, ligand-binding methodologies have revealed that competitive antagonism of dopamine D₂ receptors is a shared property of most antipsychotic drugs (Creese et al., 1976; Seeman et al., 1976). However, antipsychotics also interact with many additional monoaminergic GPCRs at clinically relevant concentrations, and these additional interactions likely contribute to the differences in the clinical profiles of these agents. The completion of the human genome project, with an estimate of nearly 600 GPCR genes, has increased the number of potential receptor interactions for existing drugs (International Human Genome Consortium, 2001), yet the functional nature and extent of these interactions have not yet been explored because of the limitations of existing methodologies. Current antipsychotic drugs have limited efficacy in many patients and possess debilitating side effects. While recent efforts have produced newer and improved antipsychotics (Meltzer and McGurk, 1999), these "atypical" agents also have broad ranges of molecular actions (Bymaster et al., 1996) and possess unique side effect profiles that were not exhibited by older agents. Therefore, an improved understanding of the functional molecular profiles of existing drugs, including antipsychotics, may identify unique drug targets to exploit or to expressly avoid.

Previous attempts to determine the molecular profiles of antipsychotic drugs have utilized ligand-binding techniques to characterize drug actions at various receptors. These studies have revealed a tremendous degree of heterogeneity in potential sites of drug/receptor interactions and have highlighted the lack of selectivity of most antipsychotic agents in routine clinical use. However, these methodologies often could not provide accurate information on agonist affinities at these receptors, nor could these techniques appreciate subtleties in agonist-independent signaling. Indeed, many drugs previously thought to be competitive antagonists actually have intrinsic activity as inverse agonists at monoaminergic receptors and can attenuate the basal, constitutive activity of these receptors (Leftkowitz et al., 1993; Milligan et al., 1995; Spalding et al., 1995). The elucidation of the physiological role of these various receptor subtypes, and the clinical relevance of inverse agonism versus competitive antagonism, are among the most relevant of current challenges in pharmacology.

Using a cell-based functional assay, we have generated detailed pharmacologic profiles of therapeutically relevant compounds against most monoaminergic GPCRs. We report the finding that nearly all clinically useful antipsychotics are potent, fully efficacious, inverse agonists at the 5-HT_2A receptor, in addition to their known activity as potent dopamine D₂ receptor antagonists. Based on this finding, we launched a drug screening and chemical-lead optimization effort that has identified novel, potent, and selective 5-HT_2A inverse agonists. We show that one of these novel 5-HT_2A inverse agonists displays a behavioral pharmacological profile similar to that of existing atypical antipsychotic agents.

	Materials and Methods

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Molecular Cloning

Cloning of the known human monoamine GPCRs was performed using PCR. In brief, oligonucleotides flanking the coding region of each receptor were synthesized based on sequences deposited to GenBank. General PCR conditions used 100 ng (~125 pmol) of each primer, 250 µM deoxynucleoside-5'-triphosphates, 5% DMSO, 80 ng of genomic DNA or 25 ng of cDNA, 1 × Pfu cloned buffer and 2.5 units of Pfu Turbo (Stratagene, San Diego, CA). The standard cycling conditions were as follows: 94 to 98°C for 5 min then 40 cycles of 94 to 98°C for 15 s, 45 to 65°C for 10 s, and 72°C for 1 min/kilobase. The clonings of the dopamine D₁ receptor (Sunahara et al., 1990), D₂ receptor short isoform (Stormann et al., 1990), D₃ receptor (MacKenzie et al., 1994), D₅ receptor (Sunahara et al., 1991), the m1 to m5 muscarinic receptors (Bonner et al., 1987, 1988), and the m5 constitutively activated mutant (m5CAM) (Spalding et al., 1995) were reported previously. The alpha subunit of Gq, and the G-protein chimera Gqi5, were gifts from Dr. B. Conklin (Gladstone Institute, University of California at San Francisco, San Francisco, CA). All receptor and G-protein constructs were sequence verified.

