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Blockade of the serotonin 5-HT(2A) G protein-coupled receptor (5-HT(2A)R) is a fundamental pharmacological characteristic of numerous antipsychotic medications, which are FDA-approved to treat schizophrenia, bipolar disorder, and as adjunctive therapies in major depressive disorder. Meanwhile, activation of the 5-HT(2A)R by serotonergic psychedelics may be useful in treating neuropsychiatric indications, including major depressive and substance use disorders. Serotonergic psychedelics and other 5-HT(2A)R agonists, however, often bind other receptors, and standard 5-HT(2A)R antagonists lack sufficient selectivity to make well-founded mechanistic conclusions about the 5-HT(2A)R-dependent effects of these compounds and the general neurobiological function of 5-HT(2A)Rs. This review discusses the limitations and strengths of currently available “selective” 5-HT(2A)R antagonists, the molecular determinants of antagonist selectivity at 5-HT(2A)Rs, and the utility of molecular pharmacological and computational methods in guiding the discovery of novel unambiguously selective 5-HT(2A)R antagonists.

The prevalence of seizures in individuals with fragile X syndrome (FXS) is ~25%; however, there are no reports of spontaneous seizures in the Fmr1 knockout mouse model of FXS. Herein, we report that 48% of adult (median age P96), Fmr1 knockout mice from our colony were found expired in their home cages. We observed and recorded adult Fmr1 knockout mice having spontaneous convulsions in their home cages. In addition, we captured by electroencephalography an adult Fmr1 knockout mouse having a spontaneous seizure-during preictal, ictal, and postictal phases-which confirmed the presence of a generalized seizure. We did not observe this phenotype in control conspecifics or in juvenile (ageP35) Fmr1 knockout mice. We hypothesized that chronic, random, noise perturbations during development caused the phenotype. We recorded decibels (dB) in our vivarium. The average was 61 dB, but operating the automatic door to the vivarium caused spikes to 95 dB. We modified the door to eliminate noise spikes, which reduced unexpected deaths to 33% in Fmr1 knockout mice raised from birth in this environment (P = 0.07). As the modifications did not eliminate unexpected deaths, we further hypothesized that building vibrations may also be a contributing factor. After installing anti-vibration pads underneath housing carts, unexpected deaths of Fmr1 knockout mice born and raised in this environment decreased to 29% (P 0.01 compared to the original environment). We also observed significant sex effects, for example, after interventions to reduce sound and vibration, significantly fewer male, but not female, Fmr1 knockout mice died unexpectedly (P 0.001). The spontaneous seizure phenotype in our Fmr1 knockout mice could serve as a model of seizures observed in individuals with FXS, potentially offering a new translationally-valid phenotype for FXS research. Finally, these observations, although anomalous, serve as a reminder to consider gene-environment interactions when interpreting data derived from Fmr1 knockout mice.

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disabilities and a plethora of neuropsychiatric symptoms. FXS is the leading monogenic cause of autism spectrum disorder (ASD), which is defined clinically by repetitive and/or restrictive patterns of behavior and social communication deficits. Epilepsy and anxiety are also common in FXS and ASD. Serotonergic neurons directly innervate and modulate the activity of neurobiological circuits altered in both disorders, providing a rationale for investigating serotonin receptors (5-HTRs) as targets for FXS and ASD drug discovery. Previously we unveiled an orally active aminotetralin, (S)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), that exhibits partial agonist activity at 5-HT1ARs, 5-HT2CRs, and 5-HT7Rs and that reduces repetitive behaviors and increases social approach behavior in wild-type mice. Here we report that in an Fmr1 knockout mouse model of FXS and ASD, FPT is prophylactic for audiogenic seizures. No FPT-treated mice displayed audiogenic seizures, compared to 73% of vehicle-treated mice. FPT also exhibits anxiolytic-like effects in several assays and increases social interactions in both Fmr1 knockout and wild-type mice. Furthermore, FPT increases c-Fos expression in the basolateral amygdala, which is a preclinical effect produced by anxiolytic medications. Receptor pharmacology assays show that FPT binds competitively and possesses rapid association and dissociation kinetics at 5-HT1ARs and 5-HT7Rs, yet has slow association and rapid dissociation kinetics at 5-HT2CRs. Finally, we reassessed and report FPT's affinity and function at 5-HT1ARs, 5-HT2CRs, and 5-HT7Rs. Collectively, these observations provide mounting support for further development of FPT as a pharmacotherapy for common neuropsychiatric symptoms in FXS and ASD.

Synthetic cathinones (SCs) are designer, psychostimulant drugs of abuse that primarily act on monoamine transporters; little is known about their off-target liability. Abuse of pyrrolidine-containi...

