Publications by authors named "Raymond G Booth"

28 Publications

  • Page 1 of 1

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

Epilepsy Res 2021 09 27;175:106677. Epub 2021 May 27.

Mercer University, College of Pharmacy, Department of Pharmaceutical Sciences, 3001 Mercer University Drive, Atlanta, GA, 30341, USA. Electronic address:

Recent preclinical and clinical studies suggest that lorcaserin, a preferential serotonin 2C receptor (5-HTR) 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-HTRs at therapeutic doses, we pretreated one group of mice with the selective 5-HTR antagonist/inverse agonist, M100907 (0.03 mg/kg), alone or before administering lorcaserin (5.6 mg/kg), to discern putative contributions of 5-HTRs to AGSs. We also assessed lorcaserin's in vitro pharmacology at human (h) and mouse (m) 5-HTRs and 5-HTRs and its in vivo interactions at m5-HTRs and m5-HTRs. 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-HTRs and m5-HTRs, and near full efficacy agonist activity at h5-HTRs and m5-HTRs; selectivity for activation of 5-HTRs over 5-HTRs was greater for human (38-fold) compared to mouse (13-fold) receptors. Lorcaserin displayed relatively low affinities at antagonist-labeled 5-HTRs and 5-HTRs, regardless of species. Lorcaserin (3 and 5.6 mg/kg) increased the 5-HTR-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-HTR 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-HTRs as well as 5-HTRs in mice at doses as low as 3 mg/kg. The similar activity at m5-HTRs and m5-HTRs suggests careful dosing of lorcaserin is necessary to selectively engage 5-HTRs 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.
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http://dx.doi.org/10.1016/j.eplepsyres.2021.106677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8296307PMC
September 2021

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

ACS Pharmacol Transl Sci 2020 Jun 21;3(3):509-523. Epub 2020 Feb 21.

Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University Health Sciences Center, Mercer University, 3001 Mercer University Drive, Atlanta, Georgia 30341, United States.

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, ()-5-(2'-fluorophenyl)--dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), that exhibits partial agonist activity at 5-HTRs, 5-HTRs, and 5-HTRs and that reduces repetitive behaviors and increases social approach behavior in wild-type mice. Here we report that in an 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 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-HTRs and 5-HTRs, yet has slow association and rapid dissociation kinetics at 5-HTRs. Finally, we reassessed and report FPT's affinity and function at 5-HTRs, 5-HTRs, and 5-HTRs. Collectively, these observations provide mounting support for further development of FPT as a pharmacotherapy for common neuropsychiatric symptoms in FXS and ASD.
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http://dx.doi.org/10.1021/acsptsci.9b00101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296548PMC
June 2020

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

Bioorg Med Chem 2020 02 12;28(3):115262. Epub 2019 Dec 12.

Center for Drug Discovery, Department of Pharmaceutical Sciences, Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115, USA.

The serotonin 5-HT 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-HT. 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-HT GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT 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-HT receptor for comparison with similar studies at the highly homologous 5-HT receptor. We report 35 new 5-SAT ligands, some with very high affinity (K ≤ 1 nM) and stereoselectivity at 5-HT + or 5-HT receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT, and, several ligands with high selectivity (up to 40-fold) at the 5-HT receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT over 5-HT receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT 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-HT vs. 5-HT GPCRs, as may be required for successful clinical translation.
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http://dx.doi.org/10.1016/j.bmc.2019.115262DOI Listing
February 2020

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

Eur J Pharmacol 2019 Apr 25;848:131-139. Epub 2019 Jan 25.

