Constitutively active mutants of the histamine H1 receptor suggest a conserved hydrophobic asparagine-cage that constrains the activation of class A G protein-coupled receptors.

The aim of this study was to create and characterize constitutively active mutant (CAM) histamine H(1) receptors (H(1)R) using random mutagenesis methods to further investigate the activation process of the rhodopsin-like family of G protein-coupled receptors (GPCRs). This approach identified position 6.40 in TM 6 as a "hot spot" because mutation of Ile6.40(420) either to Glu, Gly, Ala, Arg, Lys, or Ser resulted in highly active CAM H(1)Rs, for which almost no histamine-induced receptor activation response could be detected. The highly conserved hydrophobic amino acid at position 6.40 defines, in a computational model of the H(1)R, the asparagine cage motif that restrains the side chain of Asn7.49 of the NPxxY motif toward transmembrane domain (TM 6) in the inactive state of the receptor. Mutation of the asparagine cage into Ala or Gly, removing the interfering bulky constraints, increases the constitutive activity of the receptor. The fact that the Ile6.40(420)Arg/Lys/Glu mutant receptors are highly active CAM H(1)Rs leads us to suggest that a positively charged residue, presumably the highly conserved Arg3.50 from the DRY motif, interacts in a direct or an indirect (through other side chains or/and internal water molecules) manner with the acidic Asp2.50..Asn7.49 pair for receptor activation.

Differential modulation of inflammatory pain by a selective estrogen receptor beta agonist.

To understand the contribution of the estrogen receptor beta, the potent and selective agonist ERb-131 was evaluated in animal models of inflammatory pain. In paradigms of acute and persistent inflammatory pain, ERb-131 did not alleviate the nociception induced by either carrageenan or formalin. However, in the chronic complete Freund's adjuvant model, ERb-131 resolved both inflammatory and hyperalgesic components. Thus, ERb-131 is sufficient to alleviate chronic but not acute inflammatory pain states.

Broad modulation of neuropathic pain states by a selective estrogen receptor beta agonist.

The effects of estrogens on pain perception remain controversial. In animal models, both beneficial and detrimental effects of non-selective estrogens have been reported. ERb-131 a non-steroidal estrogen receptor beta ligand was evaluated in several pain animal models involving nerve injury or sensitization. Using functional and binding assays, ERb-131 was characterized as a potent and selective estrogen receptor beta agonist. In vivo, ERb-131 was devoid of estrogen receptor alpha activity as assessed in a rat uterotrophic assay. ERb-131 alleviated tactile hyperalgesia induced by capsaicin, and reversed tactile allodynia caused by spinal nerve ligation and various chemical insults. Moreover, ERb-131 did not influence the pain threshold of normal healthy animals. Thus, estrogen receptor beta agonism is a critical effector in attenuating a broad range of anti-nociceptive states.

Monitoring interactions between receptor tyrosine kinases and their downstream effector proteins in living cells using bioluminescence resonance energy transfer.

A limited number of whole-cell assays allow monitoring of receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. We present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase (RTK) superfamily. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTKs and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for nine RTKs derived from four subfamilies [erythroblastic leukemia viral (v-erb-b) oncogene homolog (ErbB), platelet-derived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLCgamma1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in the carboxyl terminus. RTK BRET assays are highly sensitive for quantifying ligand-independent (constitutive), agonist-induced, or antagonist-inhibited RTK activity levels. We studied the signaling properties of the PDGF receptor, alpha polypeptide (PDGFRA) isoforms (V561D; D842V and delta842-845) carrying activating mutations identified in gastrointestinal stromal tumors (GIST). All three PDGFRA isoforms are fully constitutively activated, insensitive to the growth factor PDGF-BB, but show differential sensitivity of their constitutive activity to be inhibited by the inhibitor imatinib (Gleevec). Epidermal growth factor receptor (EGFR) BRET structure-function studies identify the tyrosine residues 1068, 1114, and 1148 as the main residues mediating the interaction of EGFR with the adapter protein Grb2. The BRET technology provides an assay platform to study signaling pathway-specific RTK structure-function and will facilitate drug discovery efforts for the identification of novel RTK modulators.