There are no Food and Drug Administration approved pharmacotherapies for methamphetamine (METH) overdose, thus identifying novel drug targets to prevent this devastating adverse event is a public-health imperative. Previous research suggests that serotonin and sigma receptors may contribute to the adverse effects of METH. The present study assessed whether pretreatment with the 5-HT2A receptor antagonist M100907 or the sigma 1 (σ1) receptor antagonist BD 1047 attenuated METH-induced lethality, hyperthermia, convulsions, and seizures. Male, Swiss-Webster mice received intraperitoneal injections of M100907 (1 and 10 mg/kg), BD 1047 (10 mg/kg), or a combination of M100907 (1 mg/kg) and BD 1047 (10 mg/kg) prior to treatment with METH (78 mg/kg). Convulsions and lethality were assessed by observation, core body temperature was assessed by surgically implanted telemetric probes, and seizures were assessed by electroencephalography. M100907 reduced METH-elicited lethality from 67% to 33%, BD1047 reduced METH-elicited lethality from 67% to 50%, and combined administration of both agents eliminated lethality in all mice tested. Similarly, both agents and their combination reduced METH-elicited seizures and convulsions. None of the treatments decreased METH-induced hyperthermia. This research suggests that reducing METH-induced seizures is an important factor in reducing lethality associated with METH overdose. However, future studies should examine whether M100907 and BD 1047 modulate METH-induced hypertension and other adverse effects that may also contribute to METH overdose. Our data support the continued investigation of compounds that target 5-HT2A and σ1 receptors in METH-induced overdose, including their potential to yield emergency reversal agents.

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT(2A) receptors (5‐HT(2A)R) and muscarinic M(1) receptors (M(1)R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M(1)R (minimal displacement of [~K(d)] [3H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K(i) values at 5‐HT(2A)R. In 5‐HT(2A)R–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT(2A)R functional activity of α‐PPP, and observed it competitively antagonized 5‐HT(2A)R signaling stimulated by the 5‐HT(2)R agonist (±)‐2,5‐ dimethoxy‐4‐iodoamphetamine (DOI; K(b) = 851 nM). To assess in vivo 5‐HT(2A)R activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppressiont 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT(2A)R mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT(2A)R K(i)s > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT(2A)R in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT(2A)R antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.

Kratom alkaloids have mostly been evaluated for their opioid activity but less at other targets that could contribute to their physiological effects. Here, we investigated the in vitro and in vivo activity of kratom alkaloids at serotonin receptors (5-HTRs). Paynantheine and speciogynine exhibited high affinity for 5-HT(1A)Rs and 5-HT(2B)Rs, unlike the principal kratom alkaloid mitragynine. Both alkaloids produced antinociceptive properties in rats via an opioid receptor-independent mechanism, and neither activated 5-HT(2B)Rs in vitro. Paynantheine, speciogynine, and mitragynine induced lower lip retraction and antinociception in rats, effects blocked by a selective 5-HT(1A)R antagonist. In vitro functional assays revealed that the in vivo 5-HT(1A)R agonistic effects may be due to the metabolites 9-O-desmethylspeciogynine and 9-O-desmethylpaynantheine and not the parent compounds. Both metabolites did not activate 5-HT(2B)R, suggesting low inherent risk of causing valvulopathy. The 5-HT(1A)R agonism by kratom alkaloids may contribute to the mood-enhancing effects associated with kratom use.

The molecular characterization of bioactive molecules, for example, small molecules targeting G-protein-coupled receptors, is evolving in complexity, impacting the meaning of terms like "agonist", "antagonist", and "selective", which, in the absence of detailed definitions and scientific consensus, can be sources of confusion in the literature. We discuss this issue and offer straightforward solutions to it.

Recent preclinical and clinical studies suggest that lorcaserin, a preferential serotonin 2C receptor (5-HT(2C)R) agonist that was approved for the treatment of obesity, possesses antiepileptic properties. Here, we tested whether lorcaserin (1, 3, 5.6, 10 mg/kg) is prophylactic against audiogenic seizures (AGSs) in juvenile Fmr1 knockout mice, a mouse model of fragile X syndrome (FXS). MPEP (30 mg/kg), a non-competitive mGluR5 receptor antagonist, was used as a positive control. As lorcaserin likely engages 5-HT(2A)Rs at therapeutic doses, we pretreated one group of mice with the selective 5-HT(2A)R antagonist/inverse agonist, M100907 (0.03 mg/kg), alone or before administering lorcaserin (5.6 mg/kg), to discern putative contributions of 5-HT(2A)Rs to AGSs. We also assessed lorcaserin’s in vitro pharmacology at human (h) and mouse (m) 5-HT(2C)Rs and 5-HT(2A)Rs and its in vivo interactions at m5-HT(2C)Rs and m5-HT(2A)Rs. MPEP significantly decreased AGS prevalence (P=0.011) and lethality (P=0.038). Lorcaserin, 3 mg/kg, attenuated AGS prevalence and lethality by 14% and 32%, respectively, however, results were not statistically significant (P=0.5 and P=0.06); other doses and M100907 alone or with lorcaserin did not significantly affect AGSs. Lorcaserin exhibited full efficacy agonist activity at h5-HT(2C)Rs and m5-HT(2C)Rs, and near full efficacy agonist activity at h5-HT(2A)Rs and m5-HT(2A)Rs; selectivity for activation of 5-HT(2C)Rs over 5-HT(2A)Rs was greater for human (38-fold) compared to mouse (13-fold) receptors. Lorcaserin displayed relatively low affinities at antagonist-labeled 5-HT(2C)Rs and 5-HT(2A)Rs, regardless of species. Lorcaserin (3 and 5.6 mg/kg) increased the 5-HT(2A)R-dependent head-twitch response (HTR) elicited by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) in mice (P=0.03 and P=0.02). At 3 mg/kg, lorcaserin alone did not elicit an HTR. If mice were treated with the selective 5-HT(2C)R antagonist SB242084 (0.5 and 1 mg/kg) plus lorcaserin (3 mg/kg), a significantly increased HTR was observed, relative to vehicle (P=0.01 and P=0.03), however, the HTR was much lower than what was elicited by DOI or DOI plus lorcaserin. Lorcaserin, 3 mg/kg, significantly reduced locomotor activity on its own, an effect reversed by SB242084, and lorcaserin also dose-dependently reduced locomotor activity when administered prior to DOI (Ps

