[3H]A-585539 [(1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5- aza-2-azoniabicyclo[2.2.1]heptane], a novel high-affinity α7 neuronal nicotinic receptor agonist: Radioligand binding characterization to rat and human brain

Article abstract of DOI:10.1124/jpet.107.130062

Receptor binding was characterized for [³H](1S,4S)-2,2-dimethyl- 5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane ([ ³H]A-585539), a selective high-affinity α7 nicotinic acetylcholine receptor (nAChR) agonist with rapid

Full text of DOI:10.1124/jpet.107.130062

0022-3565/08/3241-179–187$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics
JPET 324:179–187, 2008
Vol. 324, No. 1
130062/3291911
Printed in U.S.A.
[3H]A-585539 [(1S,4S)-2,2-Dimethyl-5-(6-phenylpyridazin-3-yl)-
5-aza-2-azoniabicyclo[2.2.1]heptane], a Novel High-Affinity ␣7
Neuronal Nicotinic Receptor Agonist: Radioligand Binding
Characterization to Rat and Human Brain
David J. Anderson, William Bunnelle, Bruce Surber, Jia Du, Carol Surowy, Eliane Tribollet,
Anouk Marguerat, Daniel Bertrand, and Murali Gopalakrishnan
Neuroscience Research, Global Pharmaceutical Research & Development, Abbott Laboratories, Abbott Park, Illinois (D.J.A.,
W.B., B.S., J.D., C.S., M.G.); and Department of Neuroscience, Centre Me´ dical Universitaire,
Geneva, Switzerland (E.T., A.M., D.B.)
ABSTRACT
Receptor binding was characterized for [3H](1S,4S)-2,2-dimethyl- for [3H]MLA, and in general, a 5 to 10-fold higher affinity was
5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane observed for [3H]A-585539 binding. There was also a good
([3H]A-585539), a selective high-affinity ␣7 nicotinic acetylcholine correlation of Ki values between [3H]A-585539 binding to rat
receptor (nAChR) agonist with rapid kinetics, low nonspecific brain and human cortex. The use of a ␣7/5-hydroxytrypta-
binding, and high specific activity. At 4°C, the association was mine type-3 chimera revealed that the N-terminal domain of
1
monophasic and rapid (t
⁄2
ϭ 8.0 min); dissociation was slower ␣7 nAChR was sufficient to faithfully reproduce the phar-
(t
ϭ 64.2 min). The Kd in rat brain at 4°C was 0.063 nM, whereas macology of [3H]A-585539 binding. Autoradiographic studies
⁄2
1
at 22 and 37°C, the Kd values were 0.188 and 0.95 nM, respec- comparing [3H]A-585539 and [125I]␣-bungarotoxin revealed a
tively. In contrast, the Bmax (34 fmol/mg protein) was unaffected similar pattern of labeling in the rat. In summary, [3H]A-
by temperature. In human cortex, [3H]A-585539 bound with a 585539 was shown to have excellent binding characteristics
Kd of 0.066 nM and a Bmax of 5.8 fmol/mg protein at 4°C, in rat and human brain and represents the first high-affinity
whereas under similar conditions, specific [3H]methyllycac- ␣7 agonist radioligand with utility in the characterization of
onitine ([3H]MLA) binding was not measurable. A number of this important nAChR subtype that is targeted toward
agonist and antagonist nAChR ligands displaced binding to ameliorating cognitive deficits underlying neuropsychiatric
rat brain membranes with rank order of affinity similar to that and neurodegenerative disorders.
