JPET #68098 Lobeline Analogs with Enhanced Affinity and Selectivity for Plasmalemma and ...

51 Pages·2004·0.25 MB·English

JPET Fast Forward. Published on April 30, 2004 as DOI: 10.1124/jpet.104.068098
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JPET #68098

Lobeline Analogs with Enhanced Affinity and Selectivity for Plasmalemma D
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and Vesicular Monoamine Transporters lo
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Dennis K. Miller1, Peter A. Crooks, Guangrong Zheng, Vladimir P. Grinevich2, Seth D. Norrholm3 et.a
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and Linda P. Dwoskin tjo
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College of Pharmacy, University of Kentucky, Lexington, KY t A
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Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on April 30, 2004 as DOI: 10.1124/jpet.104.068098
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JPET #68098

Running Title: Lobeline Analog Pharmacology
Number of Pages: 39
Number of Tables: 3
Number of Figures: 8
Number of References: 40
Number of Words:
- Abstract: 250 D
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- Introduction: 639 lo
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- Discussion: 1562 fro
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Correspondence should be addressed to: et.a
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Linda P. Dwoskin, Ph.D. tjo
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College of Pharmacy ls
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University of Kentucky t A
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Lexington, KY, 40536-0082 T
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Phone: [phone] rn
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FAX: [phone] F
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e-mail: [email protected] ary
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Abbreviations: 5-HT, 5-hydroxytryptamine, serotonin; 10R-MESP, N-methyl-2R-(2R-hydroxy-2-
phenylethyl)6S-(2-phenylethen-1-yl)piperidine; 10S/10R-MEPP, N-methyl-2R-(2R/2S-hydroxy-2-
phenylethyl)6S-(2-phenylethyl)piperidine; BSA, bovine serum albumin; DA, dopamine; DAT,
dopamine transporter; DTBZ, dihydroxytetrabenazine; EDTA, disodium ethylenediamine
tetraacetate; GBR-12909, 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)4-(3-phenylpropyl)piperazine;
hDAT, human dopamine transporter; HEPES, N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic
acid]; HEK, human embryonic kidney 293 cells; HEK-hDAT, HEK-hNET, HEK-hSERT, human
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embryonic kidney 293 cells transfected with hDAT, hNET, and hSERT cDNA, respectively;
hDAT, human dopamine transporter; hNET, human norepinephrine transporter; hSERT, human
serotonin transporter; ketoalkene, N-methyl-2R-(2-oxo-2-phenylethyl)6S-(2-phenylethen-
1yl)piperidine; lobeline Tos, lobeline tosylate, lobeline-8-O-tosylate; MLA, methyllycaconitine;
MTD, N-methyl-2,6-cis-di-trans-styrylpiperidine, meso-transdiene; MTBZ,
β β
methoxytetrabenazine; [125I]RTI-55 ((-)-2 -carbomethoxy-3 -(4-iodophenyl)tropane; nAChR,
nicotinic receptor; NE, norepinephrine; NET, norepinephrine transporter; PEI,
phenylethylenimine; SERT, serotonin transporter; (-)-TTD, N-methyl-2S,6S-di-trans- D
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styrylpiperidine, trans-transdiene; TTX, tetrodotoxin; VMAT2, vesicular monoamine transporter. lo
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Recommended Section Assignment: Neuropharmacology et.a
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JPET Fast Forward. Published on April 30, 2004 as DOI: 10.1124/jpet.104.068098
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Abstract
Lobeline attenuates the behavioral effects of psychostimulants in rodents and inhibits the
function of nicotinic receptors (nAChRs), dopamine transporters (DAT) and vesicular
monoamine transporters (VMAT2). Monoamine transporters are considered valid targets for
drug development for the treatment of methamphetamine abuse. In the current study, a series
of lobeline analogs were evaluated for affinity and selectivity at these targets. None of the
analogs were more potent than nicotine at the [3H]methyllycaconitine binding site (α7* nAChR
subtype). Lobeline tosylate was equipotent with lobeline inhibiting [3H]nicotine binding, but 70- D
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fold more potent inhibiting nicotine-evoked 86Rb+ efflux, demonstrating antagonism of α4β2* loa
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nAChRs. Compared to lobeline, defunctionalized analogs, lobelane, meso-transdiene and (-)- fro
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trans-transdiene, showed dramatically reduced affinity at α4β2* nAChRs, and 15-100-fold et.a
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higher affinity (Ki= 1.95, 0.58 and 0.26 µM, respectively) at DAT. meso-Transdiene and (-)- tjou
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trans-transdiene competitively inhibited DAT function, whereas lobelane and lobeline acted .o
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noncompetitively. 10S/10R-MEPP and 10R-MESP were 2-3 orders of magnitude more potent t A
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(Ki = 0.01 and 0.04 µM, respectively) than lobeline inhibiting [3H]serotonin uptake; 10S/10R- J
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MEPP showed 600-fold selectivity for this transporter. Uptake results using hDAT and hSERT ls
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expressed in HEK-293 cells were consistent with native transporter assays. Lobelane and eb
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ketoalkene were 5-fold more potent (Ki= 0.92 and 1.35 µM, respectively) than lobeline (Ki= 5.46 ry 1
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µM) inhibiting [3H]methoxytetrabenazine binding to VMAT2 in vesicle preparations. Thus, 3
structural modification (defunctionalization) of the lobeline molecule markedly decreases affinity
for α4β2* and α7* nAChRs, while increasing affinity for neurotransmitter transporters, affording
analogs with enhanced selectivity for these transporters and providing new leads for the
treatment of psychostimulant abuse.
Keywords: dopamine, lobeline, lobelane, meso-transdiene, methamphetamine, nicotinic
receptors, norepinephrine, serotonin, transporter, vesicular monoamine transporter
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Lobeline, an alkaloid from Indian tobacco, inhibits the behavioral and neurochemical
effects of psychostimulant drugs of abuse. For example, lobeline attenuates d-amphetamine-,
methamphetamine- and nicotine-induced hyperactivity (Green et al., 2001, Miller et al., 2001,
2002), and lobeline inhibits the discriminative stimulus effects of methamphetamine (Miller et al.,
2001). Although lobeline is not self-administered, lobeline decreases methamphetamine self-
administration in rats, which is not surmounted by increasing methamphetamine unit doses
(Harrod et al., 2001, 2003). These results suggest that lobeline lacks abuse liability, while D
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decreasing the stimulant and rewarding effects of methamphetamine via a noncompetitive lo
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mechanism of action. fro
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Psychostimulant-induced behavioral activation and reinforcement are mediated, at least tjo
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in part, via interaction with neurotransmitter transporters, that regulate synaptic dopamine (DA) ls
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concentrations (Wise and Bozarth, 1987; Koob, 1992). Methamphetamine is a substrate for the t A
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DA transporter (DAT; Sulzer et al., 1995; Johnson et al., 1998) and decreases the activity of the T
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vesicular monoamine transporter (VMAT2; Brown et al., 2000, 2001). Support for a role for rn
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VMAT2 in mediating the behavioral effects of stimulant drugs is provided by studies with VMAT2 F
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knockout mice in which amphetamine-induced conditioned place preference is attenuated ary
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(Takahashi et al., 1997). While effects on DAT, SERT and/or VMAT2 may not be the only 0
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mechanisms responsible for the reinforcing properties of psychostimulants (Rocha et al., 1998;
Sora et al., 1998), these neurotransmitter transporters are considered prime targets for
developing pharmacotherapies to treat psychostimulant abuse.