RATIONALE: Synthetic cathinones (“bath salts”) are β-ketone analogs of amphetamines, yet few studies have examined their potential neurotoxic effects. OBJECTIVE: In the current study, we assessed the persistent behavioral and neurochemical effects of exposure to the second-generation synthetic cathinone α-pyrrolidinopropiophenone (α-PPP). METHODS: Male, Swiss-Webster mice were exposed to α-PPP (80mg/kg) using a binge-like dosing regimen (QID, q2h). Behavior was assessed 4–5 days after the dosing regimen, and neurochemistry was assessed the following day. Behavior was studied using the elevated plus maze, Y-maze, and novel object recognition tests. Regional levels of dopamine, serotonin, norepinephrine, and the major dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were determined in the prefrontal cortex and striatum using high-pressure liquid chromatography. Additional experiments assessed the time courses of the effects of α-PPP on locomotor activity and core temperature using telemetry. RESULTS: Exposure to α-PPP significantly impaired performance in the Y-maze, decreased overall exploratory activity in the novel object recognition test, and resulted in regionally specific depletions in monoamine neurochemistry. In contrast, it had no significant effect on elevated plus maze performance or object discrimination in the novel object recognition test. The locomotor-stimulant effects of α-PPP were comparable to cocaine (30mg/kg), and α-PPP (80mg/kg) did not induce hyperthermia. CONCLUSIONS: α-PPP exposure results in persistent changes in exploratory behavior, spatial working memory, and monoamine neurochemistry. This research highlights potential dangers of α-PPP, including potential neurotoxicity, and suggests that the mechanisms underlying the persistent untoward effects of the cathinones may be distinct from those of the amphetamines.

The serotonin 5-HT

Classic hallucinogens share pharmacology as serotonin 5-HT2A, 5-HT2B, and 5-HT2C receptor agonists. Unique among most other Schedule 1 drugs, they are generally non-addictive and can be effective tools in the treatment of addiction. Mechanisms underlying these attributes are largely unknown. However, many preclinical studies show that 5-HT2C agonists counteract the addictive effects of drugs from several classes, suggesting this pharmacological property of classic hallucinogens may be significant. Drawing from a comprehensive analysis of preclinical behavior, neuroanatomy, and neurochemistry studies, this review builds rationale for this hypothesis, and also proposes a testable, neurobiological framework. 5-HT2C agonists work, in part, by modulating dopamine neuron activity in the ventral tegmental area—nucleus accumbens (NAc) reward pathway. We argue that activation of 5-HT2C receptors on NAc shell, GABAergic, medium spiny neurons inhibits potassium Kv1.x channels, thereby enhancing inhibitory activity via intrinsic mechanisms. Together with experiments that show that addictive drugs, such as cocaine, potentiate Kv1.x channels, thereby suppressing NAc shell GABAergic activity, this hypothesis provides a mechanism by which classic hallucinogen-mediated stimulation of 5-HT2C receptors could thwart addiction. It also provides a potential reason for the non-addictive nature of classic hallucinogens.

While exploring the structure–activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT2C receptors. In HEK293 cells expressing human 5-HT2C-INI receptors, for example, (−)-trans-3′-Br-PAT and (−)-trans-3′-Cl-PAT are agonists regarding Gαq-inositol phosphate signaling, whereas (−)-trans-3′-CF3-PAT is an inverse agonist. To investigate the ligand–receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT2C receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (Ki) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC50 functional potencies of PATs. We discovered that when...

