Development of 2-Aminotetralin-Type Serotonin 5-HT1 Agonists: Molecular Determinants for Selective Binding and Signaling at 5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F Receptors.

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT1 G-protein coupled receptor subtypes (5-HT1A/1B/1D/1E/1F) share a high sequence homology, confounding development of subtype-specific ligands. This study used a 5-HT1 structure-based ligand design approach to develop subtype-selective ligands using a 5-substituted-2-aminotetralin (5-SAT) chemotype, leveraging results from pharmacological, molecular modeling, and mutagenesis studies to delineate molecular determinants for 5-SAT binding and function at 5-HT1 subtypes. 5-SATs demonstrated high affinity (Ki ≤ 25 nM) and at least 50-fold stereoselective preference ([2S] > [2R]) at 5-HT1A, 5-HT1B, and 5-HT1D receptors but essentially nil affinity (Ki > 1 µM) at 5-HT1F receptors. The 5-SATs tested were agonists with varying degrees of potency and efficacy, depending on chemotype substitution and 5-HT1 receptor subtype. Models were built from the 5-HT1A (cryo-EM), 5-HT1B (crystal), and 5-HT1D (cryo-EM) structures, and 5-SATs underwent docking studies with up to 1 µs molecular dynamics simulations. 5-SAT interactions observed at positions 3.33, 5.38, 5.42, 5.43, and 7.39 of 5-HT1 subtypes were confirmed with point mutation experiments. Additional 5-SATs were designed and synthesized to exploit experimental and computational results, yielding a new full efficacy 5-HT1A agonist with 100-fold selectivity over 5-HT1B/1D receptors. The results presented lay the foundation for the development of additional 5-HT1 subtype selective ligands for drug discovery purposes.

Effects of (-)-MBP, a novel 5-HT2C agonist and 5-HT2A/2B antagonist/inverse agonist on brain activity: A phMRI study on awake mice.

A novel serotonin ligand (-)-MBP was developed for the treatment of schizophrenia that has 5-HT2A/2B antagonist activity together with 5-HT2C agonist activity. The multi-functional activity of this novel drug candidate was characterized using pharmacological magnetic resonance imaging. It was hypothesized (-)-MBP would affect activity in brain areas associated with sensory perception. Adult male mice were given one of three doses of (-)-MBP (3.0, 10, 18 mg/kg) or vehicle while fully awake during the MRI scanning session and imaged for 15 min post I.P. injection. BOLD functional imaging was used to follow changes in global brain activity. Data for each treatment were registered to a 3D MRI mouse brain atlas providing site-specific information on 132 different brain areas. There was a dose-dependent decrease in positive BOLD signal in numerous brain regions, especially thalamus, cerebrum, and limbic cortex. The 3.0 mg/kg dose had the greatest effect on positive BOLD while the 18 mg/kg dose was less effective. Conversely, the 18 mg/kg dose showed the greatest negative BOLD response while the 3.0 mg/kg showed the least. The prominent activation of the thalamus and cerebrum included the neural circuitry associated with Papez circuit of emotional experience. When compared to vehicle, the 3.0 mg dose affected all sensory modalities, for example, olfactory, somatosensory, motor, and auditory except for the visual cortex. These findings show that (-)-MBP, a ligand with both 5-HT2A/2B antagonist and 5-HT2C agonist activities, interacts with thalamocortical circuitry and impacts areas involved in sensory perception.

Conformationally Selective 2-Aminotetralin Ligands Targeting the alpha2A- and alpha2C-Adrenergic Receptors.