Receptor Selection and Amplification (R-SAT) Assays

R-SAT (ACADIA Pharmaceuticals, San Diego, CA) assays were performed with minor modifications from that previously described (Spalding et al., 1995; Brauner-Osborne and Brann, 1996; Burstein et al., 1997a,b). Briefly, NIH-3T3 cells were grown in 96-well tissue culture plates to 70 to 80% confluence in Dulbecco's modified essential media (DMEM) supplemented with 10% calf serum and 1% penicillin/streptomycin/glutamine (PSG). Cells were transfected for 12 to 16 h with plasmid DNAs using Superfect Reagent (Qiagen, Valencia, CA) per manufacturer's protocols. R-SATs were generally performed with 1 to 50 ng/well receptor and 20 ng/well -galactosidase plasmid DNA. For G-protein coexpression studies, 4 to 20 ng/well Gq or Gqi5 was used. After overnight transfection, medium was replaced with serum-free DMEM containing 2% cyto-sf3 (Kemp Biotechnologies, Frederick, MD), and 1% PSG and varying concentrations of drug. Cells were grown in a humidified atmosphere with 5% ambient CO₂ for 4 to 6 days. Medium was removed from the plates, and -galactosidase activity was measured by the addition of o-nitrophenyl -d-galactopyranoside (in phosphate-buffered saline with 5% Nonidet P-40 detergent). The resulting colorimetric reaction was measured in a spectrophotometric plate reader (Titertek, Huntsville, AL) at 420 nM. All data were analyzed using the computer programs Excel Fit and GraphPad Prism software (San Diego, CA).

Drugs

All compounds for R-SAT studies were solubilized as 10 mM stock solutions in either water or DMSO. Working dilutions were made from 50 µM solutions in DMEM with 2% cyto-sf3, 1% PSG. All compounds were obtained from Sigma/RBI (Natick, MA) except as follows: spiperone and remoxipride (Tocris, St. Louis, MO) tiospirone (Bristol Myers Squibb, Stamford, CT), amperozide (Upjohn), chlorproethizene (Orgasynth Industries, Glasse, France), prothipendyl (Asta Medica), sultopride (IC Rom, Milan, Italy), moperone and bromperidol (Janssen Research Foundation, Beerse, Belgium), perazine (Byk Gulden, Singen, Germany), sertindole, trans-flupenthixol, and molindone (Lundbeck A/S, Copenhagen, Denmark), mesoridazine (BIOMOL Research Laboratories, Plymouth Meeting, PA), melperone (Cilag, Schaffhausen, Switzerland), while AC-90179 and M100907 were synthesized by ACADIA Pharmaceuticals. For behavioral studies, AC-90179 was dissolved in 10% Tween 80 (90% water), dizocilpine (MK-801) and d-amphetamine sulfate were dissolved in 0.9% saline, and (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) was dissolved in water. All compounds were administered in a volume of 0.1 ml/10 g of body weight, and doses were calculated based on the weight of the salt.

Compound Library and Screening

The compound library screened consisted of 130,000 diverse small organic molecules with drug-like properties. Compounds were stored at 20°C in 100% DMSO, diluted to 30 µM in water, adding 10% of the final volume to the assay plates for a 3 µM screening concentration. Coexpression of Gq was used to augment both m5 and 5-HT_2A receptor constitutive activity. After transient transfection in roller bottles, cells were trypsinized, harvested, and frozen. Cells were thawed rapidly in DMEM media contained 0.5% calf serum and 2% cyto-sf3 and then added to 96- or 384-well microtiter plates containing either test drugs or ritanserin controls. Data analysis was performed using ActivityBase (IDBS, Surrey, UK).

Behavioral Studies

Animals and Apparatus. Male non-Swiss Albino mice and male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) were housed (four mice/cage; two rats/cage) in rooms with controlled temperature and humidity and freely available water and food (Harlan Teklad, Indianapolis, IN). Mice were kept on a 12-h light/dark cycle, whereas rats were kept on a 12-h reverse light/dark cycle. For locomotor and observation experiments in mice, plastic 20- × 20- × 30-cm activity cages were equipped with photocell beams (AccuScan Instruments, Columbus, OH). Startle chambers (San Diego Instruments, San Diego, CA) were used for rat experiments (for details on startle apparatus and measures, see Mansbach et al., 1988).