Exposure of G protein-coupled receptors (GPCRs) to agonists can desensitize receptor signaling and lead to drug tolerance, whereas inverse agonists can sensitize signaling. For example, activation of serotonin 5-HT2C GPCRs is pharmacotherapeutic for obesity, but there is tolerance to the anorectic effect of the o n l y approved 5-HT2C agonist, lorcaserin. We tested the hypothesis that different agonists or inverse agonists differentially desensitize or sensitize, respectively, canonical 5-HT2C-mediated activation of phospholipase C (PLC) signaling in vitro. Lorcaserin, which displays potency and efficacy equal to 5-HT, desensitized the 5-HT2C receptor significantly more than 5-HT (p

Pimavanserin is a first-in-class selective serotonin 5-HT2A receptor inverse agonist approved for the treatment of Parkinson disease psychosis. This article discusses pimavanserin's mechanism of action, which patients are appropriate candidates for therapy, adverse reactions, and appropriate dosing.

Stereotypy (e.g., repetitive hand waving) is a key phenotype of autism spectrum disorder, Fragile X and Rett syndromes, and other neuropsychiatric disorders, and its severity correlates with cognitive and attention deficits. There are no effective treatments, however, for stereotypy. Perturbation of serotonin (5-HT) neurotransmission contributes to stereotypy, suggesting that distinct 5-HT receptors may be pharmacotherapeutic targets to treat stereotypy and related neuropsychiatric symptoms. For example, preclinical studies indicate that 5-HT7 receptor activation corrects deficits in mouse models of Fragile X and Rett syndromes, and clinical trials for autism are underway with buspirone, a 5-HT1A partial agonist with relevant affinity at 5-HT7 receptors. Herein, we report the synthesis, in vitro molecular pharmacology, behavioral pharmacology, and pharmacokinetic parameters in mice after subcutaneous and oral administration of (+)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine ((+)-5-FPT), a new, dual partial agonist targeting both 5-HT7 (Ki = 5.8 nM, EC50 = 34 nM) and 5-HT1A (Ki = 22 nM, EC50 = 40 nM) receptors. Three unique, heterogeneous mouse models were used to assess the efficacy of (+)-5-FPT to reduce stereotypy: idiopathic jumping in C58/J mice, repetitive body rotations in C57BL/6J mice treated with the NMDA antagonist, MK-801, and repetitive head twitching in C57BL/6J mice treated with the 5-HT2 agonist, DOI. Systemic (+)-5-FPT potently and efficaciously reduced or eliminated stereotypy in each of the mouse models without altering locomotor behavior on its own, and additional tests showed that (+)-5-FPT, at the highest behaviorally active dose tested, enhanced social interaction and did not cause behaviors indicative of serotonin syndrome. These data suggest that (+)-5-FPT is a promising medication for treating stereotypy in psychiatric disorders.

Syntheses were undertaken of derivatives of (2S, 4R)-(−)-trans-4-phenyl-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (4-phenyl-2-dimethylaminotetralin, PAT), a stereospecific agonist at the serotonin 5-HT2C G protein-coupled receptor (GPCR), with inverse agonist activity at 5-HT2A and 5-HT2B GPCRs. Molecular changes were made at the PAT C(4)-position, while preserving N, N-dimethyl substitution at the 2-position as well as trans-stereochemistry, structural features previously shown to be optimal for 5-HT2 binding. Affinities of analogs were determined at recombinant human 5-HT2 GPCRs in comparison to the phylogenetically closely-related histamine H1 GPCR, and in silico ligand docking studies were conducted at receptor molecular models to help interpret pharmacological results and guide future ligand design. In most cases, C(4)-substituted PAT analogs exhibited the same stereoselectivity ([−]-trans > [+]-trans) as the parent PAT across 5-HT2 and H1 GPCRs, albeit, with variable receptor selectivity. 4-(4′-substituted)-PAT analogs, however, demonstrated reversed stereoselectivity ([2S, 4R]-[+]-trans > [2S, 4R]-[−]-trans), with absolute configuration confirmed by single X-ray crystallographic data for the 4-(4′-Cl)-PAT analog. Pharmacological affinity results and computational results herein support further PAT drug development studies and provide a basis for predicting and interpreting translational results, including, for (+)-trans-4-(4′-Cl)-PAT and (−)-trans-4-(3′-Br)-PAT that were previously shown to be more potent and efficacious than their corresponding enantiomers in rodent models of psychoses, psychostimulant-induced behaviors, and compulsive feeding (‘binge-eating’).

Recent, well-controlled – albeit small-scale – clinical trials show that serotonergic psychedelics, including psilocybin and lysergic acid diethylamide, possess great promise for treating psychiatric disorders, including treatment-resistant depression. Additionally, fresh results from a deluge of clinical neuroimaging studies are unveiling the dynamic effects of serotonergic psychedelics on functional activity within, and connectivity across, discrete neural systems. These observations have led to testable hypotheses regarding neural processing mechanisms that contribute to psychedelic effects and therapeutic benefits. Despite these advances and a plethora of preclinical and clinical observations supporting a central role for brain serotonin 5-HT(2A) receptors in producing serotonergic psychedelic effects, lingering and new questions about mechanisms abound. These chiefly pertain to molecular neuropharmacology. This chapter is devoted to illuminating and discussing such questions in the context of preclinical experimental approaches for studying mechanisms of action of serotonergic psychedelics, classic and new.