Center for Drug Discovery, Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, United States; Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, United States; Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610, United States. Electronic address:

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-HT GPCRs is pharmacotherapeutic for obesity, but there is tolerance to the anorectic effect of the only approved 5-HT agonist, lorcaserin. We tested the hypothesis that different agonists or inverse agonists differentially desensitize or sensitize, respectively, canonical 5-HT-mediated activation of phospholipase C (PLC) signaling in vitro. Lorcaserin, which displays potency and efficacy equal to 5-HT, desensitized the 5-HT 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-HT 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-HT-PLC signaling. Several 5-HT agonists also were assessed for β-arrestin recruitment-lorcaserin was a 'super-agonist', but a 2-aminotetralin and aripiprazole had nil activity, suggesting they are biased towards 5-HT-PLC signaling. We observed robust positive correlations between the magnitude of 5-HT desensitization and agonist efficacy to stimulate PLC or to recruit β-arrestin. In contrast, different inverse agonists caused different magnitudes of 5-HT sensitization that did not correlate with efficacy (or potency) to inhibit constitutive 5-HT-PLC signaling.  Assessment of the 5-HT-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-HT desensitization and sensitization processes..
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http://dx.doi.org/10.1016/j.ejphar.2019.01.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767919PMC
April 2019

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

ACS Chem Neurosci 2017 01 10;8(1):28-39. Epub 2016 Oct 10.

Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.

While exploring the structure-activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT receptors. In HEK293 cells expressing human 5-HT receptors, for example, (-)-trans-3'-Br-PAT and (-)-trans-3'-Cl-PAT are agonists regarding Gα-inositol phosphate signaling, whereas (-)-trans-3'-CF-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-HT 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 (K) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (-)-trans-3'-CF-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 (K) of the antagonist radioligand [H]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.
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http://dx.doi.org/10.1021/acschemneuro.6b00124DOI Listing
January 2017

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

ACS Chem Neurosci 2015 Jul 8;6(7):1259-70. Epub 2015 Jun 8.

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.
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http://dx.doi.org/10.1021/acschemneuro.5b00099DOI Listing
July 2015

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.

Bioorg Med Chem 2015 Apr 7;23(7):1588-600. Epub 2015 Feb 7.

Department of Medicinal Chemistry and Department of Chemistry, University of Florida, Gainesville, FL 32610-0485, United States; Center for Drug Discovery, Department of Pharmaceutical Sciences and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115-5000, United States. Electronic address:

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').
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http://dx.doi.org/10.1016/j.bmc.2015.01.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363177PMC
April 2015

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

Synapse 2015 Feb 20;69(2):78-85. Epub 2014 Nov 20.

Department of Pharmacology and Toxicology, University of Texas-Medical Branch, Galveston, Texas, 77555.

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.
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http://dx.doi.org/10.1002/syn.21790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275350PMC
February 2015

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

Mol Phys 2014 Jan;112(3-4):398-407

Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610 USA ; Center for Drug Discovery, Departments of Pharmaceutical Sciences, and, Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115 USA.

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ~75% transmembrane (TM) sequence identity. Agonists for 5-HT receptors are under development for psychoses, whereas, at 5-HT receptors, antipsychotic effects are associated with antagonists-in fact, 5-HT agonists can cause hallucinations and 5-HT agonists cause cardiotoxicity. It is known that 5-HT TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function, however, ligand interactions with these residues at the 5-HT receptor has not been reported. To predict and validate molecular determinants for 5-HT-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-HT receptors.
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http://dx.doi.org/10.1080/00268976.2013.833656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979624PMC
January 2014

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.

J Pharmacol Exp Ther 2014 May 21;349(2):310-8. Epub 2014 Feb 21.

Center for Drug Discovery (C.E.C., R.G.B.), Department of Pharmaceutical Sciences (C.E.C., R.G.B.), and Department of Chemistry and Chemical Biology (R.G.B.), Northeastern University, Boston, Massachusetts; Department of Psychiatry (D.M.), Medicinal Chemistry (D.F., R.S., K.K., R.G.B.), and Psychology (N.E.R., K.L.R.), University of Florida, Gainesville, Florida.

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.
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http://dx.doi.org/10.1124/jpet.113.212373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989798PMC
May 2014

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.

J Pharmacol Exp Ther 2013 Dec 30;347(3):705-16. Epub 2013 Sep 30.

Center for Drug Discovery (C.E.C., T.C.-S., Y.L., R.G.B.), Department of Pharmaceutical Sciences (C.E.C., T.C.-S., Y.L., R.G.B.), and Department of Chemistry and Chemical Biology (R.G.B.), Northeastern University, Boston, Massachusetts; and Department of Medicinal Chemistry (M.S.K., T.C.-S.) and Department of Psychiatry (D.M.), University of Florida, Gainesville, Florida.

