Development of CVN424: A Selective and Novel GPR6 Inverse Agonist Effective in Models of Parkinson Disease.

GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.

II. In vitro evidence that (-)-OSU6162 and (+)-OSU6162 produce their behavioral effects through 5-HT2A serotonin and D2 dopamine receptors.

(-)-OSU6162 has promise for treating Parkinson's disease, Huntington's disease and schizophrenia. Behavioral tests evaluating the locomotor effects of (-) and (+)-OSU6162 on 'low activity' animals (reserpinized mice and habituated rats) and 'high activity' animals (drug naive mice and non-habituated rats) revealed that both enantiomers of OSU6162 had dual effects on behavior, stimulating locomotor activity in 'low activity' animals and inhibiting locomotor activity in 'high activity' animals. To elucidate a plausible mechanism of action for their behavioral effects, we evaluated the intrinsic actions of (-)- and (+)-OSU6162, and a collection of other antipsychotic and antiparkinsonian agents at 5-HT2A and D2 receptors in functional assays with various degrees of receptor reserve, including cellular proliferation, phosphatidyl inositol hydrolysis, GTPγS and beta-arrestin recruitment assays. We also tested for possible allosteric actions of (-)-OSU6162 at D2 receptors. Both enantiomers of OSU6162 were medium intrinsic activity partial agonists at 5-HT2A receptors and low intrinsic activity partial agonists at D2 receptors. (+)-OSU6162 had higher efficacy at 5-HT2A receptors, which correlated with its greater stimulatory activity in vivo, but (-)-OSU6162 had higher potency at D2 receptors, which correlated with its greater inhibitory activity in vivo. (-)-OSU6162 did not display any convincing allosteric properties. Both (+)- and (-)-OSU6162 were significantly less active at 27 other monoaminergic receptors and reuptake transporters tested suggesting that D2 and 5-HT2A receptors play crucial roles in mediating their behavioral effects. Compounds with balanced effects on these two receptor systems may offer promise for treating neuropsychiatric diseases.

First-Time Disclosure of CVN424, a Potent and Selective GPR6 Inverse Agonist for the Treatment of Parkinson's Disease: Discovery, Pharmacological Validation, and Identification of a Clinical Candidate.

Parkinson's disease (PD) is a chronic and progressive movement disorder with the urgent unmet need for efficient symptomatic therapies with fewer side effects. GPR6 is an orphan G-protein coupled receptor (GPCR) with highly restricted expression in dopamine receptor D2-type medium spiny neurons (MSNs) of the indirect pathway, a striatal brain circuit which shows aberrant hyperactivity in PD patients. Potent and selective GPR6 inverse agonists (IAG) were developed starting from a low-potency screening hit (EC50 = 43 µM). Herein, we describe the multiple parameter optimization that led to the discovery of multiple nanomolar potent and selective GPR6 IAG, including our clinical compound CVN424. GPR6 IAG reversed haloperidol-induced catalepsy in rats and restored mobility in the bilateral 6-OHDA-lesioned rat PD model demonstrating that inhibition of GPR6 activity in vivo normalizes activity in basal ganglia circuitry and motor behavior. CVN424 is currently in clinical development to treat motor symptoms in Parkinson's disease.

Discovery of TAK-041: a Potent and Selective GPR139 Agonist Explored for the Treatment of Negative Symptoms Associated with Schizophrenia.

The orphan G-protein-coupled receptor GPR139 is highly expressed in the habenula, a small brain nucleus that has been linked to depression, schizophrenia (SCZ), and substance-use disorder. High-throughput screening and a medicinal chemistry structure-activity relationship strategy identified a novel series of potent and selective benzotriazinone-based GPR139 agonists. Herein, we describe the chemistry optimization that led to the discovery and validation of multiple potent and selective in vivo GPR139 agonist tool compounds, including our clinical candidate TAK-041, also known as NBI-1065846 (compound 56). The pharmacological characterization of these GPR139 agonists in vivo demonstrated GPR139-agonist-dependent modulation of habenula cell activity and revealed consistent in vivo efficacy to rescue social interaction deficits in the BALB/c mouse strain. The clinical GPR139 agonist TAK-041 is being explored as a novel drug to treat negative symptoms in SCZ.

Differential regulation of muscarinic M1 receptors by orthosteric and allosteric ligands.