The ␣7 nicotinic acetylcholine receptor (nAChR) is a pen- autonomic ganglia, and adrenal chromaffin cells, and in non-
tameric, rapidly activating and desensitizing ligand-gated
ion channel expressed in the mammalian central nervous
system. The ␣7 subunit is widely expressed in the brain,
especially in regions associated with cognitive processing,
neuronal cell types. The ␣7 subtype is distinguished from
other nAChRs by its relatively high permeability to Ca2ϩ
,
rapid desensitization, and sensitivity to antagonists, such as
␣-bungarotoxin (␣-Bgt) and methyllycaconitine (MLA) (Ward
et al., 1990). Unlike other nAChR subtypes, the functional
significance of the ␣7 nAChR is not only attributable to its
electrogenic properties (i.e., modulation of neuronal excitabil-
This work was supported by Abbott Laboratories. The autoradiography
work was supported, in part, by the Swiss National Science Foundation (to
D.B.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.130062.
ity and neurotransmitter release) but also to its high Ca2ϩ
-
permeability and association with biochemical signaling path-
ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; ␣-Bgt, ␣-bungarotoxin; MLA, methyllycaconitine; A-585539, (1S,4S)-2,2-dimethyl-5-
(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane; PNU-282987, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochlo-
ride; PHA-543613, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide; (Ϯ)-AR-R17779, (Ϯ)-spiro[1-azabicyclo[2.2.2]octane-
3,5Ј-oxazolidin-2Ј-one]; A-582941, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole; SSR180711, 1,4-diazabicyclo[3.2.2]-
noname-4-carboxylic acid, 4-bromophenyl ester; BSS, balanced salt solution; BSA, bovine serum albumen; PEI, polyethylenimine; A-85380,
3-(2(S)-azetidinylmethoxy)pyridine; PNU-120596, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea; MDL 72222, 3-tropanyl-3,5-
dichlorobenzoate; TC-5280, 2(R)-(3-pyridyl)-1-azabicyclo[3.2.2]nonane; varenicline, 7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)(3)-
benzazepine; HPLC, high-performance liquid chromatography; BRL 43694, granisetron; 5-HT3, 5-hydroxytryptamine type-3; [3H]GR65630,
3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-3H3-1H-indol-3-yl)-1-propanone.
179

180
Anderson et al.
ways (reviewed in Role and Berg, 1996; Berg and Conroy, 2002; able tool in the characterization of nAChRs in skeletal mus-
Dajas-Bailador and Wonnacott, 2004). At the cellular level, cle for functional studies as a pseudo-irreversible antagonist
activation of ␣7 nAChRs can regulate interneuron excitability, and for labeling, purifying, and visualizing native nAChRs.
modulate the release of excitatory and inhibitory neurotrans- Of the cloned neuronal subunits, the ␣7, ␣8, and ␣9 subunits
mitters, and contribute to neuroprotective effects (for review are unique in that they are all sensitive to ␣-Bgt when
see, Dani and Bertrand, 2007).
expressed in Xenopus laevis oocytes. MLA is a natural nord-
Recent studies with antisense, gene knockout, and sub- iterpenoid alkaloid, identified as the principal toxic agent in
type selective ligands have provided evidence that target- Delphinium brownie (Ward et al., 1990), [3H]MLA binds to
ing the ␣7 nAChRs can lead to improvements in cognitive rat brain membranes with an affinity, pharmacological pro-
performance and sensory gating deficits in vivo. For example, file and regional distribution characteristic of ␣-Bgt-sensi-
␣7 nAChR genetic knockout mice have shown impaired per- tive, putative ␣7 subunit-containing nAChRs (Davies et al.,
formance in ethanol-induced contextual fear conditioning 1999). Although [3H]MLA displays rapid binding kinetics, it
(Wehner et al., 2004) and showed further deterioration in suffers from a relatively high degree of nonspecific binding.
hippocampus-selective associative learning and memory when Unlike the heteromeric nAChRs where a number of agonist
crossed with Tg2576 animals (Dineley et al., 2002). Selective ␣7 ligands (e.g., A-85380) have become available to characterize
nAChR agonists, such as PNU-282987 (Bodnar et al., 2005; receptors in animals and humans under various drug treat-
Hajos et al., 2005), PHA-543613 (Wishka et al., 2006), ment and disease conditions, high affinity ␣7 nAChR agonist
AR-R17779 (Levin et al., 1999; Van Kampen et al., 2004), ligands have not yet emerged.