Until recently, the pharmacological activity of lobeline was believed to primarily result
from its high affinity interaction with nicotinic acetylcholine receptors (nAChRs; Abood et al.,
1988; Reavill et al., 1990; Bhat et al., 1991; Court et al., 1994). Lobeline inhibits nAChR
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JPET Fast Forward. Published on April 30, 2004 as DOI: 10.1124/jpet.104.068098
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JPET #68098
subtypes mediating both nicotine-evoked [3H]DA release and nicotine-evoked 86Rb+ efflux (Miller
et al., 2000). However, lobeline also interacts with VMAT2 and DAT (Dwoskin and Crooks,
2002). Lobeline potently inhibits [3H]dihydroxytetrabenazine (DTBZ) binding to VMAT2 (IC =
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0.90 µM) and inhibits [3H]DA uptake (IC = 0.88 µM) into rat striatal vesicle preparations (Teng
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et al., 1997, 1998), and furthermore is ~90-fold less potent (IC = 80 µM) inhibiting [3H]DA
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uptake into rat striatal synaptosomes (Teng et al., 1997). In addition to inhibiting DAT and
VMAT2 function, high concentrations of lobeline (10-50 µM) increase [3H]serotonin (5-HT)
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release from rat hippocampal slices in a mecamylamine-insensitive manner (Lendvai et al., o
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1996), suggesting that lobeline interacts with SERT. Thus, in addition to interacting with ad
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nAChRs, lobeline also inhibits VMAT2 more potently than DAT or SERT, suggesting that from
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VMAT2 may be a critical target for its pharmacological activity. t.a
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Consistent with the observation that lobeline is not self-administered in rats (Harrod et .o
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al., 2003), lobeline does not evoke DA release, but stimulates dihydroxyphenylacetic acid t A
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overflow (Teng et al., 1997), which likely results from alterations in presynaptic DA storage via J
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an interaction of lobeline with VMAT2 (Dwoskin and Crooks, 2002). Furthermore, lobeline als
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inhibits d-amphetamine- and methamphetamine-evoked DA release from superfused rat striatal e
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slices (Miller et al., 2001; Krishnamurthy et al., 2004). These results are consistent with the ry
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effect of lobeline to inhibit methamphetamine self-administration (Harrod et al., 2001). These 2
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preclinical data suggest that lobeline has potential as a pharmacotherapy for psychostimulant
abuse.