BACKGROUND: Activation of muscarinic M1 receptors is mediated via interaction of orthosteric agonists with the acetylcholine binding site or via interaction of allosteric agonists with different site(s) on the receptor. The focus of the present study was to determine if M1 receptors activated by allosteric agonists undergo the same regulatory fate as M1 receptors activated by orthosteric agonists. RESULTS: The orthosteric agonists carbachol, oxotremorine-M and pilocarpine were compared to the allosteric agonists AC-42, AC-260584, N-desmethylclozapine and xanomeline. All ligands activated M1 receptors and stimulated interaction of the receptors with beta-arrestin-1. All ligands reduced cell surface binding and induced the loss of total receptor binding. Receptor internalization was blocked by treatment with hypertonic sucrose indicating that all ligands induced formation of clathrin coated vesicles. However, internalized receptors recycled to the cell surface following removal of orthosteric, but not allosteric agonists. Whereas all ligands induced loss of cell surface receptor binding, no intracellular vesicles could be observed after treatment with AC-260584 or xanomeline. Brief stimulation of M1 receptors with AC-260584 or xanomeline resulted in persistent activation of M1 receptors, suggesting that continual receptor signaling might impede or delay receptor endocytosis into intracellular vesicles. CONCLUSION: These results indicate that allosteric agonists differ from orthosteric ligands and among each other in their ability to induce different regulatory pathways. Thus, signaling and regulatory pathways induced by different allosteric ligands are ligand specific.

Identification and characterization of novel small-molecule protease-activated receptor 2 agonists.

We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide], each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca(2+) mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH(2) had similar potency, whereas SLIGRL-NH(2) was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.

Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator.

Because of the limitations and liabilities of current testosterone therapies, non-steroidal tissue-selective androgen receptor modulators may provide a clinically meaningful advance in therapy. Using a functional cell-based assay AC-262536 was identified as a potent and selective AR ligand, with partial agonist activity relative to the natural androgen testosterone. A 2-week chronic study in castrated male rats indicated that AC-262536 significantly improves anabolic parameters in these animals, especially in stimulating the growth of the levator ani and in suppressing elevated LH levels. In sharp contrast to testosterone, AC-262536 has weak androgenic effects, as measured by prostate and seminal vesicle weights. Thus, AC-262536 represents a novel class of selective androgen receptor modulators (SARMs) with beneficial anabolic effects.

The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation.

The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensin AT(1) receptor signalling is illustrated by the common use of angiotensin AT(1) receptor-inverse agonists in clinical practice. It is well established that rodent orthologues of the angiotensin AT(1) receptor can selectively signal through G protein-dependent and -independent mechanisms in recombinant expression systems, primary cells and in vivo. The in vivo work clearly demonstrates profoundly different cellular consequences of angiotensin AT(1) receptor signalling in the cardiovascular system, suggesting pharmacological potential for drugs which specifically affect a subset of angiotensin AT(1) receptor actions. However, it is currently unknown whether the human angiotensin AT(1) receptor can signal through G protein-independent mechanisms - and if so, what the physiological impact of such signalling is. We have performed a detailed pharmacological analysis of the human angiotensin AT(1) receptor using a battery of angiotensin analogues and registered drugs targeting this receptor. We show that the human angiotensin AT(1) receptor signals directly through G protein-independent pathways and supports NIH3T3 cellular proliferation. The realization of G protein-independent signalling by the human angiotensin AT(1) receptor has clear pharmacological implications for development of drugs with pathway-specific actions and defined biological outcomes.

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.

Glutamate receptor genes: susceptibility factors in schizophrenia and depressive disorders?

Schizophrenia, depression, and bipolar disorder are three major neuropsychiatric disorders that are among the leading causes of disability and have enormous economic impacts on our society. Although several neurotransmitter systems have been suggested to play a role in their etiology, we still have not identified any gene or molecular mechanism that might lead to genetic susceptibility for or protection against these neuropsychiatric disorders. The glutamatergic receptor system, and in particular the N-methyl-D-aspartate (NMDA) receptor complex, has long been implicated in their etiology. I review the current molecular evidence that supports a critical role for the glutamatergic receptor system in schizophrenia and the potential involvement of this receptor system in depression and bipolar disorder. It is likely that mutations in glutamate receptor genes might alter the risk of developing one of these disorders. Potential future research directions designed to identify these mutations and to elucidate their effect on mental health will be discussed.

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.

AC-260584, an orally bioavailable M(1) muscarinic receptor allosteric agonist, improves cognitive performance in an animal model.

The recent discovery of allosteric potentiators and agonists of the muscarinic M(1) receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC(50) 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M(1) receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M(1) receptor over the M(2), M(3), M(4) and M(5) muscarinic receptor subtypes. Using GTPgammaS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M(1) receptor activation since it was not observed in M(1) knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M(1) receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M(1) selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimer's disease.

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.

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.