Integrative functional assays, chemical genomics and high throughput screening: harnessing signal transduction pathways to a common HTS readout.

Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.

Characterization of the Mas-related gene family: structural and functional conservation of human and rhesus MrgX receptors.

Recently, a large family of G-protein-coupled receptors called Mas-related genes (Mrgs), which is selectively expressed in small-diameter sensory neurons of dorsal root ganglia, was described. A subgroup of human Mrg receptors (MrgX1-X4) is not found in rodents and this has hampered efforts to define the physiological roles of these receptors. MrgX receptors were cloned from rhesus monkey and functionally characterized alongside their human orthologs. Most of the human and rhesus MrgX receptors displayed high constitutive activity in a cellular proliferation assay. Proliferative responses mediated by human or rhesus MrgX1, or rhesus MrgX2 were partially blocked by pertussis toxin (PTX). Proliferative responses mediated by rhesus MrgX3 and both human and rhesus MrgX4 were PTX insensitive. These results indicate that human and rhesus MrgX1 and MrgX2 receptors activate both Gq- and Gi-regulated pathways, while MrgX3 and MrgX4 receptors primarily stimulate Gq-regulated pathways. Peptides known to activate human MrgX1 and MrgX2 receptors activated the corresponding rhesus receptors in cellular proliferation assays, Ca(2+)-mobilization assays, and GTP-gammaS-binding assays. Cortistatin-14 was selective for human and rhesus MrgX2 receptors over human and rhesus MrgX1 receptors. BAM22 and related peptides strongly activated human MrgX1 receptors, but weakly activated rhesus MrgX1, human MrgX2, and rhesus MrgX2 receptors. These data suggest that the rhesus monkey may be a suitable animal model for exploring the physiological roles of the MrgX receptors.

Discovery of a potent, orally available, and isoform-selective retinoic acid beta2 receptor agonist.

4'-Octyl-4-biphenylcarboxylic acid (1g, AC-55649) was identified as a highly isoform-selective agonist at the human RARbeta2 receptor in a functional intact cell-based screening assay. The subsequent hit to lead optimization transformed the lipophilic, poorly soluble hit into a more potent and orally available compound (2, AC-261066) with retained beta2 selectivity and greatly improved physiochemical properties. Being an isoform-selective RARbeta2 receptor agonist that discriminates between nuclear receptor isoforms having identical ligand binding domains, 2 will be useful as a pharmacological research tool but also a valuable starting point for drug development.

Identification of novel subtype selective RAR agonists.

Drugs targeting retinoid receptors have been developed to treat a variety of therapeutic indications, but their success has been limited in part due to lack of selectivity. A novel functional cell-based assay R-SATtrade mark was employed to screen a small molecule chemical library and identify a variety of novel RAR agonists with various subtype selectivities, including RARbeta/gamma and RARgamma selective agonists. A novel class of synthetic compounds that distinguishes between the different RARbeta isoforms is described. This pharmacophore displays anti-proliferative activity and induces differentiation in a neuronal cell line, consistent with a classical retinoid mechanism of action while providing unique subtype selectivity. These novel subtype selective RAR agonists could serve as powerful tools to probe into subtype and isoform-specific retinoid function.

Discovery of the first nonpeptide agonist of the GPR14/urotensin-II receptor: 3-(4-chlorophenyl)-3-(2- (dimethylamino)ethyl)isochroman-1-one (AC-7954).

A functional cell-based screen identified 3-(4-chlorophenyl)-3-(2-(dimethylamino)ethyl)isochroman-1-one hydrochloride (AC-7954, 1) as a nonpeptidic agonist of the urotensin-II receptor. Racemic 1 had an EC50 of 300 nM at the human UII receptor and was highly selective. Testing of the enantiopure (+)- and (-)- 1 revealed that the UII receptor activity of racemic 1 resides primarily in (+)-1. Being a selective nonpeptidic druglike UII receptor agonist, (+)-1 will be useful as a pharmacological research tool and a potential drug lead.

Functional screening of drug target genes: m1 muscarinic acetylcholine receptor phenotypes in degenerative dementias.