Many important physiological processes are mediated by alpha2A- and alpha2C-adrenergic receptors (α2Rs), a subtype of class A G protein-coupled receptors (GPCRs). However, α2R signaling is poorly understood, and there are few approved medications targeting these receptors. Drug discovery aimed at α2Rs is complicated by the high degree of binding pocket homology between α2AR and α2CR, which confounds ligand-mediated selective activation or inactivation of signaling associated with a particular subtype. Meanwhile, α2R signaling is complex and it is reported that activating α2AR is beneficial in many clinical contexts, while activating α2CR signaling may be detrimental to these positive effects. Here, we report on a novel 5-substituted-2-aminotetralin (5-SAT) chemotype that, depending on substitution, has diverse pharmacological activities at α2Rs. Certain lead 5-SAT analogues act as partial agonists at α2ARs, while functioning as inverse agonists at α2CRs, a novel pharmacological profile. Leads demonstrate high potency (e.g., EC50 < 2 nM) at the α2AR and α2CRs regarding Gαi-mediated inhibition of adenylyl cyclase and production of cyclic adenosine monophosphate (cAMP). To help understand the molecular basis of 5-SAT α2R multifaceted functional activity, α2AR and α2CR molecular models were built from the crystal structures and 1 µs molecular dynamics (MD) simulations and molecular docking experiments were performed for a lead 5-SAT with α2AR agonist and α2CR inverse agonist activity, i.e., (2S)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), in comparison to the FDA-approved (for opioid withdrawal symptoms) α2AR/α2CR agonist lofexidine. Results reveal several interactions between FPT and α2AR and α2CR amino acids that may impact the functional activity. The computational data in conjunction with experimental in vitro affinity and function results provide information to understand ligand stabilization of functionally distinct GPCR conformations regarding α2AR and α2CRs.

Oxycodone self-administration and reinstatement in male and female squirrel monkeys: Effects of alternative reinforcer availability.

The use of non-drug alternative reinforcers has long been utilized as a component of therapeutic interventions for the management of substance use disorder; however, the conditions under which alternative reinforcers are most effective are not well characterized. This study evaluated the impact of varying the magnitude of an alternative reinforcer on oxycodone self-administration and reinstatement in male and female squirrel monkeys. Subjects (n=4/sex) were trained under concurrent second-order schedules of reinforcement for intravenous oxycodone (0.001-0.1mg/kg/inj) on one lever, and sweetened condensed milk (5, 10, 20, 30% in water) on another. Oxycodone-primed reinstatement was evaluated by administering 0.32mg/kg oxycodone prior to sessions in which saline was available on the drug-paired lever. During oxycodone self-administration sessions, milk availability decreased oxycodone self-administration and preference in a concentration-dependent manner; low milk concentrations were more effective at decreasing oxycodone’s reinforcing potency in males. During reinstatement tests, milk significantly attenuated oxycodone-primed responding in both males and females; low milk concentrations were more effective at decreasing the priming effects of oxycodone in females. That alternative reinforcers differentially impacted self-administration and reinstatement in a sex-dependent manner suggests that treatment strategies that utilize alternative reinforcers may be more effective in males or females depending on when they are implemented.

FPT, a 2-Aminotetralin, Is a Potent Serotonin 5-HT1A, 5-HT1B, and 5-HT1D Receptor Agonist That Modulates Cortical Electroencephalogram Activity in Adult Fmr1 Knockout Mice.

There are no approved medicines for fragile X syndrome (FXS), a monogenic, neurodevelopmental disorder. Electroencephalogram (EEG) studies show alterations in resting-state cortical EEG spectra, such as increased gamma-band power, in patients with FXS that are also observed in Fmr1 knockout models of FXS, offering putative biomarkers for drug discovery. Genes encoding serotonin receptors (5-HTRs), including 5-HT1A, 5-HT1B, and 5-HT1DRs, are differentially expressed in FXS, providing a rationale for investigating them as pharmacotherapeutic targets. Previously we reported pharmacological activity and preclinical neurotherapeutic effects in Fmr1 knockout mice of an orally active 2-aminotetralin, (S)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT). FPT is a potent (low nM), high-efficacy partial agonist at 5-HT1ARs and a potent, low-efficacy partial agonist at 5-HT7Rs. Here we report new observations that FPT also has potent and efficacious agonist activity at human 5-HT1B and 5-HT1DRs. FPT's Ki values at 5-HT1B and 5-HT1DRs were <5 nM, but it had nil activity (>10 µM Ki) at 5-HT1FRs. We tested the effects of FPT (5.6 mg/kg, subcutaneous) on EEG recorded above the somatosensory and auditory cortices in freely moving, adult Fmr1 knockout and control mice. Consistent with previous reports, we observed significantly increased relative gamma power in untreated or vehicle-treated male and female Fmr1 knockout mice from recordings above the left somatosensory cortex (LSSC). In addition, we observed sex effects on EEG power. FPT did not eliminate the genotype difference in relative gamma power from the LSSC. FPT, however, robustly decreased relative alpha power in the LSSC and auditory cortex, with more pronounced effects in Fmr1 KO mice. Similarly, FPT decreased relative alpha power in the right SSC but only in Fmr1 knockout mice. FPT also increased relative delta power, with more pronounced effects in Fmr1 KO mice and caused small but significant increases in relative beta power. Distinct impacts of FPT on cortical EEG were like effects caused by certain FDA-approved psychotropic medications (including baclofen, allopregnanolone, and clozapine). These results advance the understanding of FPT's pharmacological and neurophysiological effects.

