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Jason C. Parrish et al- Differential phospholipase C activation by phenylalkylamine serotonin 5-HT2A receptor agonists

Jason C. Parrish et al- Differential phospholipase C activation by phenylalkylamine serotonin 5-HT2A receptor agonists

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02/06/2013

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Differential phospholipase C activation by phenylalkylamineserotonin 5-HT
2A
receptor agonists
Jason C. Parrish, Michael R. Braden, Emily Gundy and David E. Nichols
 Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, PurdueUniversity, West Lafayette, Indiana, USA
Abstract
Experiments compared a series of phenethylaminehallucinogens with their phenylisopropylamine analogues forbinding affinity and ability to stimulate serotonin 5-HT
2A
receptor-mediated hydrolysis of phosphatidyl inositol in cellsexpressing cloned rat and human 5-HT
2A
receptors. The (±)phenylisopropylamine analogues had significantly higherintrinsic activities for 5-HT
2A
receptor-mediated hydrolysis ofphosphatidyl inositol compared to their phenethylamine ana-logues. With respect to the effects of the stereochemistry ofthe phenylisopropylamines, those with the (
) absolute con-figuration at the alpha carbon had higher intrinsic activities forhydrolysis of phosphatidyl inositol in a cell line expressing thehuman 5-HT
2A
receptor compared to those with the (
)absolute configuration. In virtual docking studies comparingthe (
)- and (
)-phenylisopropylamines with their phenethyl-amine analogues, there were distinct differences in the ori-entations of key ligand binding domain residues that havebeen identified as important by previous mutagenesis studies.In conclusion, our data support the hypothesis that phenyl-isopropylamines have higher hallucinogenic potency than theirphenethylamine analogues primarily because they havehigher intrinsic activities at 5-HT
2A
receptors. Although virtualligand binding led to significant perturbations of certain keyresidues, our results emphasize the conclusion reached byothers that overall three-dimensional structural microdomainswithin the receptor must be considered.
Keywords:
5-HT
2A
, hallucinogen, phenethylamine, phenyl-isopropylamine, phospholipase C, serotonin.
J. Neurochem.
(2005)
95,
1575–1584.
Since Arthur Heffter’s 1897 discovery that mescaline (3,4,5-trimethoxyphenethylamine) was the active constituent of thehallucinogenic peyote cactus,
Lophophora williamsii
(Heff-ter 1898; Perrine 2001), the biochemical mechanism(s)responsible for altered states of consciousness (ASC)induced by hallucinogens is still incompletely understood.Although today hallucinogens (psychedelics) remain largely pharmacological curiosities, much has been learned about their pharmacology in the past 100 years (Nichols 2004).All hallucinogens have in common relatively high affinityfor the serotonin 5-HT
2A
receptor subtype, and it is nowwidely thought that the effects of hallucinogens are depend-ent on activation of brain 5-HT
2A
receptors, particularly infrontal cortex and thalamus (Nichols 2004). The recent demonstration that the hallucinogenic effect of the indole-amine psilocybin in human subjects is blocked by ketanserin,a selective 5-HT
2A
receptor antagonist, provides compellingevidence for a 5-HT
2A
receptor agonist-mediated mechanismof action (Vollenweider 
et al 
. 1998).Although mescaline is the least potent of the hallucino-gens, it has served as the template for the synthesis of numerous phenethylamine (PEA) analogues with variousefficacies for producing ASC. In the earliest such report,adding an alpha-methyl group to the structure of mescaline provided the phenylisopropylamine (PIA) analogue,
Received April 30, 2005; revised manuscript received July 13, 2005;accepted July 23, 2005.Address correspondence and reprint requests to Dr David E. Nichols,Department of Medicinal Chemistry and Molecular Pharmacology,School of Pharmacy and Pharmaceutical Sciences, 575 Stadium MallDrive, Purdue University, West Lafayette, IN 47907–2091, USAE-mail: drdave@pharmacy.purdue.edu
 Abbreviations used 
: 2C-B, 4-bromo-2,5-dimethoxyphenethylamine;2C-I, 4-iodo-2,5-dimethoxyphenethylamine; 2C-T2, 4-ethylthio-2,5-di-methoxyphenethylamine; 2C-TFM, 4-trifluoromethyl-2,5-dimethoxy- phenethylamine; 5-HT, 5-hydroxytryptamine, serotonin; Aleph-2,4-ethylthio-2,5-dimethoxyphenylisopropylamine; ASC, altered states of consciousness; DOB, 4-bromo-2,5-dimethoxyphenylisopropylamine;DOI, 4-iodo-2,5-dimethoxyphenylisopropylamine; DOTFM, 4-trifluoro-methyl-2,5-dimethoxyphenylisopropylamine; PEA, phenethylamine;PIA, phenylisopropylamine; PLC, phospholipase C; TFMfly, 4-trifluoro-methyl-benzoditetrahydrofuranylisopropylamine; TM, transmembrane;TMA, trimethoxyphenylisopropylamine.