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.
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http://dx.doi.org/10.1124/jpet.113.208637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836315PMC
December 2013

Molecular Determinants for Ligand Binding at Serotonin 5-HT and 5-HT GPCRs: Experimental Affinity Results Analyzed by Molecular Modeling and Ligand Docking Studies.

Int J Quantum Chem 2012 Dec;112(24):3807-3814

Department of Medicinal Chemistry. College of Pharmacy, University of Florida, Gainesville, Florida 32610.

Ligands that activate the serotonin 5-HT G protein-coupled receptor (GPCR) may be therapeutic for psychoses, addiction, and other neuropsychiatric disorders. Ligands that are antagonists at the closely related 5-HT GPCR also may treat neuropsychiatric disorders; in contrast, 5-HT activation may cause hallucinations. 5-HT-specific agonist drug design is challenging because 5-HT 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-HT GPCRs, 5-HT, and 5-HT homology models were built from the -adrenergic GPCR crystal structure and equilibrated in a lipid phosphatidyl choline bilayer performing molecular dynamics simulations. Ligand docking studies at the 5-HT receptor models were conducted with the (2, 4)- and (2, 4)-enantiomers of the novel 5-HT agonist/5-HT antagonist -4-phenyl--dimethyl-2-aminotetralin (PAT) and its 4'-chlorophenyl congners. Results indicate PAT-5-HT molecular interactions especially in TM domain V are important for the (2, 4) enantiomer, whereas, TM domain VI and VII interactions are more important for the (2, 4) enantiomer.
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http://dx.doi.org/10.1002/qua.24237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3729958PMC
December 2012

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

Neuropharmacology 2013 Sep 9;72:274-81. Epub 2013 May 9.

Department of Psychiatry, College of Medicine, University of Florida, Gainesville, FL, USA.

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.
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http://dx.doi.org/10.1016/j.neuropharm.2013.04.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696402PMC
September 2013

Support for 5-HT2C receptor functional selectivity in vivo utilizing structurally diverse, selective 5-HT2C receptor ligands and the 2,5-dimethoxy-4-iodoamphetamine elicited head-twitch response model.

Neuropharmacology 2013 Jul 23;70:112-21. Epub 2013 Jan 23.

University of Florida, Department of Medicinal Chemistry, PO Box 1000485, 1600 SW Archer Road, Gainesville, FL 32610-0485, USA.

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.
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http://dx.doi.org/10.1016/j.neuropharm.2013.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754837PMC
July 2013

Molecular determinants of ligand binding at the human histamine H receptor: Site-directed mutagenesis results analyzed with ligand docking and molecular dynamics studies at H homology and crystal structure models.

J Chem Pharm Res 2012 Jun;4(6):2937-2951

Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610 US ; Northeastern University Center for Drug Discovery, Boston, Massachusetts 02115, USA.

The human histamine H G-protein coupled receptor (GPCR) is an important drug target for inflammatory, sleep, and other neuropsychiatric disorders. To delineate molecular determinants for ligand binding for drug discovery purposes, human H receptor models were built by homology to the crystal structure of the human β adrenoceptor (βAR) and from the recently reported crystal structure of the human H receptor complex with doxepin at 3.1 Å (PDB code 3RZE). Ligand affinity of histamine and the H antagonists mepyramine and (2S, 4R)-(-)-trans-4-phenyl-2-N, N-dimethylaminotetralin (PAT) at wild type and point-mutated (D3.32A, Y3.33A, W4.56A, F5.47A, W6.48A, Y6.51A, F6.52A, F6.55A, Y7.43A) human H receptors were determined experimentally and results analyzed by ligand docking and molecular dynamic studies at WT and point-mutated H receptor models. Differences in ligand binding affinities correlated to differences in ligand binding modes at models built according to homology or crystal structure, indicating, both models are accurate templates for predicting ligand affinity for H drug design.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085681PMC
June 2012

Drug discovery targeting human 5-HT(2C) receptors: residues S3.36 and Y7.43 impact ligand-binding pocket structure via hydrogen bond formation.

Eur J Pharmacol 2011 Dec 12;673(1-3):1-12. Epub 2011 Oct 12.