"Selective" serotonin 5-HT2A receptor antagonists.

Blockade of the serotonin 5-HT2A G protein-coupled receptor (5-HT2AR) 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-HT2AR by serotonergic psychedelics may be useful in treating neuropsychiatric indications, including major depressive and substance use disorders. Serotonergic psychedelics and other 5-HT2AR agonists, however, often bind other receptors, and standard 5-HT2AR antagonists lack sufficient selectivity to make well-founded mechanistic conclusions about the 5-HT2AR-dependent effects of these compounds and the general neurobiological function of 5-HT2ARs. This review discusses the limitations and strengths of currently available "selective" 5-HT2AR antagonists, the molecular determinants of antagonist selectivity at 5-HT2ARs, and the utility of molecular pharmacology and computational methods in guiding the discovery of novel unambiguously selective 5-HT2AR antagonists.

A new class of 5-HT2A /5-HT2C receptor inverse agonists: Synthesis, molecular modeling, in vitro and in vivo pharmacology of novel 2-aminotetralins.

BACKGROUND AND PURPOSE: The 5-HT receptor subtypes 5-HT2A and 5-HT2C are important neurotherapeutic targets, though, obtaining selectivity over 5-HT2B and H1 receptors is challenging. Here, we delineated molecular determinants of selective binding to 5-HT2A and 5-HT2C receptors for novel 4-phenyl-2-dimethylaminotetralins (4-PATs). EXPERIMENTAL APPROACH: We synthesized 42 novel 4-PATs with halogen or aryl moieties at the C(4)-phenyl meta-position. Affinity, function, molecular modeling and 5-HT2A receptor mutagenesis studies were performed to understand structure-activity relationships at 5-HT2 -type and H1 receptors. Lead 4-PAT-type 5-HT2A /5-HT2C receptor inverse agonists were compared with pimavanserin, a selective 5-HT2A /5-HT2C receptor inverse agonist approved to treat Parkinson's disease-related psychosis, in the mouse head twitch response and locomotor activity assays, models relevant to antipsychotic drug development. KEY RESULTS: Most 4-PAT diastereomers in the (2S,4R)-configuration bound non-selectively to 5-HT2A , 5-HT2C and H1 receptors, with >100-fold selectivity over 5-HT2B receptors, whereas diastereomers in the (2R,4R)-configuration bound preferentially to 5-HT2A over 5-HT2C receptors and had >100-fold selectivity over 5-HT2B and H1 receptors. Results suggest that G2385.42 and V2355.39 in 5-HT2A receptors (conserved in 5-HT2C receptors) are important for high affinity binding, whereas interactions with T1945.42 and W1584.56 determine H1 receptor affinity. The 4-PAT analog (2S,4R)-4-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, (2S,4R)-2k, a potent and selective 5-HT2A /5-HT2C receptor inverse agonist, had activity like pimavanserin in the mouse head twitch response assay but was distinct in not suppressing locomotor activity. CONCLUSIONS AND IMPLICATIONS: The novel 4-PAT chemotype can yield selective 5-HT2A /5-HT2C receptor inverse agonists for antipsychotic drug development by optimizing ligand-receptor interactions in transmembrane domain 5. Chirality can be exploited to attain selectivity over H1 receptors, which may circumvent sedative effects.

Evaluation of lorcaserin as an anticonvulsant in juvenile Fmr1 knockout mice.