Aripiprazole was the first antipsychotic developed to possess agonist properties at dopamine D2 autoreceptors, a groundbreaking strategy that presented a new vista for schizophrenia drug discovery. The dopamine D2 receptor is the crucial target of all extant antipsychotics, and all developed prior to aripiprazole were D2 receptor antagonists. Extensive blockade of these receptors, however, typically produces extrapyramidal (movement) side effects which plagued first-generation antipsychotics, such as haloperidol. Second-generation antipsychotics, such as clozapine, with unique polypharmacology and D2 receptor binding kinetics, have significantly lower risk of movement side effects, but can cause myriad additional ones, such as severe weight gain and metabolic dysfunction. Aripiprazole’s polypharmacology—characterized by its unique agonist activity at dopamine D2, D3 and serotonin 5-HT1A receptors as well as antagonist activity at serotonin 5-HT2A receptors—translates to successful reduction of positive, negative, and cognitive symptoms of schizophrenia, while also mitigating risk of weight gain and movement side effects. New observations, however, link aripiprazole to compulsive behaviors in a small group of patients, an unusual side effect for antipsychotics. In this review, we discuss the chemical synthesis, pharmacology, pharmacogenomics, drug metabolism, and adverse events of aripiprazole, and we present a current understanding of aripiprazole’s neurotherapeutic mechanisms, as well as the history and importance of aripiprazole to neuroscience.

The serotonin 5-HT7 G protein-coupled receptor (GPCR) is a proposed pharmacotherapeutic target for a variety of central and peripheral indications, albeit, there are no approved drugs selective for binding 5-HT7. We previously reported that a lead analog based on the 5-substituted-N,N-disubstituted-1,2,3,4-tetrahydronaphthalen-2-amine (5-substituted-2-aminotetralin, 5-SAT) scaffold binds with high affinity at the 5-HT7 GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT1A GPCR. Herein, we report the synthesis of novel 5-SAT analogs to develop a 3-dimensional quantitative structure—affinity relationship (3D-QSAR) at the human 5-HT7 receptor for comparison with similar studies at the highly homologous 5-HT1A receptor. We report 35 new 5-SAT ligands, some with very high affinity (Ki ≤ 1 nM) and stereoselectivity at 5-HT7 + or 5-HT1A receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT7, and, several ligands with high selectivity (up to 40-fold) at the 5-HT1A receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT7 over 5-HT1A receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT1A selectivity. In silico receptor homology modeling studies, supplemented with molecular dynamics simulations and binding free energy calculations, were used to rationalize experimentally-determined receptor selectivity and stereoselective affinity results. The data from these studies indicate that the 5-SAT chemotype, previously shown to be safe and efficacious in rodent paradigms of neurodevelopmental and neuropsychiatric disorders, is amenable to structural modification to optimize affinity at serotonin 5-HT7 vs. 5-HT1A GPCRs, as may be required for successful clinical translation.

During translational studies to develop 4-phenyl-2-dimethylaminotetralin (PAT) compounds for neuropsychiatric disorders, the (2R,4S)-trans-(+)- and (2S,4R)-trans-(-)-enantiomers of the analog 6-hydroxy-7-chloro-PAT (6-OH-7-Cl-PAT) demonstrated unusual pharmacology at serotonin (5-HT) 5-HT2 G protein-coupled receptors (GPCRs). The enantiomers had similar affinities (Ki) at human (h) 5-HT2A receptors (≈ 70 nM). In an in vivo mouse model of 5-HT2A receptor activation [(±)-(2,5)-dimethoxy-4-iodoamphetamine (DOI)-elicited head twitch], however, (-)-6-OH-7-Cl-PAT was about 5-fold more potent than the (+)-enantiomer at attenuating the DOI-elicited response. It was discovered that (+)-6-OH-7-Cl-PAT (only) had ≈ 40-fold-lower affinity at mouse (m) compared with h5-HT2A receptors. Molecular modeling and computational ligand docking studies indicated that the 6-OH moiety of (+)- but not (-)-6-OH-7-Cl-PAT could form a hydrogen bond with serine residue 5.46 of the h5-HT2A receptor. The m5-HT2A as well as m5-HT2B, h5-HT2B, m5-HT2C, and h5-HT2C receptors have alanine at position 5.46, obviating this interaction; (+)-6-OH-7-Cl-PAT also showed ≈ 50-fold lower affinity than (-)-6-OH-7-Cl-PAT at m5-HT2C and h5-HT2C receptors. Mutagenesis studies confirmed that 5-HT2A S5.46 is critical for (+)- but not (-)-6-OH-7-Cl-PAT binding, as well as function. The (+)-6-OH-7-Cl-PAT enantiomer showed partial agonist effects at h5-HT2A wild-type (WT) and m5-HT2A A5.46S point-mutated receptors but did not activate m5-HT2A WT and h5-HT2A S5.46A point-mutated receptors, or h5-HT2B, h5-HT2C, and m5-HT2C receptors; (-)-6-OH-7-Cl-PAT did not activate any of the 5-HT2 receptors. Experiments also included the (2R,4S)-trans-(+)- and (2S,4R)-trans-(-)-enantiomers of 6-methoxy-7-chloro-PAT to validate hydrogen bonding interactions proposed for the corresponding 6-OH analogs. Results indicate that PAT ligand three-dimensional structure impacts target receptor binding and translational outcomes, supporting the hypothesis that GPCR ligand structure governs orthosteric binding pocket molecular determinants and resulting pharmacology.