Procedures

Observation for Head Twitches. Mice were treated with 2.5 mg/kg DOI i.p. The dose of DOI was chosen based on pilot dose-effect curves, which revealed that the lowest doses consistently produced a significant behavioral effect. Five minutes later, mice were treated with AC-90179 s.c. and placed into activity cages. Ten minutes later, mice were observed using a repeated sampling technique. Each mouse was observed for 10 s and rated for presence (1) or absence (0) of head twitch behavior for a total of six observations in 15 min and a total head twitch score of 0 to 6. Each dose combination was tested in a separate group of animals (n = 8), and the experimenter was blind to drug conditions. Head twitch scores were averaged followed by analysis of variance (ANOVA) and post hoc Dunnett's t test comparisons.

Locomotor Activity. For hyperactivity experiments, mice were treated with 0.3 mg/kg dizocilpine or 3.0 mg/kg d-amphetamine i.p. 15 min before the session. The doses of d-amphetamine and dizocilpine were chosen based on pilot dose-effect curves, which revealed that the lowest doses consistently produced a significant behavioral effect. Five minutes after pretreatment, mice were treated with AC-90179 s.c. and placed into the activity cages. For spontaneous activity, AC-90179 was administered alone. Locomotor data were collected during a 15-min session without habituation in a lit room. Each dose combination was tested in a separate group of animals (n = 8). Distance traveled (cm) was calculated and averaged followed by ANOVA and post hoc Dunnett's t test comparisons.

Startle Testing. Rats were tested and groups (n = 10) matched for levels of startle reactivity and prepulse inhibition (PPI; Mansbach et al., 1988). Two days later, test sessions started and consisted of a 5-min acclimation period with a constant background noise (65 dB), followed by 60 presentations of acoustic stimuli to measure acoustic startle responses. The 60 trials consisted of 22 40-ms presentations of a 120-dB broadband pulse, 10 20-ms presentations of each prepulse intensity (68, 71, and 77 dB) 100 ms prior to a 40-ms presentation of a 120-dB broadband pulse, and 8 NOSTIM trials in which no stimuli were delivered to assess general motor activation in the rats. Thirty minutes before testing, rats were treated with sterile water (s.c.), risperidone (1.0 mg/kg, i.p.), or AC-90179 (s.c.). Five minutes later, rats were administered DOI (0.5 mg/kg, s.c.) or 0.9% saline (s.c.). One week later, rats were administered the same pretreatment drug or vehicle and crossed over to receive the treatment opposite to that they received the previous week. Startle magnitudes and percentage of PPI for the three prepulse intensities were calculated as described elsewhere (Bakshi et al., 1994) and ANOVAs with repeated measures performed.