Recent preclinical and clinical studies suggest that lorcaserin, a preferential serotonin 2C receptor (5-HT2CR) 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-HT2ARs at therapeutic doses, we pretreated one group of mice with the selective 5-HT2AR antagonist/inverse agonist, M100907 (0.03 mg/kg), alone or before administering lorcaserin (5.6 mg/kg), to discern putative contributions of 5-HT2ARs to AGSs. We also assessed lorcaserin's in vitro pharmacology at human (h) and mouse (m) 5-HT2CRs and 5-HT2ARs and its in vivo interactions at m5-HT2CRs and m5-HT2ARs. 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 also did not significantly affect AGSs. Lorcaserin exhibited full efficacy agonist activity at h5-HT2CRs and m5-HT2CRs, and near full efficacy agonist activity at h5-HT2ARs and m5-HT2ARs; selectivity for activation of 5-HT2CRs over 5-HT2ARs was greater for human (38-fold) compared to mouse (13-fold) receptors. Lorcaserin displayed relatively low affinities at antagonist-labeled 5-HT2CRs and 5-HT2ARs, regardless of species. Lorcaserin (3 and 5.6 mg/kg) increased the 5-HT2AR-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-HT2CR antagonist SB 242084 (0.5 or 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 SB 242084, and lorcaserin also dose-dependently reduced locomotor activity when administered prior to DOI (Ps<0.002). These data suggest that lorcaserin may engage 5-HT2CRs as well as 5-HT2ARs in mice at doses as low as 3 mg/kg. The similar activity at m5-HT2CRs and m5-HT2ARs suggests careful dosing of lorcaserin is necessary to selectively engage 5-HT2CRs in vivo. In conclusion, lorcaserin was ineffective at preventing AGSs in Fmr1 knockout mice. Lorcaserin may not be a suitable pharmacotherapy for seizures in FXS.

(S)-5-(2'-Fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, a Serotonin Receptor Modulator, Possesses Anticonvulsant, Prosocial, and Anxiolytic-like Properties in an Fmr1 Knockout Mouse Model of Fragile X Syndrome and Autism Spectrum Disorder.

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.

Synthesis of novel 5-substituted-2-aminotetralin analogs: 5-HT1A and 5-HT7 G protein-coupled receptor affinity, 3D-QSAR and molecular modeling.

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.

Ligand-directed serotonin 5-HT2C receptor desensitization and sensitization.

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 only 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<0.05). Agonist chemotypes such as 2-aminotetralins, with similar potency but lower efficacy than 5-HT, produced little 5-HT2C desensitization. The piperazine agonist 1-(3-chlorophenyl)piperazine (mCPP), with lower potency but similar efficacy as 5-HT, elicited desensitization indistinguishable from 5-HT, while the piperazine agonist aripiprazole, with lower potency and efficacy, did not desensitize 5-HT2C-PLC signaling. Several 5-HT2C agonists also were assessed for ß-arrestin recruitment-lorcaserin was a 'super-agonist', but a 2-aminotetralin and aripiprazole had nil activity, suggesting they are biased towards 5-HT2C-PLC signaling. We observed robust positive correlations between the magnitude of 5-HT2C desensitization and agonist efficacy to stimulate PLC or to recruit ß-arrestin. In contrast, different inverse agonists caused different magnitudes of 5-HT2C sensitization that did not correlate with efficacy (or potency) to inhibit constitutive 5-HT2C-PLC signaling.  Assessment of the 5-HT2C-S407A point-mutated receptor indicated this residue's involvement in ligand-dependent desensitization, but we did not observe a role for protein kinase C.These data show that ligand structure uniquely impacts 5-HT2C desensitization and sensitization processes..

Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5-HT2C Receptor Underlying the Pharmacology of Distinct Ligands.

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 residue S5.43, N6.55, or both are mutated to alanine, (-)-trans-3'-CF3-PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (-)-trans-3'-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (KD) of the antagonist radioligand [3H]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.

An Orally Active Phenylaminotetralin-Chemotype Serotonin 5-HT7 and 5-HT1A Receptor Partial Agonist that Corrects Motor Stereotypy in Mouse Models.

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.

Novel 4-substituted-N,N-dimethyltetrahydronaphthalen-2-amines: synthesis, affinity, and in silico docking studies at serotonin 5-HT2-type and histamine H1 G protein-coupled receptors.

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').

Serotonin-2C receptor agonists decrease potassium-stimulated GABA release in the nucleus accumbens.