Department of Medicinal Chemistry, University of Florida, P.O. Box 100485, 1600 SW Archer Road, Gainesville, FL 32610-0485, USA.

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.
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http://dx.doi.org/10.1016/j.ejphar.2011.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224194PMC
December 2011

A novel potential therapeutic avenue for autism: design, synthesis and pharmacophore generation of SSRIs with dual action.

Bioorg Med Chem Lett 2011 Nov 21;21(22):6714-23. Epub 2011 Sep 21.

College of Pharmacy, Qatar University, PO Box 2713, Doha, Qatar.

Autism symptoms are currently modulated by Selective Serotonin Reuptake Inhibitors (SSRIs). SSRIs slow onset of action limits their efficiency. The established synergistic activity of SSRIs and 5HT(1B/1D) autoreceptors antagonists motivated us to incorporate SSRIs and 5HT(1B/1D) antagonists in one 'hybrid' molecule. A library of virtual 'hybrid' molecules was designed using the tethering technique. A pharmacophore model was generated derived from 16 structurally diverse SSRIs (K(i)=0.013-5000 nM) and used as 3D query. Compounds with fit values (≥2) were chosen for synthesis and subsequent in vitro biological evaluation. Our pharmacophore model is a promising milestone to a class of SSRIs with dual action.
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http://dx.doi.org/10.1016/j.bmcl.2011.09.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104666PMC
November 2011

New Approach to 4-Phenyl-β-aminotetralin from 4-(3-Halophenyl)tetralen-2-ol Phenylacetate.

Tetrahedron Lett 2009 Sep;50(36):5107-5109

Department of Medicinal Chemistry, PO Box 100485, College of Pharmacy, University of Florida, Gainesville, FL 32610-0485, USA.

Mixed trifluoroacetyl phenylacetyl anhydride and 3-halostyrenes (fluoro, chloro, and bromo) or vinylcycloalkanes (cyclohexyl, cyclooctyl), undergo cascade Friedel-Crafts cycli-acylalkylation, enolization, and O-acylation to give 4-substituted tetralen-2-ol phenylacetates, without additional solvent in good yields. Base alcoholysis of 4-phenyltetralen-2-ol phenylacetate reveals the tetral-2-one for asymmetric transfer hydrogenation. Bromophenyltetralen-2-ol phenylacetate undergoes Suzuki coupling, and provides a short route to trans-4-phenyl-β-aminotetralin.
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http://dx.doi.org/10.1016/j.tetlet.2009.06.099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758692PMC
September 2009

(1R, 3S)-(-)-trans-PAT: a novel full-efficacy serotonin 5-HT2C receptor agonist with 5-HT2A and 5-HT2B receptor inverse agonist/antagonist activity.

Eur J Pharmacol 2009 Aug 3;615(1-3):1-9. Epub 2009 May 3.

Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610-0485, United States.

The serotonin 5-HT(2A), 5-HT(2B), and 5-HT(2C) G protein-coupled receptors signal primarily through G alpha(q) to activate phospholipase C (PLC) and formation of inositol phosphates (IP) and diacylglycerol. The human 5-HT(2C) receptor, expressed exclusively in the central nervous system, is involved in several physiological and psychological processes. Development of 5-HT(2C) agonists that do not also activate 5-HT(2A) or 5-HT(2B) receptors is challenging because transmembrane domain identity is about 75% among 5-HT(2) subtypes. This paper reports 5-HT(2) receptor affinity and function of (1R,3S)-(-)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (PAT), a small molecule that produces anorexia and weight-loss after peripheral administration to mice. (-)-Trans-PAT is a stereoselective full-efficacy agonist at human 5-HT(2C) receptors, plus, it is a 5-HT(2A)/5-HT(2B) inverse agonist and competitive antagonist. The K(i) of (-)-trans-PAT at 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors is 410, 1200, and 37 nM, respectively. Functional studies measured activation of PLC/[(3)H]-IP formation in clonal cells expressing human 5-HT(2) receptors. At 5-HT(2C) receptors, (-)-trans-PAT is an agonist (EC(50) = 20 nM) comparable to serotonin in potency and efficacy. At 5-HT(2A) and 5-HT(2B) receptors, (-)-trans-PAT is an inverse agonist (IC(50) = 490 and 1,000 nM, respectively) and competitive antagonist (K(B) = 460 and 1400 nM, respectively) of serotonin. Experimental results are interpreted in light of molecular modeling studies indicating the (-)-trans-PAT protonated amine can form an ionic bond with D3.32 of 5-HT(2A) and 5-HT(2C) receptors, but, not with 5-HT(2B) receptors. In addition to probing 5-HT(2) receptor structure and function, (-)-trans-PAT is a novel lead regarding 5-HT(2C) agonist/5-HT(2A) inverse agonist drug development for obesity and neuropsychiatric disorders.
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http://dx.doi.org/10.1016/j.ejphar.2009.04.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749987PMC
August 2009