Structural modification of the lobeline molecule has afforded compounds with differing affinities
for nAChRs (Flammia et al., 1999), however, these analogs have not been evaluated for their
activity at neurotransmitter transporters. The present study evaluates a series of lobeline
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JPET Fast Forward. Published on April 30, 2004 as DOI: 10.1124/jpet.104.068098
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JPET #68098
α β α
analogs for their activities at 4 2* nAChRs, 7*nAChRs, DAT, SERT, NET and VMAT2, with
the aim of identifying analogs with high affinity and selectivity for these target sites. The exact
subunit composition, stoichiometry and arrangement of native nAChRs remain to be elucidated,
which is indicated by an asterisk (*) following the subunit designation (Lukas et al., 1999). Such
analogs may be useful candidates for probing specific targets for elucidating the underlying
neurochemical mechanism(s) responsible for lobeline-induced inhibition of the behavioral
effects of methamphetamine.
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JPET Fast Forward. Published on April 30, 2004 as DOI: 10.1124/jpet.104.068098
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Methods

Animals. Male Sprague-Dawley rats (200-250 g upon arrival) were purchased from
Harlan Laboratories (Indianapolis, IN) and were housed two per cage with ad libitum access to
food and water in the Division of Laboratory Animal Resources at the College of Pharmacy at
the University of Kentucky. Experimental protocols involving the animals were in accordance
with the NIH Guide for the Care and Use of Laboratory Animals, and were approved by the
Institutional Animal Care and Use Committee at the University of Kentucky.
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Chemicals. [3H]DA (dopamine, dihydroxyphenylethylamine 3,4-ethyl-2-[N-3H]; specific ed
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activity, 25.6 Ci/mmol), (±)-[3H]MLA (methyllycaconitine, [1α,4(S),6β,14α,16β]-20-ethyl- jp
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1,6,14,16-tetramethoxy-4[[[2-([3-3H]methyl-2,5-dioxo-1-pyrrolidinyl)benzoyl]oxy]methyl]- sp
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aconitane-7,8-diol); specific activity, 25.4 Ci/mmol), S(-)-[3H]nicotine (S(-)[N-methyl-3H]; specific rn
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activity, 80 Ci/mmol), [3H]NE (norepinephrine, levo-[7-3H]; specific activity 27.5 Ci/mmol), rg
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β β t A
86RbCl (specific activity, 55.2 mCi/mmol), [125I]RTI-55 ((-)-2 -carbomethoxy-3 -(4- SP
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iodophenyl)tropane; specific activity, 2200 Ci/mmol) and [3H]5-HT (serotonin, hydroxytryptamine ou
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creatine sulfate 5-[1,2-3H(N)]; specific activity, 27.5 Ci/mmol) were purchased from Perkin Elmer o
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Life Sciences (Boston, Massachusetts). [3H]MTBZ ((±)-methoxytetrabenazine [O-methyl-3H]; bru
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specific activity 56.8 Ci/mmol) was a generous gift from Dr. Michael Kilbourn (Department of , 2
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Radiology, University of Michigan Medical School, Ann Arbor, MI). Bovine serum albumin
(BSA), catechol, dopamine, disodium ethylenediamine tetraacetate (EDTA), ethylene glycol-
bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), fluoxetine HCl, 1-(2-[bis(4-
fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR-12909 HCl), N-[2-
hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid] (HEPES), S(-)-nicotine ditartrate (nicotine),
nomifensine maleate, pargyline HCl, polyethylenimine (PEI), serotonin, tetrodotoxin (TTX),
tris[hydroxymethyl]aminomethane hydrochloride (Trizma HCl), tris[hydroxymethyl]-
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aminomethane base (Trizma) and tropolone were purchased from Sigma Chemical Co. (St.
Louis, MO). α-D-Glucose, L-ascorbic acid and potassium phosphate monobasic were
purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI), AnalaR- BHD Ltd. (Poole,
U.K.) and Mallinckrodt (Paris, KY), respectively. Lobeline hemisulfate [2S,6R,8S-(-)-lobeline]
was purchased from ICN Biochemicals Inc. (Aurora, OH). All other commercially obtained
chemicals were purchased from Fisher Scientific (Pittsburgh, PA).