 Journal of Neurochemistry
, 2005,
95
, 15751584 doi:10.1111/j.1471-4159.2005.03477.x
Ó
2005 International Society for Neurochemistry,
J. Neurochem.
(2005)
95
, 1575–1584
1575
 
3,4,5-trimethoxyamphetamine (TMA) (Hey 1947). Thisanalogue had roughly twice the potency of mescaline(Shulgin
et al 
. 1961). Following the synthesis of TMA,numreous ring-substituted PEAs and PIAs were prepared andtested for biological activity. In general, PIA analogues werefound to be more potent and have longer durations of actionwhen compared to PEA analogues (Glennon
et al 
. 1983), but the reason(s) for the difference in hallucinogenic potency hasnot been elucidated. The addition of the alpha-methyl groupincreases hydrophobicity and also will likely retard metabolicdeamination, but this explanation is not adequate to explainthe potency differences, and fails to account for differencesobserved using
in vitro
experiments.A recent study has systematically examined the differ-ence in efficacy between PEAs and their correspondingPIA analogues. In that work, voltage clamp techniqueswere used to measure current generated in response to phenylalkylamine hallucinogens using
Xenopus laevis
oocyte membranes transiently expressing the rat 5-HT
2A
receptor (Acuna-Castillo
et al 
. 2002). PEAs were found to be virtually non-efficacious whereas their correspondingPIA analogues were reported to be weak partial agonists.The authors proposed that an antagonist or weak partialagonist mechanism of action for hallucinogens might still be tenable. More recently, using the same oocyte assaysystem, these workers have reported that PEAs areantagonists at the transiently expressed rat 5-HT
2A
receptor (Villalobos
et al 
. 2004). They concluded that their resultscast doubt on the notion that phenylalkylamines halluci-nogens are full or partial 5-HT
2A
agonists. The fact that a 5-HT
2A
receptor antagonist blocks the effects of halluci-nogens in man, however (Vollenweide
et al 
. 1998),indicates that the
Xenopus
oocyte model may not reflect 5-HT
2A
receptor-mediated signaling relevant to human psychopharmacology.AlthoughthePEAsareachiral,additionofthemethylgrouptothealphasidechaincarbonaffordsthePIAs,whichhavetwooptical isomers. It is a well-accepted tenet of pharmacologythat the optical isomers (enantiomers) of biologically activemolecules have different pharmacological properties. Most  biological targets, often macromolecular proteins, are chiral,stereospecific and/or stereoselective in their actions. Thedifference in potency (the eudismic ratio) between the moreactive enantiomer (eutomer) and the less active enantiomer (distomer) can typically vary from several-fold to severalhundred-fold,dependingonthechemicalstructure.OfthePIAenantiomers, the (
 R
)-isomers [(
 R
)-(–)-PIAs, e.g. (
 R
)-4-bromo-2,5-dimethoxyphenylisopropylamine(DOB),Fig. 1]aremore potent 
in vitro
(Dyer 
et al 
.1973;Cheng
et al 
.1974),inanimalmodels (Harris
et al 
. 1977; Young and Glennon 1996), and intheir ability to induce ASC in humans (Shulgin 1973; Shulginand Shulgin 1991). We reasoned that further study of the pharmacological differences between the enantiomers of selected PIAs and their corresponding PEAs might provideadditional insight into the underlying mechanism of action for hallucinogenic phenethylamines.The present work focuses on the role of the alpha-methylin PIAs in 5-HT
2A
receptor activation. We describe herein the pharmacological characterization of a small series of substi-tuted phenethylamine 5-HT
2A
agonists (Fig. 1) in activatingthe phospholipase C (PLC) signaling pathway in both thecloned rat and human 5-HT
2A
receptors. We then docked andminimized one set of alpha-methyl enantiomers (
 R
)-DOBand (
)-DOB, along with their achiral homologue 4- bromo-2,5-dimethoxyphenethylamine (2C-B) (Fig. 1) into a homology model of the human 5-HT
2A
receptor that was
Fig. 1
Phenethylamine (PEA) and phenyl-isopropylamine (PIA) compounds used inthis study. 4-Trifluoromethyl-2,5-dimethoxy-phenylisopropylamine (DOTFM) and 4-ethylthio-2,5-dimethoxyphenylisopropylamine(Aleph-2) were not available as the pureenantiomers and were only tested as theracemic (±) materials.