BACKGROUND AND OBJECTIVES: A number of recent studies surveying single nucleotide polymorphisms within the exonic regions of human genes have revealed a significant number of such variants, including many non-synonymous variants. This highlights the need to directly identify, within individual clinically well-defined patients, those variants that alter protein function as well as structure. We report on the development of a novel phenotypic screening process that combines high-throughput molecular cloning techniques with functional expression utilizing the cell-based assay R-SAT. METHODS: We applied the phenotypic screening process to an analysis of the m1 muscarinic acetylcholine receptor (CHRM1) gene in a cohort of 74 individuals, including 48 diagnosed with neurodegenerative disease, primarily Alzheimer disease, who have been stratified according to their clinical response to the acetylcholinesterase inhibitor donepezil. Phenotypic screening of the CHRM1 gene involved PCR-based amplification from genomic DNA and heterologous expression in mammalian cells. RESULTS: Phenotypic screening yielded functional responses to the agonist carbachol displaying a mean potency (-pEC(50)+/- standard deviation) of 5.8 +/- 0.2, which did not differ from that observed with expression of the wild-type receptor gene (6.0 +/- 0.3). No altered levels of constitutive receptor activity were observed. Dideoxy sequencing did not reveal any non-synonymous variants in the coding exon of this gene within this clinical cohort, while detecting three synonymous variants. CONCLUSION: The results confirm that the m1 receptor gene (CHRM1) is not highly polymorphic in the human population, suggesting that genetic variation within the coding exon of this gene is not a contributing factor to the clinical variability observed during treatment of dementia with cholinergic enhancement therapies.

Pharmacology of polymorphic variants of the human 5-HT1A receptor.

The 5-HT1A receptor is a critical mediator of serotonergic (5-HT) function. We have identified 13 potential single nucleotide polymorphisms resulting in amino acid changes throughout the human 5-HT1A receptor. The pharmacological profiles of these 13 polymorphic variants were then characterized using a high-throughput assay based on ligand-dependent transformation of NIH/3T3 cells. The majority of the polymorphic variants displayed wild-type pharmacological profiles in response to a panel of well-established agonists at the 5-HT1A receptor. However, the A50V polymorphic variant, which had an alanine to valine substitution in transmembrane 1, exhibited a loss of detectable response to 5-HT. Interestingly, all other agonists tested, including buspirone, lisuride, and (+)8-OH-DPAT, exhibited efficacies similar to that of the wild-type receptor. The competitive antagonist, methiothepin, also displayed a 19-fold decrease in potency at the A50V variant receptor. However, both 5-HT and methiothepin were able to compete for [3H]WAY-100635 binding to the A50V variant with affinities similar to the wild-type receptor. Moreover, the Bmax of [3H]WAY-100635 binding was 14-fold lower for the A50V variant than for the wild-type receptor. Thus, the A50V receptor variant exhibited ligand-specific functional alterations in addition to lower expression levels. These data suggest a previously unappreciated role for transmembrane 1 in mediating 5-HT response at the 5-HT1A receptor. Furthermore, individuals that potentially harbor the A50V polymorphism might display aberrant affective behaviors and altered responses to drugs targeting the 5-HT1A receptor.

Discovery of novel peptide/receptor interactions: identification of PHM-27 as a potent agonist of the human calcitonin receptor.

Many naturally occurring peptides exhibit a high degree of promiscuity across G-protein coupled receptor subtypes. The degree to which this phenomenon occurs, and its physiological significance is not well characterized. In addition, many 'orphan' peptides exist for which there are no known receptors. Therefore, to identify novel interactions between biologically active peptides and G-protein coupled receptors, a library of nearly 200 peptides was screened against the human calcitonin (hCTr), human Parathyroid Hormone (PTH1R), human Corticotropin Releasing Factor (CRF1), and the human Glucagon-like peptide (GLP1) receptors using a cell-based functional assay (Receptor Selection and Amplification Technology). Functional profiling revealed that the 'orphan peptide' PHM-27 selectively activated the hCTr; no activity was observed at the PTH1, CRF1, or GLP1 receptors. PHM-27 was a potent agonist at the hCTr, with similar efficacy as human calcitonin, and a potency of 11 nM. These results were confirmed in cyclic AMP assays. Responses to calcitonin and PHM-27 could be suppressed by the antagonist salmon calcitonin (8-32). In competition binding studies, salmon calcitonin (8-32), calcitonin, and PHM-27 were each able to inhibit (125)I-calcitonin from cell membranes containing transiently expressed hCTr. These results indicate that the orphan peptide PHM-27 is a potent agonist at the hCTr.