Effect of (-)-trans-PAT, a novel 5-HT2C receptor agonist, on intake of palatable food in mice.

Pharmacol Biochem Behav 2008 Nov 19;91(1):176-80. Epub 2008 Jul 19.

Department of Psychology, University of Florida, Gainesville, USA.

(1R,3S)-(-)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (PAT) is a novel compound that has full-efficacy agonist activity at human 5-HT2C receptors and inverse agonist/antagonist activity at 5HT2A and 5HT2B receptors. In the present paper we describe its effects on food intake in non-deprived C57BL/6 mice adapted to eating a palatable dessert meal each day. PAT showed a dose-related inhibition of food intake with a 50% inhibitory dose of 4.2 mg/kg. The dose-effect curve was similar to that obtained using WAY-161503. Abnormal behaviors were not observed by casual inspection following administration of PAT. The anorectic effect of PAT was additive with that of amphetamine. When PAT, or PAT+amphetamine, were injected 2 h before access to food, most of the anorectic activity had dissipated, indicating that PAT has a biologically effective period of about 1 h. Four daily injections of PAT were associated with some, but not complete loss of the initial anorectic effect; this differs from the rapid tolerance that has been reported to fenfluramine anorexia and suggests that different mechanism(s) are involved in the loss of anorexia.
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http://dx.doi.org/10.1016/j.pbb.2008.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712337PMC
November 2008

Role of PKA and PKC in histamine H1 receptor-mediated activation of catecholamine neurotransmitter synthesis.

Neurosci Lett 2006 Oct 15;407(3):249-53. Epub 2006 Sep 15.

Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, NC 25799-7360, USA.

Activation of the histamine H1 receptor stimulates tyrosine hydroxylase (TH) to increase catecholamine neurotransmitter synthesis in mammalian brain and adrenal tissues. Histamine non-selectively activates both H1-linked phospholipase (PL) C/inositol phosphates (IP)/diacylglycerol (DAG) signaling and adenylyl cyclase (AC)/adenosine 3',5'-cyclic monophosphate (cAMP) signaling, confounding determination of signaling events involved in H(1)-mediated TH activation. This research uses two new functionally-selective H1 agonists, cis-PAB and trans-PAT, that selectively activate H1/PLC/IP/DAG and H1/AC/cAMP signaling, respectively, to characterize H(1)-mediated activation of TH in rat striatum and bovine adrenal chromaffin (BAC) cells. Histamine, cis-PAB, and trans-PAT produced a two-fold maximal TH activation by an H1 receptor mechanism in rat striatum and BAC cells. Histamine is more potent and efficacious in BAC cells (EC50 approximately 0.2 microM, Emax approximately 200% basal) versus rat striatum (EC50 approximately 0.4 microM; Emax approximately 150%). Cis-PAB and trans-PAT are more potent in rat striatum (EC50 approximately 0.1 microM for both agonists) versus BAC cells (EC50 approximately 1.0 microM for both), with similar efficacy in both preparations (Emax approximately 160% for both agonists). Signaling studies in BAC cells revealed that protein kinase (PK) A but not PKC is involved in H1 -mediated TH activation by trans-PAT and histamine, while, both PKA and PKC are involved for cis-PAB. Results for cis-PAB suggest H1/PLC/IP/DAG/PKC signaling activates PKA, downstream of cAMP formation, indicating apparent direct activation of PKA by PKC.
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http://dx.doi.org/10.1016/j.neulet.2006.08.051DOI Listing
October 2006

Novel ligands for the human histamine H1 receptor: synthesis, pharmacology, and comparative molecular field analysis studies of 2-dimethylamino-5-(6)-phenyl-1,2,3,4-tetrahydronaphthalenes.