Classic hallucinogens share pharmacology as serotonin 5-HT2A, 5-HT2B, and 5-HT2C receptor agonists. Unique among most other Schedule 1 drugs, they are generally non-addictive and can be effective tools in the treatment of addiction. Mechanisms underlying these attributes are largely unknown. However, many preclinical studies show that 5-HT2C agonists counteract the addictive effects of drugs from several classes, suggesting this pharmacological property of classic hallucinogens may be significant. Drawing from a comprehensive analysis of preclinical behavior, neuroanatomy, and neurochemistry studies, this review builds rationale for this hypothesis, and also proposes a testable, neurobiological framework. 5-HT2C agonists work, in part, by modulating dopamine neuron activity in the ventral tegmental area—nucleus accumbens (NAc) reward pathway. We argue that activation of 5-HT2C receptors on NAc shell, GABAergic, medium spiny neurons inhibits potassium Kv1.x channels, thereby enhancing inhibitory activity via intrinsic mechanisms. Together with experiments that show that addictive drugs, such as cocaine, potentiate Kv1.x channels, thereby suppressing NAc shell GABAergic activity, this hypothesis provides a mechanism by which classic hallucinogen-mediated stimulation of 5-HT2C receptors could thwart addiction. It also provides a potential reason for the non-addictive nature of classic hallucinogens.

While exploring the structure-activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT

Ligands that activate the serotonin 5-HT2C G protein-coupled receptor (GPCR) may be therapeutic for psychoses, addiction, and other neuropsychiatric disorders. Ligands that are antagonists at the closely related 5-HT2A GPCR also may treat neuropsychiatric disorders; in contrast, 5-HT2A activation may cause hallucinations. 5-HT2C-specific agonist drug design is challenging because 5-HT2 GPCRs share 80% transmembrane (TM) homology, same second messenger signaling, and no crystal structures are reported. To help delineate molecular determinants underlying differential binding and activation of 5-HT2 GPCRs, 5-HT2A, and 5-HT2C homology models were built from the β2-adrenergic GPCR crystal structure and equilibrated in a lipid phosphatidyl choline bilayer performing molecular dynamics simulations. Ligand docking studies at the 5-HT2 receptor models were conducted with the (2R, 4S)- and (2S, 4R)-enantiomers of the novel 5-HT2C agonist/5-HT2A/2B antagonist trans-4-phenyl-N,N-dimethyl-2-aminotetralin (PAT) and its 4'-chlorophenyl congners. Results indicate PAT-5-HT2 molecular interactions especially in TM domain V are important for the (2R, 4S) enantiomer, whereas, TM domain VI and VII interactions are more important for the (2S, 4R) enantiomer.

Activation of the serotonin (5-hydroxytryptamine, 5-HT) 5HT2C G protein-coupled receptor (GPCR) is proposed as novel pharmacotherapy for obesity and neuropsychiatric disorders. In contrast, activation of the 5-HT2A and 5-HT2B GPCRs is associated with untoward hallucinogenic and cardiopulmonary effects, respectively. There is no crystal structure available to guide design of 5-HT2C receptor-specific ligands. For this reason, a homology model of the 5-HT2C receptor was built based on the crystal structure of the human β2 adrenoceptor GPCR to delineate molecular determinants of ligand–receptor interactions for drug design purposes. Computational and experimental studies were carried out to validate the model. Binding of N(CH3)2-PAT [(1R, 3S)-(−)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene], a novel 5-HT2C agonist/5-HT2A/2B inverse agonist, and its secondary [NH(CH3)-PAT] and primary (NH2-PAT) amine analogs were studied at the 5-HT2C wild type (WT) and D3.32A, S3.36A, and Y7.43A 5-HT2C point-mutated receptors. Reference ligands included the tertiary amines lisuride and mesulergine and the primary amine 5-HT. Modeling results indicated that 5-HT2C residues D3.32, S3.36, and Y7.43 play a role in ligand binding. Experimental ligand binding results with WT and point-mutated receptors confirmed the impact of D3.32, S3.36, and Y7.43 on ligand affinity.

Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed brain sites, through mechanisms that include the release of norepinephrine and acetylcholine within the amygdala. Thus, CREB antisense injections may affect memory by interfering with mechanisms of modulation, rather than storage, of memory. In the present experiment, rats received bilateral intra-amygdala infusions of CREB antisense (2 nmol/1 μL) 6 h prior to inhibitory avoidance training. In vivo microdialysis samples were collected from the right amygdala before, during, and following training. CREB antisense produced amnesia tested at 48 h after training. In addition, CREB antisense infusions dampened the training-related release of norepinephrine, and to a lesser extent of acetylcholine, in the amygdala. Furthermore, intra-amygdala infusions of the β-adrenergic receptor agonist clenbuterol administered immediately after training attenuated memory impairments induced by intra-amygdala injections of CREB antisense. These findings suggest that intra-amygdala treatment with CREB antisense may affect processes involved in modulation of memory in part through interference with norepinephrine and acetylcholine neurotransmission in the amygdala.