Psychosis of Parkinson's disease: serotonin 2A receptor inverse agonists as potential therapeutics.

Parkinson's disease (PD) is a movement disorder characterized by progressive degeneration of central dopaminergic systems. Current therapies designed to augment dopaminergic neurotransmission effectively treat the motoric aspects of the disease, however, with prolonged use, they produce a range of treatment-limiting side effects. Of these, neuropsychiatric abnormalities including hallucinosis and psychosis are common, disabling and refractory to most current therapies. This review describes the clinical syndrome of psychosis in PD and data regarding the efficacy and tolerability of existing antipsychotic agents, and presents the scientific rationale for the development of serotonin 2A receptor inverse agonists as potential therapeutic agents for treatment-induced psychosis of PD.

PET analysis of the 5-HT2A receptor inverse agonist ACP-103 in human brain.

The mechanisms underlying the clinical properties of atypical antipsychotics have been postulated to be mediated, in part, by interactions with the 5-HT2A receptor. Recently, it has been recognized that clinically effective antipsychotic drugs are 5-HT2A receptor inverse agonists rather than neutral antagonists. In the present study, which is part of the clinical development of the novel, selective 5-HT2A receptor inverse agonist ACP-103, we applied positron emission tomography (PET) with the radioligand [11C]N-methylspiperone ([11C]NMSP) to study the relationship between oral dose, plasma level, and uptake of ACP-103 in living human brain. The safety of drug administration was also assessed. Four healthy volunteers were examined by PET at baseline, and after the oral administration of various single doses of ACP-103. Two subjects each received 1, 5, and 20 mg doses, and two subjects each received 2, 10, and 100 mg doses, respectively. ACP-103 was well tolerated. Detectable receptor binding was observed at very low ACP-103 serum levels. Cortical [11C]NMSP binding was found to be dose-dependent and fitted well to the law of mass action. A reduction in binding was detectable after an oral dose of ACP-103 as low as 1 mg, and reached near maximal displacement following the 10-20 mg dose. In conclusion, administration of ACP-103 to healthy volunteers was found to be safe and well tolerated, and single oral doses as low as 10 mg were found to fully saturate 5-HT2A receptors in human brain as determined by PET.

Identification of novel selective V2 receptor non-peptide agonists.

Peptides with agonist activity at the vasopressin V(2) receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. Of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V(1b) receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V(2) receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT((R))), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V(2) receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V(2) receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V(2) receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V(2) receptor agonist deficiency.

ACP-103, a 5-hydroxytryptamine 2A receptor inverse agonist, improves the antipsychotic efficacy and side-effect profile of haloperidol and risperidone in experimental models.

Dopamine D(2) receptor antagonism contributes to the therapeutic action of antipsychotic drugs (APDs) but also produces undesirable side effects, including extrapyramidal motor deficits, cognitive dulling, and prolactinemia. The introduction of atypical APDs was a significant advancement in the treatment of schizophrenia. Whereas these agents are D(2) receptor antagonists, they are also potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonists, a feature that may explain their improved efficacy and tolerability. Recently, we reported that N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103), a novel selective 5-HT(2A) receptor inverse agonist that fails to bind D(2) receptors, is active in several models predictive of antipsychotic activity. Using ACP-103, we tested the hypothesis that combining high levels of 5-HT(2A) inverse agonism with low levels of D(2) antagonism would result in a favorable interaction, such that antipsychotic efficacy could be achieved with reduced D(2) receptor-related adverse effects. Here we show that ACP-103 1) potently inhibited head-twitching produced by the 5-HT(2A/2C) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine, 2) increased the potency of haloperidol against amphetamine-induced hyperactivity, 3) interacted synergistically with haloperidol or risperidone to suppress hyperactivity induced by the N-methyl-d-aspartate receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and, by contrast, 4) attenuated haloperido-l- or risperidone-induced prolactinemia. ACP-103 also attenuated catalepsy produced by haloperidol or risperidone. However, the doses that were required for this effect were higher than would be expected for a 5-HT(2A) receptor-mediated mechanism. These data indicate that utilizing ACP-103 as an adjunctive therapy to currently used APDs may result in enhanced antipsychotic efficacy while reducing adverse effects including those attributable to D(2) receptor antagonism.