Bioorg Med Chem 2006 Oct 16;14(19):6640-58. Epub 2006 Jun 16.

Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA.

This paper reports the synthesis of a novel series of (+/-)-2-dimethylamino- 5- and 6-phenyl-1,2,3,4-tetrahydronaphthalene derivatives (5- and 6-APTs), and, corresponding affinity, functional activity, and, molecular modeling studies with regard to drug design targeting the human histamine H1 receptor. The 5-APTs have 2- to 4-fold higher H1 receptor affinity than the endogenous agonist histamine. The chemical nature of a meta-substituent on the 5-APT pendant phenyl moiety does not significantly affect H1 affinity. In contrast, analogous meta-substitution for the 6-APTs increases H1 affinity up to 100-fold. The new APTs do not activate H1 receptor-linked intracellular signaling and apparently are competitive H1 antagonists. A new model that establishes structural parameters for binding to the human H1 receptor by APTs and other ligands was developed using 3-D QSAR (CoMFA). The model predicts H1 ligand binding with a higher degree of external predictability compared to a previously reported model. The APTs also were examined for activity at human serotonin 5-HT2A and 5-HT2C receptors, which are phylogenetically closely related to the H1 receptor. 5-APT and m-Cl-6-APT were identified as novel agonists that selectively activate 5-HT2C receptors. It is concluded that the lipophilic (brain-penetrating) APT molecular scaffold may have pharmacotherapeutic potential in neuropsychiatric diseases.
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http://dx.doi.org/10.1016/j.bmc.2006.05.077DOI Listing
October 2006

Neuronal nitric oxide modulates morphine antinociceptive tolerance by enhancing constitutive activity of the mu-opioid receptor.

Biochem Pharmacol 2005 Feb 7;69(4):679-88. Epub 2005 Jan 7.

Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360, USA.

NO is a key mediator of morphine antinociceptive tolerance. This work was conducted to evaluate the specific effects of NO on mu-opioid receptor activity. To investigate the effects of morphine- and L-arginine (the NO precursor)-induced increases in NO, five groups of rats were treated with saline, l-arginine (100-, 300-, or 500-mg/kg/h), or morphine 3-mg/kg/h for 8h on Day 1; brain tissue was collected on Day 2. To evaluate the effects of additional increases in NO on morphine-induced alterations of the mu-opioid receptor, six groups of rats were treated with 8-h intravenous infusions for two consecutive days as per the following scheme (Day 1:Day 2): saline:saline (control); saline:morphine 3-mg/kg/h (tolerant); L-arginine 500-mg/kg/h:saline (NO control); L-arginine 100-mg/kg/h:morphine 3-mg/kg/h; L-arginine 300-mg/kg/h:morphine 3-mg/kg/h; and L-arginine 500-mg/kg/h:morphine 3-mg/kg/h (supertolerant). Brain tissue was collected at the end of Day 2. The time course of effects on morphine-induced receptor alterations due to increased NO also was evaluated. Brain tissue was analyzed for changes in radioligand (agonist and antagonist) binding and [(35)S]GTPgammaS binding (agonist and antagonist). In the absence of agonist exposure, NO produced an alteration in the mu-opioid receptor that increased receptor activity. In the presence of agonist, NO increased constitutive activation of the mu-opioid receptor and reduced the ability of a selective mu-opioid agonist to activate the mu-opioid G-protein-coupled receptor; these molecular effects occurred in a time course consistent with the development of antinociceptive tolerance. This work establishes important NO-induced alterations in mu-opioid receptor functionality, which directly lead to the development of opioid antinociceptive tolerance.
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http://dx.doi.org/10.1016/j.bcp.2004.11.004DOI Listing
February 2005

Ligand-directed functional heterogeneity of histamine H1 receptors: novel dual-function ligands selectively activate and block H1-mediated phospholipase C and adenylyl cyclase signaling.