1576
J. C. Parish
et al.
Ó
2005 International Society for Neurochemistry,
J. Neurochem.
(2005)
95
, 1575–1584
 
developed based on an
in silico
activated model of bovinerhodopsin (Chambers and Nichols 2002).
Experimental procedures
Materials
All agonists used in this study were synthesized in our laboratory(e.g. see Nichols
et al 
. 1973). Drugs were fully characterized bymelting point, NMR, mass spectrometric, TLC, and elementalanalysis. Enantiomers were analyzed for optical rotation and valuesmatched those reported.
Cell culture methods
The GF-6 cell line stably expressing the rat 5-HT
2A
receptor (5500 fmol/mg) was the kind gift of Dr David Julius of theDepartment of Pharmacology, University of California, SanFrancisco, CA, USA. A549 cells, stably expressing the human5-HT
2A
receptor (150 fmol/mg protein) (A20 cells) were the kindgift of Dr Ulrike Weyer-Czernilofsky of the Ernst Boehringer Institute, Bender and Co., Vienna, Austria. Both GF-6 and A20 cellswere maintained in culture flasks at 37
°
C with 5% CO
2
inDulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis,MO, USA) supplemented with 10% (v/v) fetal bovine serum(Atlanta Biologicals, Lawrenceville, GA, USA), 2 m
M L
-glutamine(Gibco, Grand Island, NY), 100 units/mL penicillin (Gibco, GrandIsland, NY), 100
l
g/mL streptomycin (Gibco), and 300 mg/LG-418 m (Sigma-Aldrich). Cells were passaged at 90–95% conflu-ency and discarded after approximately 30 passages or when theyfailed to respond to 5-HT.
Establishing a stable high-expression human 5-HT
2A
R cell line
The h5-HT
2A
receptor gene insert was excised from pcDNA3.1-h5HT
2A
R (Guthrie cDNA resource center, Sayre, PA, USA; http:// www.cdna.org) with
Pme
I and
Xho
I and subcloned into the pBudCE4 vector (Invitrogen, Carlsbad, CA, USA), that was cut with
Not 
I, blunted with T4 DNA polymerase in the presence of dNTPs, and then cut with
Xho
I (New England Biolabs, Beverly,MA, USA). Insert and vector were purified, quantified, ligated, andamplified utilizing standard methods. Orientation, identity, andsequence were verified by primer-directed sequencing (Retrogen,San Diego, CA, USA) utilizing custom synthesized EF-1
a
forwardand BGH reverse primers (Integrated DNATechnologies, Coralville,IA). HEK-293 cells were transfected with 3.5
l
g pBudCE4-h5HT
2A
R utilizing the Fugene-6 lipofection reagent (Roche Bio-molecules, Indianapolis, IN, USA) in a 3:1 (w : v) ratio, and stableclones were selected as per the package insert instructions in the presence of 60
l
g/mL Zeocin (Invitrogen). Cells were maintained inculture flasks at 37
°
C with 5% CO
2
in Dulbecco’s modified Eagle’smedium supplemented with 10% fetal bovine serum, 2 m
ML
-glutamine, 100 units/mL penicillin, 100
l
g/mL streptomycin,and 30
l
g/mL Zeocin. Colonies of stable Hh2A clones survivingselection and maintenance with Zeocin were then assayed for h5HT
2A
R expression by saturation isotherm binding.