The serotonin 5-HT2C receptor has shown promise in vivo as a pharmacotherapeutic target for alcoholism. For example, recently, a novel 4-phenyl-2-N,N-dimethylaminotetralin (PAT) drug candidate, that demonstrates 5-HT2C receptor agonist activity together with 5-HT2A/2B receptor inverse agonist activity, was shown to reduce operant responding for ethanol after peripheral administration to rats. Previous studies have shown that the 5-HT2C receptor is found throughout the mesoaccumbens pathway and that 5-HT2C receptor agonism causes activation of ventral tegmental area (VTA) GABA neurons. It is unknown what effect 5-HT2C receptor modulation has on GABA release in the nucleus accumbens core (NAcc). To this end, microdialysis coupled to capillary electrophoresis with laser-induced fluorescence was used to quantify extracellular neurotransmitter concentrations in the NAcc under basal and after potassium stimulation conditions, in response to PAT analogs and other 5-HT2C receptor modulators administered by reverse dialysis to rats. 5-HT2C receptor agonists specifically attenuated stimulated GABA release in the NAcc while 5-HT2C antagonists or inverse agonists had no effect. Agents with activity at 5-HT2A receptors had no effect on GABA release. Thus, in contrast to results reported for the VTA, current results suggest 5-HT2C receptor agonists decrease stimulated GABA release in the NAcc, and provide a possible mechanism of action for 5HT2C -mediated negative modulation of ethanol self-administration.

Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: Receptor homology modeling, ligand docking, and molecular dynamics results validated by experimental studies.

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ~75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses, whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists-in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function, however, ligand interactions with these residues at the 5-HT2C receptor has not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modeling, ligand docking, and molecular dynamics (MD) simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

A novel aminotetralin-type serotonin (5-HT) 2C receptor-specific agonist and 5-HT2A competitive antagonist/5-HT2B inverse agonist with preclinical efficacy for psychoses.

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.

Molecular pharmacology and ligand docking studies reveal a single amino acid difference between mouse and human serotonin 5-HT2A receptors that impacts behavioral translation of novel 4-phenyl-2-dimethylaminotetralin ligands.

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.

Molecular and behavioral pharmacology of two novel orally-active 5HT2 modulators: potential utility as antipsychotic medications.

BACKGROUND: Desired serotonin 5HT2 receptor pharmacology for treatment of psychoses is 5HT2A antagonism and/or 5HT2C agonism. No selective 5HT2A antagonist has been approved for psychosis and the only approved 5HT2C agonist (for obesity) also activates 5HT2A and 5HT2B receptors, which can lead to clinical complications. Studies herein tested the hypothesis that a dual-function 5HT2A antagonist/5HT2C agonist that does not activate 5HT2B receptors would be suitable for development as an antipsychotic drug, without liability for weight gain. METHODS: The novel compounds (+)- and (-)-trans-4-(4'-chlorophenyl)-N,N-dimethyl-2-aminotetralin (p-Cl-PAT) were synthesized, characterized in vitro for affinity and functional activity at human 5HT2 receptors, and administered by intraperitoneal (i.p.) and oral (gavage) routes to mice in behavioral paradigms that assessed antipsychotic efficacy and effects on feeding behavior. RESULTS: (+)- and (-)-p-Cl-PAT activated 5HT2C receptors, with (+)-p-Cl-PAT being 12-times more potent, consistent with its higher affinity across 5HT2 receptors. Neither p-Cl-PAT enantiomer activated 5HT2A or 5HT2B receptors at concentrations up to 300-times greater than their respective affinity (Ki), and (+)-p-Cl-PAT was shown to be a 5HT2A competitive antagonist. When administered i.p. or orally, (+)- and (-)-p-Cl-PAT attenuated the head-twitch response (HTR) in mice elicited by the 5HT2 agonist (-)-2,5-dimethoxy-4-iodoamphetamine (DOI) and reduced intake of a highly palatable food in non-food-deprived mice, with (+)-p-Cl-PAT being more potent across behavioral assays. CONCLUSIONS: The novel in vitro pharmacology of (+)-p-Cl-PAT (5HT2A antagonism/5HT2C agonism without activation of 5HT2B) translated in vivo to an orally-active drug candidate with preclinical efficacy to treat psychoses without liability for weight gain.