	Results

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As part of an ongoing effort to enable a universal functional assay for GPCR subtypes, we have cloned all of the known monoaminergic receptor subtypes and transiently expressed most of them in NIH-3T3 cells to determine their functional responses and pharmacological profiles for a large series of existing therapeutic agents. Data from concentration-response experiments with reference full agonist and full inverse agonist compounds for selected receptors are shown in Fig. 1. We focused our initial efforts on Gq-coupled receptors to take advantage of the observation that coexpression of the alpha subunit of Gq can be used to augment constitutive signaling (Burstein et al., 1997b), allowing the determination of inverse agonist pharmacology. The histamine H₁, muscarinic m5, and serotonin 5-HT_2B and 5-HT_2C (VGV) receptors display minimal endogenous basal activity. The coexpression of Gq resulted in varying degrees of constitutive activity for these receptors (Fig. 1, A-D). In contrast, the 5-HT_2A receptor displayed modest degrees of basal activity (Fig. 1E). Consistent with previous observations in rat (Niswender et al., 1999), the human 5-HT_2C receptor displays a range of constitutive signaling from the minimal degree seen with the fully edited VGV isoform, the intermediate degree observed with the partially edited VSV isoform, to the profoundly activated unedited INI isoform of this receptor (Fig. 1, D, F, and G). We controlled for both endogenous receptor and non-receptor-mediated inhibition or promotion of cellular growth by assaying all compounds against cells expressing the -galactosidase marker gene alone and cells expressing unrelated receptors. The constitutive activity of the various receptor subtypes demonstrated in this study, and the inverse agonist activity of the compounds tested, could have been the result of inclusion of trace amounts of agonists in the media used to culture the NIH-3T3 cells as part of the assay. To address this concern, we replaced calf serum with synthetic medium that is free of monoamines. To definitively prove that endogenous serotonin was not present, we also coexpressed the serotonin transporter (Blakely et al., 1994), which abolished all 5-HT-mediated signaling yet did not affect the inverse agonist responses observed at the 5-HT_2A receptor (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Constitutive activity of monoamine receptors. Representative concentration response experiments for the human H1 (A), m5 (B), 5-HT2B (C), 5-HT2C-VGV (D) 5-HT2A, (E), 5-HT2C-VSV (F), and 5-HT2C-INI (G) receptors as determined using R-SAT. The y-axis is the percentage of response defined as Full Agonist Response  Full Inverse Agonist Response; the x-axis is the negative logarithm of drug concentration. Curves with circles represent agonist determinations, while those with triangles represent inverse agonists. For those receptors without significant constitutive activity, coexpression of Gq was used, and the effects are shown with open circles (agonists) and open triangles (inverse agonists). For the m5 receptor, the m5CAM was used instead of Gq coexpression. Reference full agonists and full inverse agonists used were serotonin and ritanserin for 5-HT2 receptors, histamine and mepyramine for H1 receptors, and carbachol and atropine for m5 CAM. EC50 values for these receptors are reported as mean ± standard deviation of three to eight replicate experiments and are as follows: H1, 210 ± 3.3 nM agonist; H1 and Gq, 46 ± 15 nM agonist, 3.3 ± 1 nM inverse agonist; m5, 25 ± 3 nM agonist; m5CAM, 8.3 ± 2 nM agonist, 7.4 ± 3 nM inverse agonist; 5-HT2A, 29 ± 12 nM agonist, 0.67 ± 0.3 nM inverse agonist; 5-HT2B, 37 ± 8 nM agonist; 5-HT2B and Gq, 9.2 ± 7 nM agonist, 1.9 ± 1 nM inverse agonist; 5-HT2C (VGV), 42 ± 8 nM agonist; 5-HT2C (VSV), 12 ± 5 nM agonist, 11 ± 3 nM inverse agonist; and 5-HT2C (INI), 2.1 ± 2 nM agonist, 390 ± 28 nM inverse agonist. The percentage of constitutive activity was determined by (Basal Response  Full Inverse Agonist Response)/(Full Agonist Response  Full Inverse Agonist Response) and were as follows: H1, none; H1(+Q), 30 ± 6%; m5, none; m5CAM, 51 ± 2%; 5-HT2A, 24 ± 3%; 5-HT2B, none; 5-HT2B(+Q), 58 ± 6%; 5-HT2C (VGV), 2.6 ± 1%; 5-HT2C (VSV), 53 ± 6%; and 5-HT2C (INI), 87 ± 5%.

A library of 640 clinically relevant compounds (Sigma/RBI) was screened for intrinsic activity at the three 5-HT₂ receptor subtypes and at m5CAM (Spalding et al., 1995). Known serotonergic agonists showed activity at only the three 5-HT₂ receptor subtypes, while known muscarinic agonists were selectively active at the m5 receptor. Interestingly, many dopaminergic compounds were identified as 5-HT_2A receptor agonists, including commonly used drugs such as pergolide and bromocriptine. Many serotonergic reference competitive antagonists including ritanserin, ketanserin, and methiothepin displayed inverse agonist activity at all three serotonin receptor subtypes, but not at the m5 receptor, where known muscarinic inverse agonists such as atropine, benztropine, and trihexyphenidyl were identified. Most compelling was the finding that nearly every known antipsychotic in this library was identified as a 5-HT_2A receptor inverse agonist (17/18 with raclopride as the exception). This result was not seen with the closely related 5-HT_2B or the 5-HT_2C receptors, where only a significantly smaller subset of these compounds was active. Four antipsychotics in this library (clozapine, loxapine, thioridazine, and chlorpromazine) were identified as muscarinic receptor inverse agonists, whereas most of the tricyclic antidepressants displayed this activity (data not shown).