The present experiments examined the effects of injecting glucose into the dorsal hippocampus or dorsolateral striatum on learning rates and on strategy selection in rats trained on a T-maze that can be solved by using either a hippocampus-sensitive place or striatum-sensitive response strategy. Percentage strategy selection on a probe trial (Pcrit) administered after rats achieved criterion (nine of 10 correct choices) varied by group. All groups predominately exhibited a response strategy on a probe trial administered after overtraining, i.e., after 90 trials. In experiment 1, rats that received intrahippocampal glucose injections showed enhanced acquisition of the T-maze and showed increased use of response solutions at Pcrit compared with that of unimplanted and artificial cerebral spinal fluid (aCSF)-treated groups. These findings suggest that glucose enhanced hippocampal functions to accelerate the rate of learning and the early adoption of a response strategy. In experiment 2, rats that received intrastriatal glucose injections exhibited place solutions early in training and reached criterion more slowly than did aCSF controls, with learning rates comparable to those of unoperated and operated-uninjected controls. Relative to unoperated, operated-uninjected and glucose-injected rats, rats that received intrastriatal aCSF injections showed enhanced acquisition of the T-maze and increased use of response solutions at Pcrit. The unexpected enhanced acquisition seen after striatal aCSF injections suggests at least two possible interpretations: (1) aCSF impaired striatal function, thereby releasing competition with the hippocampus and ceding control over learning to the hippocampus during early training trials; and (2) aCSF enhanced striatal functioning to facilitate striatal-sensitive learning. With either interpretation, the results indicate that intrastriatal glucose injections compensated for the aCSF-induced effect. Finally, enhanced acquisition regardless of treatment was accompanied by rapid adoption of a response solution for the T-maze.

Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (−)-trans-(2S,4R)-4-(3′[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (−)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (−)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (−)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (−)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (−)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (−)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders.

The serotonin (5-HT) 7 G protein-coupled receptor (GPCR) is the most recent 5-HT receptor cloned, and is expressed in both the periphery and the CNS. Reports propose 5-HT7 receptors are involved in a wide range of CNS functions, including modulation of circadian rhythms, sleep, pain, body temperature, and cognition. 5-HT7 receptors couple to Gαs, leading to activation of adenylyl cyclases and production of cAMP. Due to paucity of available, selective 5-HT7 ligands, the complexity of CNS processes and to a limited number of studies examining 5-HT7, however, there is no consensus about 5-HT7 receptor CNS neuropharmacological function. We have begun an iterative 5-HT7 receptor structure-based ligand synthesis program to develop novel 5-HT7-targeting phenylaminotetralin (PAT) compounds. A molecular model of the 5-HT7 receptor was built by homology to the recently reported structure of the 5-HT1B GPCR, and computational ligand docking studies were undertaken. Computational results were validated by experimenta...

Ligands that activate the serotonin 5-HT2C G protein-coupled receptor (GPCR) may be therapeutic for psychoses, addiction, and other neuropsychiatric disorders. Ligands that are antagonists at the closely related 5-HT2A GPCR also may treat neuropsychiatric disorders; in contrast, 5-HT2A activation may cause hallucinations. 5-HT2C-specific agonist drug design is challenging because 5-HT2 GPCRs share 80% transmembrane (TM) homology, same second messenger signaling, and no crystal structures are reported. To help delineate molecular determinants underlying differential binding and activation of 5-HT2 GPCRs, 5-HT2A, and 5-HT2C homology models were built from the β2-adrenergic GPCR crystal structure and equilibrated in a lipid phosphatidyl choline bilayer performing molecular dynamics simulations. Ligand docking studies at the 5-HT2 receptor models were conducted with the (2R, 4S)- and (2S, 4R)-enantiomers of the novel 5-HT2C agonist/5-HT2A/2B antagonist trans-4-phenyl-N,N-dimethyl-2-aminotetralin (PAT) and its 4′-chlorophenyl congners. Results indicate PAT–5-HT2 molecular interactions especially in TM domain V are important for the (2R, 4S) enantiomer, whereas, TM domain VI and VII interactions are more important for the (2S, 4R) enantiomer.