SSR180711 (Biton et al., 2007), and A-582941 (Bitner et
During the course of our in-house efforts, we discovered
al., 2007), improve performance in models thought to cap- several bicycloheptane analogs with high-binding affinities
ture sensory-gating deficit, short-term working memory, toward displacement of [3H]MLA. In this study, we describe
and memory consolidation domains of cognitive function. [3H]A-585539 as the first high-affinity agonist radioligand
Moreover, sensory gating and cognitive performance are with excellent binding characteristics across both rodent and
known to be impaired in rodents with decreased expression human brain.
or following pharmacological blockade of ␣7 nAChRs (Luntz-
Leybman et al., 1992; Felix and Levin, 1997; Stevens et al.,
Materials and Methods
Synthesis of [3H]A-585539. [3H]Methyl iodide in toluene (250
1998; Levin and Rezvani, 2002). Consistent with this finding,
the expression of the ␣7 protein is reduced in the brains of
patients with Alzheimer’s disease (Burghaus et al., 2000) and
mCi in 0.1 ml, 85 Ci/mmol; American Radiolabeled Chemicals, Inc.,
schizophrenia (Freedman et al., 1995). The latter phenomenon St. Louis, MO) was combined with a solution of (1S,4S)-2-methyl-5-
(6-phenylpyridazin-3-yl)-2,5-diazabicyclo[2.2.1]heptane (Schrimpf et
al., 2007 ) in dichloromethane (0.788 mg, 2.96 micromole in 0.45 ml).
The vial was capped, and the mixture was allowed to react overnight
at room temperature. Methanol was added, and the solvents were
evaporated to yield [3H]A-585539 (Schrimpf et al., 2007 ) at a total
activity of 42 mCi. The product was taken up in methanol for high-
performance liquid chromatography (HPLC) purification. The struc-
ture of [3H]A-585539 along with that of [3H]MLA is illustrated in
Fig. 1.
has been correlated with polymorphisms in the promoter region
of the ␣7 gene in schizophrenic patients where such polymor-
phisms have been shown to result in decreased gene transcrip-
tion (Freedman et al., 1997; Leonard et al., 2002). These and
other observations have attracted considerable attention to the
␣7 nAChR as a drug target in recent years, the hypothesis being
that augmenting ␣7 nAChR function could ameliorate the cog-
nitive deficits associated with neuropsychiatric and neurode-
generative diseases (Rezvani and Levin, 2001; Martin et al.,
For purification by HPLC, ϳ7 mCi of [3H]A-585539 was evapo-
2004). Accordingly, selective agonists and positive allosteric rated to dryness, and the residue was dissolved in 4.5 ml of acetoni-
trile/water/trifluoroacetic acid (15:85:0.1). This solution was injected
(0.9 ml per run) onto a Phenomenex Luna C18(2) column (5 ␮m, 250
mm ϫ 4.6 mm i.d.; Phenomenex, Torrance, CA) using an Agilent
HPLC system (Agilent Technologies, Palo Alto, CA). [3H]A-585539
was eluted by a gradient mobile phase from 10 to 20% B in 20 min
where mobile phase A ϭ 0.1% trifluoroacetic acid in water and
mobile phase B ϭ 0.1% trifluoroacetic acid in acetonitrile at a flow
rate of approximately 1 ml/min. Peak detection and chromatograms
were obtained with an Agilent variable wavelength UV detector set
at 275 nm. The fractions containing [3H]A-585539 were collected at
modulators of ␣7 nAChRs are being discovered and devel-
oped for the treatment of cognitive deficits associated with
neuropsychiatric and neurodegenerative disease, such as
schizophrenia and Alzheimer’s disease.
Although the ␣7 nAChR continues to be explored as a
target for a number of central nervous system indications,
developments in high-affinity pharmacological tools have
largely been antagonists, such as the snake toxin peptide,
␣-Bgt, and the alkaloid MLA and the corresponding radioli-
gands [125I]␣-Bgt and [3H]MLA. ␣-Bgt has been an invalu- approximately 14 min using an Agilent fraction collector. The frac-
Fig. 1. Structures of [3H]A-585539
and [3H]MLA.