A detailed pharmacological profiling of 40 antipsychotics was generated at these three 5-HT₂ receptors, as well as at the histamine H₁ and dopamine D₂ receptor. The results of these experiments are shown in Table 1. In general, the pharmacology of these compounds correlates well with published values derived from receptor binding techniques (Creese et al., 1976; Seeman et al., 1976; Leysen et al., 1978; Hals et al., 1988; Roth et al., 1992; Stockmeier et al., 1993). As expected, nearly all compounds tested were potent competitive antagonists of the dopamine D₂ receptor. Surprisingly, nearly all of these compounds were also potent 5-HT_2A receptor inverse agonists. The majority of these compounds were full inverse agonists (>90% relative efficacy compared with ritanserin), whereas those few compounds that displayed partial inverse agonist activity also displayed lower 5-HT_2A potencies. Of the six antipsychotics that lacked 5-HT_2A inverse agonist activity, five of these retained intrinsic activity at this receptor as very low-potency (>1 µM EC₅₀) agonists, of which four belong to a single chemical class, the substituted benzamides. Only a single agent, perazine, was a potent partial agonist at this site, with an EC₅₀ of 140 ± 40 nM with 40% efficacy relative to serotonin. With the exception of tiapride, all of the remaining non-5-HT_2A inverse agonists display appreciable potency as D₂ receptor competitive antagonists (0.7-136 nM; Table 1). Extensive profiling of neuropsychiatric agents from different clinical and chemical classes has elucidated competitive antagonists lacking negative intrinsic activity at the 5-HT₂ receptors, yet all competitive antagonists of the histamine H₁ receptor behave as inverse agonists (data not shown). Lastly, broader profiling of these 40 antipsychotics against most of the remaining monoaminergic GPCRs did not reveal any other in vitro molecular activity that correlates with efficacy of this class of compounds (unpublished observations).

A library of chemically diverse organic compounds was screened for 5-HT_2A receptor inverse agonists. All compounds were tested at a 3 µM concentration, and ritanserin (100 nM) was used as a positive control. Of the 130,000 compounds tested in this assay, 530 initial hits were confirmed as 5-HT_2A inverse agonists. These compounds were subsequently tested at additional doses (3000, 300, 30, and 3 nM) to determine their relative potencies, and as inverse agonists at the m5 receptor to eliminate compounds that display nonspecific inhibitory responses. Of the confirmed hits in the initial screen, 230 compounds had potencies between 300 and 30 nM, while 96 compounds had potencies less than 30 nM and were at least 100-fold selective for 5-HT_2A relative to m5. A number of chemical classes emerged, including two related to known butyrophenone and tricyclic antipsychotics, as well as multiple novel series containing piperidine and piperazine moieties. One such structural series was explored further by assaying 1400 additional analogs for 5-HT_2A receptor inverse agonist activity. Of these analogs, 176 compounds had activity at 3 µM, of which 22 compounds had potencies less than 300 nM. Additional synthetic medicinal chemistry efforts led to the development of a defined structure-activity relationship with a number of potent analogs within multiple structural series. One lead compound, AC-90179 [log P_ki of 8.82 ± 0.18 (n = 10)], the structure of which is shown in Fig. 2A, was chosen to undergo receptor selectivity profiling. This compound was functionally profiled as both an agonist, and as either a competitive antagonist or inverse agonist, at 32 of the 35 known monoaminergic GPCRs (all known human receptor subtypes except the alpha_1A/D, 5-HT_5A, and dopamine D₄ receptors). AC-90179 displays nearly 100-fold selectivity against the 5-HT_2B receptor [6.87 ± 0.24 (n = 9)], 5-HT_2C (INI) [6.94 ± 0.38 (n = 10)], and 5-HT₆ receptor [6.8 ± 0.18 (n = 2)] as inverse agonists. This compound lacks activity (defined as > 1 µM EC₅₀) at all other receptors tested (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Antipsychotic-like behavioral effects of AC-90179. A, head twitch score as a function of AC-90179 dose, and the chemical structure of AC-90179. Filled triangles, AC-90179 or vehicle in combination with 2.5 mg/kg DOI; filled circle, vehicle dosed alone. Each point represents the mean (n = 8-24), and vertical lines represent standard error of the mean. Asterisks indicate statistical significance (p < 0.05) compared with the respective vehicle control. B, distance traveled (cm) in the locomotor cages as a function of AC-90179 dose. Filled inverted triangles, AC-90179 or vehicle in combination with 3 mg/kg d-amphetamine; filled squares, AC-90179 or vehicle in combination with 0.3 mg/kg dizocilpine; open circles, AC-90179 or vehicle alone. Each point represents the mean (n = 8-24), and vertical lines represent standard error of the mean. Asterisks indicate statistical significance (p < 0.05) compared with the respective vehicle control. C, percentage of prepulse inhibition as a function of AC-90179 pretreatment dose or vehicle and risperidone pretreatment controls. Black columns, the means of saline treated rats; gray columns, DOI (0.5 mg/kg)-treated rats. Error bars represent the S.E.M. The ANOVA on the %PPI data from the AC-90179 groups revealed an overall effect of treatment [F(1,37) = 27.73, p = < 0.01] and a treatment by pretreatment interaction [F(3,37) = 8.22, p < 0.01] (C). The inset shows the effects on startle magnitude by AC-90179 or risperidone in saline or DOI-treated rats.