There are seemingly conflicting data in the literature regarding the role of serotonin (5-HT) 5-HT2C receptors in the mouse head-twitch response (HTR) elicited by the hallucinogenic 5-HT2A/2B/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). Namely, both 5-HT2C receptor agonists and antagonists, regarding 5-HT2C receptor-mediated Gq-phospholipase C (PLC) signaling, reportedly attenuate the HTR response. The present experiments tested the hypothesis that both classes of 5-HT2C receptor compounds could attenuate the DOI-elicited-HTR in a single strain of mice, C57Bl/6J. The expected results were considered in accordance with ligand functional selectivity. Commercially-available 5-HT2C agonists (CP 809101, Ro 60-0175, WAY 161503, mCPP, and 1-methylpsilocin), novel 4-phenyl-2-N,N-dimethyl-aminotetralin (PAT)-type 5-HT2C agonists (with 5-HT2A/2B antagonist activity), and antagonists selective for 5-HT2A (M100907), 5-HT2C (SB-242084), and 5-HT2B/2C (SB-206553) receptors attenuated the DOI-elicited-HTR. In contrast, there were differential effects on locomotion across classes of compounds. The 5-HT2C agonists and M100907 decreased locomotion, SB-242084 increased locomotion, SB-206553 resulted in dose-dependent biphasic effects on locomotion, and the PATs did not alter locomotion. In vitro molecular pharmacology studies showed that 5-HT2C agonists potent for attenuating the DOI-elicited-HTR also reduced the efficacy of DOI to activate mouse 5-HT2C receptor-mediated PLC signaling in HEK cells. Although there were differences in affinities of a few compounds at mouse compared to human 5-HT2A or 5-HT2C receptors, all compounds tested retained their selectivity for either receptor, regardless of receptor species. Results indicate that 5-HT2C receptor agonists and antagonists attenuate the DOI-elicited-HTR in C57Bl/6J mice, and suggest that structurally diverse 5-HT2C ligands result in different 5-HT2C receptor signaling outcomes compared to DOI.

Specific activation of serotonin (5-HT) 5-HT(2C) G protein-coupled receptors may be therapeutic for obesity and neuropsychiatric disorders. Mutagenesis coupled with computational and molecular modeling experiments based on the human β₂ adrenergic receptor structure was employed to delineate the interactions of different ligands at human 5-HT(2C) residues D3.32, S3.36 and Y7.43. No binding of the tertiary amine radioligand ([³H]-mesulergine) could be detected when the 5-HT(2C) D3.32 residue was mutated to alanine (D3.32A). The S3.36A point-mutation greatly reduced affinity of primary amine ligands, modestly reduced affinity of a secondary amine, and except for the 5-HT(2C)-specific agonist N(CH₃)₂-PAT, affinity of tertiary amines was unaffected. Molecular modeling results indicated that the primary amines form hydrogen bonds with the S3.36 residue, whereas, with the exception of N(CH₃)₂-PAT, tertiary amines do not interact considerably with this residue. The Y7.43A point-mutation greatly reduced affinity of 5-HT, yet reduced to a lesser extent the affinity of tryptamine that lacks the 5-hydroxy moiety present in 5-HT; modeling results indicated that the 5-HT 5-hydroxy moiety hydrogen bonds with Y7.43 at the 5-HT(2C) receptor. Additional modeling results showed that 5-HT induced a hydrogen bond between Y7.43 and D3.32. Finally, modeling results revealed two low-energy binding modes for 5-HT in the 5-HT(2C) binding pocket, supporting the concept that multiple agonist binding modes may stabilize different receptor active conformations to influence signaling. Ligand potencies for modulating WT and point-mutated 5-HT(2C) receptor-mediated phospholipase C activity were in accordance with the affinity data. Ligand efficacies, however, were altered considerably by the S3.36A mutation only.

Hallucinogenic serotonin 2A (5-HT(2A)) receptor partial agonists, such as (+ or -)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), induce a frontal cortex-dependent head-twitch response (HTR) in rodents, a behavioral proxy of a hallucinogenic response that is blocked by 5-HT(2A) receptor antagonists. In addition to 5-HT(2A) receptors, DOI and most other serotonin-like hallucinogens have high affinity and potency as partial agonists at 5-HT(2C) receptors.We tested for involvement of 5-HT(2C) receptors in the HTR induced by DOI.Comparison of 5-HT(2C) receptor knockout and wild-type littermates revealed an approximately 50% reduction in DOI-induced HTR in knockout mice. Also, pretreatment with either the 5-HT(2C) receptor antagonist SB206553 or SB242084 eradicated a twofold difference in DOI-induced HTR between the standard inbred mouse strains C57BL/6J and DBA/2J, and decreased the DOI-induced HTR by at least 50% in both strains. None of several measures of 5-HT(2A) receptors in frontal cortex explained the strain difference, including 5-HT(2A) receptor density, Galpha(q) or Galpha(i/o) protein levels, phospholipase C activity, or DOI-induced expression of Egr1 and Egr2. 5-HT(2C) receptor density in the brains of C57BL/6J and DBA/2J was also equivalent, suggesting that 5-HT(2C) receptor-mediated intracellular signaling or other physiological modulators of the HTR may explain the strain difference in response to DOI.We conclude that the HTR to DOI in mice is strongly modulated by 5-HT(2C) receptor activity. This novel finding invites reassessment of hallucinogenic mechanisms involving 5-HT(2) receptors.