[3H]A-585539 Binding to ␣7 nAChRs
181
binding isotherms, 16 concentrations of [3H]A-585539 (0.01–2 nM)
in quadruplicate and homogenate containing 100 to 200 ␮g of
protein were incubated in a final volume of 500 ␮l for 90 min at
4°C. Nonspecific binding was determined in the presence of 30 ␮M
MLA. For concentration-inhibition assays, seven log-dilution con-
centrations of test compounds in duplicate, homogenate contain-
ing 100 to 200 ␮g of protein, and 0.5 nM [3H]A-585539 were
incubated in a final volume of 500 ␮l for 60 min at 4°C. Nonspe-
cific binding in quadruplicate was determined in the presence of
10 ␮M MLA. Bound radioactivity was collected by vacuum filtra-
tion onto Millipore MultiScreen harvest plates FB (Millipore Cor-
poration, Billerica, MA) presoaked with 0.3% polyethyleneimine
using a Packard cell harvester. The filters were then rapidly
rinsed with 2 ml of ice-cold BSS. PerkinElmer MicroScint-20
scintillation cocktail (40 ␮l) was added to each well, and bound
radioactivity was determined using a PerkinElmer TopCount in-
strument. Binding studies were also performed using human em-
bryonic kidney-293 cells expressing a ␣7/5-hydroxytryptamine
type-3 (5-HT3) chimera containing ligand-binding domain of ␣7
nAChR and the transmembrane/pore-forming region of 5-HT3 re-
ceptor. Using reverse transcriptase-polymerase chain reaction,
the coding sequence for the N-terminal 224 amino acids of human
␣7 nAChR (protein AAA83561) and that for the C-terminal 242
amino acids of human 5-HT3 (5-hydroxytryptamine; serotonin)
receptor (protein AAP35868) were amplified with overlapping
ends. Recombinant polymerase chain reaction using these two
overlapping fragments yielded the open-reading frame of the chi-
meric receptor. Human embryonic kidney-293 cells were trans-
fected, and stable transfectants were maintained using standard
procedures. To harvest, the cells were rinsed with phosphate-
buffered saline, scraped from flasks, and centrifuged at 1000g.
Pellets were homogenized with a Polytron homogenizer at a set-
ting of 7 for 30 s in 30 volumes of BSS-Tris buffer and diluted to
yield approximately 5 ␮g of protein per well. Binding conditions
were similar to those described above.
Fig. 2. Saturation binding isotherms of [3H]A-585539. Binding of [3H]A-
585539 to membrane-enriched fractions from rat brain (A) and human
frontal cortex (B). Membranes were incubated for 90 min at 4°C with a
range of 0.01 to 2 nM [3H]A-585539. Shown are specific binding (quadru-
plicates) and nonspecific binding (duplicates), which was measured in the
presence of 10 ␮M MLA. Figures shown are one of eight independent
experiments for each tissue. Data from saturation binding experiments
were analyzed by nonlinear regression, and the Kd and Bmax values are
shown in Table 1.
[3H]MLA Binding. Assay conditions were similar to those for
[3H]A-585539 binding. Membrane-enriched fractions from rat brain
were thawed, washed, and resuspended in 30 volumes of BSS-Tris
buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 50
mM Tris-Cl, pH 7.4, 22°C). Samples containing 100 to 200 ␮g of
protein, 5 nM [3H]MLA (25 Ci/mmol; Tocris Bioscience, Ellisville,
MO), and 0.1% bovine serum albumin (BSA; Sigma, St. Louis, MO)
were incubated in a final volume of 500 ␮l for 60 min at 22°C. Seven
log-dilution concentrations of each compound were tested in dupli-
cate. Nonspecific binding was determined in the presence of 10 ␮M
MLA. Bound radioactivity was isolated by vacuum filtration onto
Millipore MultiScreen harvest plates FB presoaked with 2% BSA.
The plates were rinsed and counted as above.
Light Microscopic Autoradiography. Two 2-month-old adult
rats of the Sprague-Dawley strain were killed by decapitation under
pentobarbital anesthesia (50 mg/kg body weight i.p., following Eth-
ical Rules of Animal Rights, Geneva, Switzerland); brains were dis-
sected out and frozen by immersion in 2-methylbutane at Ϫ25°C.