To characterize the behavioral profile of a selective 5-HT_2A receptor inverse agonist, AC-90179 was tested in head twitch, locomotor, and PPI behavioral models. DOI-treated (2.5 mg/kg, i.p., 15 min) mice exhibited an average head twitch score of 2.6 (±0.3 S.E.M.). AC-90179 (0.1-30 mg/kg, s.c, 10 min) caused a dose-related decrease in DOI-induced head twitches with a minimum effective dose of 1 mg/kg and with higher doses completely eliminating head twitch behavior (Fig. 2A). In the locomotor experiments (Fig. 2B), mice traveled an average of 794 cm (±122 S.E.M.) after vehicle administration. Dizocilpine (0.3 mg/kg, i.p., 15 min) and d-amphetamine (3.0 mg/kg, i.p., 15 min) caused increases in distance traveled with averages of 2625 (±312) and 3367 (±532) cm, respectively. AC-90179 (0.3-10 mg/kg, s.c., 10 min) attenuated the hyperactivity induced by dizocilpine, but not by d-amphetamine. The minimum effective dose against dizocilpine was 1 mg/kg, whereas AC-90179 reduced spontaneous locomotor activity only at the highest dose tested (30 mg/kg).

DOI significantly disrupted PPI, and AC-90179 was effective in restoring this disruption, especially at the higher doses. AC-90179 did not affect PPI on its own, with no significant effect of pretreatment (p > 0.05) on percentage of PPI. The ANOVA on the PPI data from the risperidone comparison also revealed a significant effect of treatment [F(1,18) = 14.08, p < 0.01] and a treatment by pretreatment interaction [F(1,18) = 24.48, p < 0.01]. As predicted, risperidone was also effective in restoring PPI in DOI-treated rats, while having no effect on PPI by itself (p > 0.05). Since there were no significant interactions with prepulse intensity, the data were collapsed across the three prepulse intensities for graphical purposes (Fig. 2C). Since there was a significant pretreatment by treatment interaction, pair-wise 2-way repeated measures ANOVAs were conducted on the saline- and DOI-treated groups. In the vehicle-treated rats, there was no effect of AC-90179 (p > 0.025) or risperidone (p > 0.025) on PPI. In the DOI-treated groups, there were significant effects of AC-90179 [F(3,37) = 5.68, p < 0.01] and risperidone [F(1,18) = 16.73, p < 0.01] on percentage of PPI. The ANOVA on startle magnitude from the AC-90179 groups revealed significant effects of pretreatment [F(3,37) = 2.89, p = 0.048] and treatment [F(1,37) = 10.27, p < 0.01] on startle magnitude, but no treatment by pretreatment interaction (p > 0.05; Fig. 2C, inset). Risperidone, on the other hand, had no effect on startle magnitude (p > 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

These data represent the first description of the functional profile of a large set of antipsychotics at multiple receptor subtypes. Two mechanistic correlations have emerged from this analysis. Potency values from the R-SAT assay reconfirm the previously reported correlation between D₂ receptor competitive antagonism and antipsychotic efficacy and establish a correlation between inverse agonism at 5-HT_2A receptors and efficacy that is nearly as complete. No such correlation can be made between these compounds and the highly homologous 5-HT_2B and 5-HT_2C receptor subtypes, or with any other monoamine receptor subtype tested to date. The compounds that fail to display 5-HT_2A inverse agonist activity (i.e., substituted benzamides) have relatively potent D₂ receptor competitive antagonist activity. Taken together, these data argue that competitive antagonism of D₂ receptors and inverse agonism of 5-HT_2A receptors are independent mechanisms of antipsychotic efficacy.