J Pharmacol Exp Ther 2004 Oct 28;311(1):274-81. Epub 2004 May 28.

Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA.

The autacoid and neurotransmitter histamine activates the H(1) G protein-coupled receptor (GPCR) to stimulate predominantly phospholipase C (PLC)/inositol phosphate (IP) signaling and, to a lesser extent, adenylyl cyclase (AC)/cAMP signaling in a variety of mammalian cells and tissues, as well as H(1)-transfected clonal cell lines. This study reports that two novel H(1) receptor ligands developed in our laboratory, (-)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (trans-PAT) and (+/-)-cis-5-phenyl-7-dimethylamino-5,6,7,8-tetrahydro-9H-benzocycloheptane (cis-PAB), activate H(1) receptors to selectively stimulate AC/cAMP formation and PLC/IP formation, respectively, in Chinese hamster ovary cells transfected with guinea pig H(1) receptor cDNA. trans-PAT and cis-PAB also are shown to be functionally selective antagonists of H(1)-linked PLC/IP and AC/cAMP signaling, respectively. Whereas cis-PAB H(1) receptor activity is shown to be typically competitive, trans-PAT displays a complex interaction with the H(1) receptor that is not competitive regarding antagonism of saturation binding by the standard H(1) antagonist radioligand [(3)H]mepyramine or H(1)/PLC/IP functional activation by histamine. trans-PAT, however, does competitively block H(1)/PLC/IP functional activation by cis-PAB. Molecular determinants for trans-PAT versus cis-PAB differential binding to H(1) receptors, which presumably leads to differential activation of AC/cAMP versus PLC/IP signaling, likely involves stereochemical factors as well as more subtle steric influences. Results suggest the trans-PAT and cis-PAB probes will be useful to study molecular mechanisms of ligand-directed GPCR multifunctional signaling. Moreover, because most untoward cardiovascular-, respiratory-, and gastrointestinal H(1) receptor-mediated effects proceed via the PLC/IP pathway, PAT-type agonists that selectively enhance H(1)-mediated AC/cAMP signaling provide a mechanistic basis for exploiting H(1) receptor activation for drug design purposes.
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http://dx.doi.org/10.1124/jpet.104.070086DOI Listing
October 2004

Domain swapping in the human histamine H1 receptor.

J Pharmacol Exp Ther 2004 Oct 24;311(1):131-8. Epub 2004 May 24.

Leiden/Amsterdam Center for Drug Research, Faculty of Sciences, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands.

G-protein-coupled receptors (GPCRs) represent the largest family of receptors involved in transmembrane signaling. Although these receptors were generally believed to be monomeric entities, accumulating evidence supports the presence of GPCRs in multimeric forms. Here, using immunoprecipitation as well as time-resolved fluorescence resonance energy transfer to assess protein-protein interactions in living cells, we unambiguously demonstrate the occurrence of dimerization of the human histamine H(1) receptor. We also show the presence of domain-swapped H(1) receptor dimers in which there is the reciprocal exchange of transmembrane domain TM domains 6 and 7 between the receptors present in the dimer. Mutation of aspartate(107) in transmembrane (TM) 3 or phenylalanine(432) in TM6 to alanine results in two radioligand-binding-deficient mutant H(1) receptors. Coexpression of H(1)D(107) A and H(1)F(432)A, however, results in a reconstituted radioligand binding site that exhibits a pharmacological profile that corresponds to the wild-type H(1) receptor. Interestingly, the H(1) receptor radioligands [(3)H]mepyramine and [(3)H]-(-)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene show differential saturation binding values (B(max)) for wild-type H(1) receptors but not for the radioligand binding site that is formed upon coexpression of H(1) D(107)A and H(1) F(432)A receptors, suggesting the presence of different H(1) receptor populations.
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http://dx.doi.org/10.1124/jpet.104.067041DOI Listing
October 2004

A novel phenylaminotetralin radioligand reveals a subpopulation of histamine H(1) receptors.

J Pharmacol Exp Ther 2002 Jul;302(1):328-36

Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, 27599-7360, USA.