Radioligand assays
Membrane preparations, saturation isotherm, and competition binding assays were performed as previously described (Chambers
et al 
. 2003). [
3
H]Ketanserin and [
125
I]4-iodo-2,5-dimethoxyphenyl-isopropylamine (DOI) were used for saturation assays.
Inositol phosphate accumulation
After treatment with phenylalkylamine agonists, GF-6 or A20 cellswere assayed for the accumulation of phosphoinositides as previously described in detail (Kurrasch-Orbaugh
et al 
. 2003).These cells provided a comparison of both the rat and human 5-HT
2A
receptor responses.
Computational modeling/virtual docking
Ligand structures were drawn and energy minimized (Powellmethod, 0.01 kcal/mol*A gradient termination, MMFF94s forcefield, MMFF94 charges, 1000 maximum iterations) using the Sybylmodeling program (Tripos, St. Louis, MO, USA). The
in silico-
activated homology model of the h5-HT
2A
receptor was prepared as previously described (Chambers and Nichols 2002). Virtual doc-kings of energy minimized ligands to the h5-HT
2A
receptor were performed using the program GOLD (Cambridge CrystallographicData Center, Cambridge, UK) and scored using GOLDScore withdefault settings except for constraints. The GOLDScore fitnessalgorithm was constrained to orientations containing ligand–proteininteractions implicated by site-directed mutagenesis and previousmodeling (Chambers and Nichols 2002) to possibly be essential for  binding. Distance constraints of 0.2–0.3 nm (2–3 A˚) were se between the side chain carbonyl carbon of D155
(3.32)
and the aminenitrogen of the ligand (Wang
et al 
. 1993; Kristiansen
et al 
. 2000);from S159
(3.36)
and T160
(3.37)
to the ligand 2-position oxygen(Almaula 
et al 
. 1996); and between S239
(5.43)
and the ligand 5- position oxygen (Johnson
et al 
. 1997; Shapiro
et al 
. 2000). Thehighest ranked docking output structures were merged with the h5-HT
2A
receptor model and analyzed with Sybyl.Merged ligand–receptor structures were energy minimized as a subset based on the ligand molecule (aggregates set to monomers>0.6 nm (6 A˚) radius from the ligand, monomers >1.2 nm (12 A˚)radius ignored, Powell method, 0.1 kcal/mol*A gradient termin-ation, MMFF94s force field, MMFF94 charges, distance dielectricof 4, 1000 maximum iterations). Constraints for subsequent molecular dynamics simulations and minimizations in Sybyl weredefined as above for GOLD; however, hydrogen bond constraintswere defined as a distance range constraint of 0.1–0.2 nm (1–2 A˚) between each polar residue’s hydrogen and the appropriate oxygenatom on the ligand. Constrained molecular dynamics simulationswere then run on the energy-minimized ligand–receptor structures(constraints, force field, charges and dielectric as outlined above,aggregates as above plus backbone atoms, NTV ensemble at 300K,Boltzmann distribution of initial velocities, 5000 steps of 1 fs, and5 fs snapshots). Structures with lowest potential energy after the first 1000 fs equilibration period were then energy minimized as outlinedabove with defined constraints.
Data analysis
GraphPad Prism Software (GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, California, USA, http:// www.graphpad.com) was used to calculate
i
values for radioliganddisplacement, EC
50
s, and intrinsic activities for PLC activation. Theradioligand displacement data were best fit to a one-site model and
 K 
i
values were calculated from EC
50
s using the concentration of 
Phenylalkylamines at the 5-HT2A receptor 
1577
Ó
2005 International Society for Neurochemistry,
J. Neurochem.
(2005)
95
, 1575–1584

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