While a number of groups have heterologously expressed 5-HT_2A receptors, none has been able to determine whether classic receptor competitive antagonists actually possess negative intrinsic activity at this receptor (Leysen et al., 1978; Roth et al., 1992; Stockmeier et al., 1993; Kehne et al., 1996; Grotewiel and Sanders-Bush, 1999). Mutagenic studies have yielded 5-HT_2A receptors that display constitutive activity, and a small number of antipsychotics were shown to possess partial inverse agonist activity (Egan et al., 1998a,b). However, the limited pharmacology conducted on these mutated receptors precluded the ability to recognize inverse agonism of 5-HT_2A receptors as a common efficacy mechanism of this class of compounds. That many antipsychotics are potent 5-HT_2A receptor competitive antagonists has been appreciated for some time (Leysen et al., 1978), and subsequent investigations into the mechanism of action of clozapine and related atypical agents have indicated the importance of mixed 5-HT_2A and dopamine D₂ receptor competitive antagonism (Stockmeier et al., 1993). Since the clinical dosing of antipsychotics is often limited by their D₂ receptor-induced extrapyramidal side effects, aspects of their clinical profiles can be predicted based on their in vitro potency ratios using this receptor as a basis for comparison. Figure 3 graphically depicts the in vitro potencies of these 40 compounds as inverse agonists of the 5-HT_2A, 5-HT_2B, 5-HT_2C, and H₁ receptor subtypes compared with their potencies as competitive dopamine D₂ receptor antagonists. Many of these compounds are selective (defined as > 10-fold relative potency) for the D₂ receptor over all other sites. These D₂-selective compounds, including haloperidol, thiothixene, and fluphenazine, have the highest propensity to induce extrapyramidal side effects. None of the atypical agents displayed a preference for D₂, as they were either equipotent at D₂ and 5-HT_2A, or preferred 5-HT_2A sites (Fig. 3A). Only three compounds, sertindole, M100907, and amperozide, displayed 5-HT_2A receptor selectivity compared with D₂. While these compounds have been shown to possess antipsychotic efficacy, and two are widely considered to be atypical, their clinical profiles are currently not well established (Axelsson et al., 1991; van Kammen et al., 1996). This analysis confirms not only the critical role of 5-HT_2A receptors in defining atypicality of an antipsychotic (Stockmeier et al., 1993), but also shows that a small number of existing clinically efficacious agents are selective 5-HT_2A inverse agonists. Compounds with H₁ receptor selectivity, namely clozapine and perlapine (Fig. 3C), are known to produce significant sedation in routine clinical use, and are often used in agitated patients with prominent sleep disturbances. That so many antipsychotics possess H₁ receptor inverse agonist properties, yet only a small subset of these cause weight gain, argues against this site being responsible for this particular side effect of these drugs. Interestingly, not a single compound tested was selective for the 5-HT_2C receptor (Fig. 3D). Finally, these data do not support the recent hypothesis that inverse agonism of 5-HT_2C receptors defines an atypical clinical profile (Herrick-Davis et al., 2000).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Correlations of antipsychotic activity at D2 versus 5-HT2A, 5-HT2B, 5-HT2C, and H1 receptors. Comparison of the potency of a series of antipsychotics as D2 receptor competitive antagonists versus 5-HT2A, 5-HT2B, 5-HT2C, and H1 receptor inverse agonists. Inverse agonist values are expressed as EC50 values, while competitive antagonist values are expressed as Ki values. All values are in nanomolar units and are derived from data presented in Table 2. Filled line, a one to one correlation in receptor potencies; dashed lines, 10-fold selectivity for D2 or the various monoamine receptors, respectively. A, the 5-HT2A receptor; B, the 5-HT2B receptor; C, the histamine H1; and D, the 5-HT2C receptor. All compounds are coded according to their designation in Table 2, where any with potencies above 1 µM were excluded from the analysis.