The lobeline analogs, ketoalkene (N-methyl-2R-(2-oxo-2-phenylethyl)6S-(2-phenylethen- D
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1yl)piperidine), 10R-MESP (N-methyl-2R-(2R-hydroxy-2-phenylethyl)6S-(2-phenylethen-1- loa
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yl)piperidine), 10S/10R-MEPP (N-methyl-2R-(2R/2S-hydroxy-2-phenylethyl)6S-(2- fro
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phenylethyl)piperidine), lobelanidine, lobelanine, MTD (N-methyl-2,6-cis-di-trans- et.a
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styrylpiperidine, meso-transdiene), (-)-TTD (N-methyl-2S,6S-di-trans-styrylpiperidine, trans- tjo
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transdiene), lobelane and lobeline tosylate (lobeline-8-O-tosylate) were synthesized by ls.o
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structural modification of the lobeline molecule (Zheng et al., 2004) and are illustrated in Fig. 1. t A
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The structures of the lobeline analogs were verified by 1H- and 13C-NMR spectroscopy, mass T
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spectrometry and in some cases X-ray crystallography. rna
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[3H]Nicotine Binding Assay. Striata from 2-4 rats were homogenized using a Tekmar ary
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polytron in 10 vol of ice-cold modified Kreb’s-HEPES buffer (20 mM HEPES, 118 mM NaCl, 4.8 02
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mM KCl, 2.5 mM CaCl , 1.2 mM MgSO , pH 7.5). Homogenates were incubated (5 min at 37
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0C) and centrifuged (29,000 x g for 20 min at 4 0C). Resulting pellets were resuspended in 10
vol of ice-cold MilliQ water, incubated (5 min at 37 0C) and centrifuged (29,000 x g for 20 min at
4 0C). Resulting pellets were again resuspended in 10 vol of ice-cold 10% Kreb’s-HEPES
buffer, then incubated and centrifuged as described. Final pellets were stored at -70 0C in fresh
10% Kreb’s-HEPES buffer until use. Upon assay, pellets were resuspended in 10% Kreb’s-
HEPES buffer, incubated and centrifuged as previously described. Final pellets were
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JPET #68098
resuspended in ice-cold MilliQ water (2.0 ml) to provide ~200 µg protein/100 µl membrane
suspension. Inhibition of specific [3H]nicotine binding by lobeline and its analogs was assessed
using a previously described method (Crooks et al., 1995). Briefly, assays were performed in
triplicate in a final vol of 200 µl Kreb’s-HEPES buffer containing 250 mM Tris (pH 7.5, 4 0C).
Reactions were initiated by addition of 100 µl of membrane suspension to tubes containing 50 µl
Kreb’s-HEPES buffer or 1 of 9 concentrations (0.1 nM – 1 mM, final concentration) of nicotine,
lobeline or analog and 50 µl [3H]nicotine (3 nM, final concentration). Nonspecific binding was
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determined in triplicate in the presence of nicotine (10 µM). Following incubation (90 min at 4 o
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0C), reactions were terminated by dilution of samples with ice-cold Kreb’s-HEPES buffer ad
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followed by immediate filtration through Whatman GF/B glass fiber filters (presoaked in 0.5% m
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PEI) using a cell harvester (MP-43RS, Brandel Inc., Gaithersburg, MD). Filters were processed t.a
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and radioactivity determined by liquid scintillation spectroscopy (B1600TR scintillation counter; u
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Packard/Perkin-Elmer Life Sciences; Boston, MA). .org
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[3H]MLA Binding Assay. Whole rat brain (minus cortex, striatum and cerebellum) was J
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homogenized in 20 vol of ice-cold hypotonic buffer (2 mM HEPES, 14.4 mM NaCl, 0.15 mM ls
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KCl, 0.2 mM CaCl2 and 0.1 mM MgSO4, pH 7.5). Homogenates were incubated at 37 0C for 10 ebru
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min and centrifuged (25,000 x g for 15 min at 4 0C). Pellets were washed 3 times by ry 1
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resuspension in 20 vol of buffer, followed by centrifugation. Final pellets were resuspended in 3
incubation buffer to provide ~150 µg protein/100 µl membrane suspension. Binding assays
were performed in duplicate, in a final vol of 250 µl incubation buffer, containing 20 mM HEPES,
144 mM NaCI, 1.5 mM KCl, 2 mM CaCl , 1 mM MgSO and 0.05% BSA, pH 7.5. Assays were
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initiated by the addition of 100 µl membrane suspension to 150 µl of sample containing 2.5 nM
[3H]MLA and 1 of 8 concentrations (30 nM - 100 µM, final concentration) of lobeline or analog,
and incubated for 2 hr at room temperature. Nonspecific binding was determined in the
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