Identification of the atypical L-type Ca2+ channel blocker diltiazem and its metabolites as ghrelin receptor agonists.

Using a high-throughput functional screen, the atypical L-type Ca2+ channel blocker diltiazem was discovered to be an agonist at the human ghrelin (GHSR1a) receptor. In cellular proliferation, Ca2+ mobilization, and bioluminescence resonance energy transfer (BRET-2) assays, diltiazem was a partial agonist at GHSR1a receptors, with 50 to 80% relative efficacy compared with the GHSR1a peptide agonist GHRP-6, and high nanomolar to low micromolar potency, depending upon the assay. Seven of the known primary metabolites of diltiazem were synthesized, and three of them (MA, M1, and M2) were more efficacious and/or more potent than diltiazem at GHSR1a receptors, with a rank order of agonist activity of M2 > M1 > MA > diltiazem, whereas M4 and M6 metabolites displayed weak agonist activity, and the M8 and M9 metabolites were inactive. Binding affinities of diltiazem and these metabolites to GHSR1a receptors followed a similar rank order. In vivo tests showed that diltiazem and M2 each stimulated growth hormone release in male Sprague-Dawley neonatal rats, although to a lesser degree than GHRP-6. Thus, diltiazem and chemical analogs of diltiazem represent a new class of GHSR1a receptor agonists. The possible contributions of GHSR1a receptor activation to the clinical actions of diltiazem are discussed in the context of the known beneficial cardiovascular effects of ghrelin.

Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer.

We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC50 of epidermal growth factor (EGF)=10.1+/-0.09], consistent with previous pharmacological methods for measuring EGFR activation. We applied EGFR BRET assays to study the characteristics of somatic EGFR mutations that were recently identified in lung cancer. In agreement with recent reports, we detected constitutively active mutant EGFR isoforms, which predominantly signal through the phosphatidylinositol-3-kinase/Akt pathway. The EGFR inhibitors Iressa or Tarceva are severalfold more potent in inhibiting constitutive activity of mutant EGFR isoforms compared with wild-type EGFR. Notable, our results reveal that most of the mutant EGFR isoforms tested were significantly impaired in their response to EGF. The highest level of constitutive activity and nearly complete loss of epidermal growth factor responsiveness was detected in isoforms that carry the activating mutation L858R and the secondary resistance mutation T790M. In summary, our study reveals that somatic mutations in EGFR quantitatively differ in pharmacology and signaling properties, which suggest the possibility of differential clinical responsiveness to treatment with EGFR inhibitors. Furthermore, we demonstrate that the EGFR BRET assays are a useful tool to study the pharmacology of ligand-induced interaction between EGFR and signaling pathway-specifying adapter proteins.

Structural requirements of transmembrane domain 3 for activation by the M1 muscarinic receptor agonists AC-42, AC-260584, clozapine, and N-desmethylclozapine: evidence for three distinct modes of receptor activation.

Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M(1) muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M(1). In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M(1) but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M(1) receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.

Pharmacological and behavioral profile of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103), a novel 5-hydroxytryptamine(2A) receptor inverse agonist.

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonist ACP-103 competitively antagonized the binding of [(3)H]ketanserin to heterologously expressed human 5-HT(2A) receptors with a mean pK(i) of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC(50) of 8.7. ACP-103 demonstrated lesser affinity (mean pK(i) of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC(50) 7.1 in R-SAT) at human 5-HT(2C) receptors, and lacked affinity and functional activity at 5-HT(2B) receptors, dopamine D(2) receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT(2A) receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT(2A) receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.