The present observations lend support to the hypothesis that selective 5-HT_2A inverse agonists devoid of D₂ receptor competitive antagonism will have antipsychotic efficacy in humans. Since chronic blockade of D₂ receptors is responsible for severe motor and cognitive side effects, compounds that can maintain antipsychotic efficacy without D₂ blockade would probably result in novel agents with truly unique clinical features. Although clinical experience with selective 5-HT_2A inverse agonists is limited, sertindole and amperozide appear to have a lower liability for these side effects than even the mixed atypical agents (Axelsson et al., 1991; van Kammen et al., 1996). One could speculate that selective 5-HT_2A inverse agonists will also have unique efficacy profiles, with distinct advantages in maintenance therapy, and perhaps improved efficacy in patient subgroups that display heightened serotonergic or diminished glutaminergic tone (see discussion below). That AC-90179 attenuated DOI-induced head twitches in mice and PPI disruptions in rats is consistent with a 5-HT_2A receptor mechanism of action in vivo and with antipsychotic-like efficacy. The attenuations by AC-90179 of both the hyperactivity and the disruption of PPI produced by dizocilpine are similar to the effects observed with M100907 (Martin et al., 1997; Varty et al., 1999). That AC-90179 attenuated dizocilpine-induced but not amphetamine-induced hyperactivity is consistent with an atypical-like antipsychotic profile. Furthermore, the 30-fold potency separation between activity against DOI or dizocilpine-induced effects in mice and spontaneous locomotor activity in rats suggest a wide therapeutic index for efficacy versus motor side effects. These data support the notion that selective 5-HT_2A receptor inverse agonists, such as AC-90179, will be efficacious and lack the side effects of compounds in current use.

The observation that some existing therapeutic agents possess negative intrinsic activity at various receptors raises intriguing possibilities as to the role of constitutive receptor activity in vivo. Studies with transgenic mice that were designed by overexpression to increase adrenergic receptor tone in their cardiovascular systems have revealed that -blockers with negative intrinsic efficacy are physiologically distinct from those that lack this molecular property (Bond et al., 1995; Nagaraja et al., 1999). Native histamine H₃ receptors display significant constitutive activity in vivo, and physiological distinctions between competitive antagonists and inverse agonists were noted (Morisset et al., 2000). Prevailing theories as to the pathophysiological basis of schizophrenia center on the finding that antipsychotics are D₂ and 5-HT_2A receptor competitive antagonists. Results of studies designed to document elevated dopaminergic or serotonergic neurotransmitter levels in the central nervous system of schizophrenics have been mixed to date. The present findings argue that levels of constitutive receptor activity may be the critical determinant of neurotransmitter tone in the central nervous system. Recent studies concerning the role of 5-HT_2A receptors and their relationship to glutamatergic systems have concluded that elevated 5-HT_2A receptor tone, and the attenuation of such tone, is a critical property for efficacy as an antipsychotic (Martin et al., 1998). Similarly, as the number of human disorders known to be caused by dominant mutations in GPCRs increases (Birnbaumer, 1995), it becomes reasonable to suggest that activating mutations in 5-HT_2A receptors may be causative or predisposing to neuropsychiatric disease, or modulate response to treatment with antipsychotics (Arranz et al., 2000). Lastly, the creation of a transgenic mouse model that exhibits increased 5-HT_2A receptor activity may provide an excellent preclinical model of antipsychotic efficacy.

The discovery of novel therapeutic compounds used to occur without knowledge of an actual molecular target, and it was frequently based on the serendipitous observation of clinical efficacy of a parent chemical structure. Antipsychotics are a good example, as the prototypical agent chlorpromazine was developed as an anesthetic adjuvant, but later was found to be effective in the management of human psychoses (Deniker, 1990). A similarly serendipitous path led to the initial discovery of antidepressants, anxiolytics, and mood stabilizers. We have explored the functional nature of the interaction between existing drugs and many of their potential targets. This evidence-based approach has elucidated the importance of inverse agonism at an established target as an efficacy mechanism for antipsychotics and has led to a successful drug discovery effort that exploits the observation of negative intrinsic activity of GPCRs. Similar evidence-based approaches to related agents like antidepressants may lead to the development of improved therapeutics for many neuropsychiatric disorders.