Previously, (-)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene ([-]-trans-H(2)-PAT) was shown to activate stereospecifically histamine H(1) receptors coupled to modulation of tyrosine hydroxylase activity in guinea pig and rat forebrain in vitro and in vivo. Furthermore, the novel radioligand [(3)H](-)-trans-H(2)-PAT was shown to label selectively H(1) receptors in guinea pig and rat brain with high affinity (K(D), ~0.1 and 0.5 nM, respectively) and a B(max) about 50 and 15%, respectively, of that observed for the H(1) antagonist radioligand [(3)H]mepyramine. In the current study, [(3)H](-)-trans-H(2)-PAT-labeled cloned guinea pig and human H(1) receptors in Chinese hamster ovary (CHO) cell membranes with high affinity (K(D), ~0.08 and 0.23 nM, respectively) and a B(max) about 15% of that observed for [(3)H]mepyramine. The binding of H(2)-PAT to H(1) receptors in both CHO-H(1) cell lines was stereoselective with the (-)-trans-isomer having affinity (K(i), ~1.5 nM) about 4-, 20-, and 50-times higher than the (-)-cis-, (+)-trans-, and (+)-cis-isomers, respectively; the affinity of (-)-trans-H(2)-PAT was unaffected by excess GTP. In functional assays, (-)-trans-H(2)-PAT was a full antagonist of histamine H(1)-mediated stimulation of phospholipase C (PLC) and [(3)H]inositol phosphates (IP) formation in CHO-H(1) cells, a full inverse agonist of constitutively active H(1) receptors in COS-7-H(1) cells, and a full competitive antagonist (pA(2) = 9.2) of histamine H(1)-mediated contraction of guinea pig ileum. It is concluded that (-)-trans-H(2)-PAT is an antagonist at H(1) receptors coupled to PLC/IP formation and smooth muscle contraction. Meanwhile, the observation that [(3)H](-)-trans-H(2)-PAT labels only a subpopulation of H(1) receptors and that (-)-trans-H(2)-PAT activates H(1) receptors coupled to modulation of tyrosine hydroxylase suggests that there may be post-translational H(1) receptor heterogeneity.
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http://dx.doi.org/10.1124/jpet.302.1.328DOI Listing
July 2002

Functional selectivity of dopamine receptor agonists. I. Selective activation of postsynaptic dopamine D2 receptors linked to adenylate cyclase.

J Pharmacol Exp Ther 2002 Jun;301(3):1166-78

Department of Psychiatry, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7160, USA.

Dihydrexidine (DHX), the first high-affinity D(1) dopamine receptor full agonist, is only 10-fold selective for D(1) versus D(2) receptors, having D(2) affinity similar to the prototypical agonist quinpirole. The D(2) functional properties of DHX and its more D(2) selective analog N-n-propyl-dihydrexidine (PrDHX) were explored in rat brain and pituitary. DHX and PrDHX had binding characteristics to D(2) receptors in rat striatum typical of D(2) agonists, binding to both high- and low-affinity sites and being sensitive to guanine-nucleotides. Consistent with these binding data, both DHX and PrDHX inhibited forskolin-stimulated cAMP synthesis in striatum with a potency and intrinsic activity equivalent to that of quinpirole. Unexpectedly, however, DHX and PrDHX had little functional effect at D(2) receptors expressed on dopaminergic neurons that mediate inhibition of cell firing, dopamine release, or dopamine synthesis. Quantitative receptor competition autoradiography demonstrated that DHX bound to D(2) receptors in striatum (predominantly postsynaptic receptor sites) with equal affinity as D(2) sites in the substantia nigra (autoreceptor sites). The data from these experiments, coupled with what is known about the location of specific dopamine receptor isoforms, lead to the hypothesis that DHX, after binding to D(2L) and D(2S) receptors, causes agonist-typical functional changes only at some of these receptors. This phenomenon (herein termed "functional selectivity") suggests that drugs may be targeted not only at specific receptor isoforms but also at separate functions mediated by a single isoform, yielding novel approaches to drug discovery.
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http://dx.doi.org/10.1124/jpet.301.3.1166DOI Listing
June 2002
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