Serial sections (14 ␮m) were cut through the diencephalons in a
cryostat, thaw-mounted on gelatin-chrome-alum-coated slides, and
stored at Ϫ80°C until use. Two series of sections were incubated with
[3H]A-585539 at 25 and 50 nM respectively. Four series of adjacent
tions were combined, and the solvents were evaporated in vacuo. The
residue was dissolved in absolute ethanol (2 ml) to give 0.7 mCi.
[3H]A-585539 was assayed using an Agilent 1100 series HPLC
system consisting of a quaternary pump, an autosampler, and a
photodiode array UV detector. A Packard Radiomatic A 500 radio-
activity detector (PerkinElmer Life and Analytical Sciences,
Waltham, MA) was connected to the HPLC system. For radiodetec-
tion, a 500-␮l flow cell and a 3:1 ratio of Ultima-Flo M scintillation
cocktail to HPLC mobile phase were used. The analyses were per-
formed using a Phenomenex Luna C18(2) column (5 ␮m, 250 mm ϫ
4.6 mm i.d.). The mobile phase consisted of a gradient starting with
10% B and ramping to 20% B in 20 min followed by ramping to 90%
B in 1 min and hold at 90% B for 9 min, where mobile phase A ϭ 0.1%
trifluoroacetic acid in water and mobile phase B ϭ 0.1% trifluoro-
acetic acid in acetonitrile. The flow rate was set at approximately 1
ml/min, and the UV detection was set at 275 nm. The radiochemical
purity of [3H]A-585539 was found to be Ͼ98%. The specific activity
was determined to be 62.78 Ci/mmol by mass spectroscopy.
TABLE 1
Kd and Bmax values from ͓3H͔A-585539 saturation binding analysis
Tissue
Temperature
Kd
Bmax
n
[3H]A-585539 Binding. [3H]A-585539 (62.8 Ci/mmol) binding
was determined using membrane-enriched fractions (Sullivan and
Anderson, 1998) from rat brain (minus cerebellum) or human cortex
(ABS Inc., Wilmington, DE). Pellets were thawed at 4°C, washed,
and resuspended with a Polytron homogenizer at a setting of 7 in 30
volumes of BSS-Tris buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl2,
2 mM MgCl2, and 50 mM Tris-Cl, pH 7.4, 4°C). For saturation
oC
nM
fmol/mg protein
Rat brain
4
22
37
4
0.063 Ϯ 0.004
0.188 Ϯ 0.013
0.945 Ϯ 0.103
0.066 Ϯ 0.006
0.80 Ϯ 0.12
33.7 Ϯ 1.1
31.7 Ϯ 1.5
8
5
2
8
4
42.1 Ϯ 2.2
Human cortex
␣7/5-HT3 chimera
5.8 Ϯ 0.5
10,500 Ϯ 1000
4

182
Anderson et al.
Excel or Assay Explorer. Ki values were calculated from the IC50
values using the Cheng-Prusoff equation, where Ki ϭ IC50/(1 ϩ
[Ligand]/Kd).
Results
Saturation Binding. Specific binding of [3H]A-585539 to
rat brain membranes was saturable, rapid, and represented
80 to 95% of total binding over the concentration range (0.01
to 2 nM) examined (Fig. 2A; Table 1). In saturation-binding
isotherms, nonlinear regression analysis of specific binding
revealed an apparent Kd of 0.063 Ϯ 0.004 nM and a Bmax of
33.7 Ϯ 1.1 fmol/mg protein (n ϭ 8) at 4°C. Nonspecific bind-
ing was less than 10% of total binding at concentrations up to
Fig. 3. Kinetics of [3H]A-585539 binding to rat brain membranes. A,
dissociation of [3H]A-585539. Samples in quadruplicate were equilibrated
with 0.1 nM [3H]A-585539 for 90 min at 4°C (closed symbols, solid line) or
at 22°C (open symbols, dotted line). The radioligand then was displaced
with 10 ␮M unlabeled A-585539. Dissociation was terminated at various
time intervals, as indicated, by rapid filtration onto 96-well glass fiber
filter plates. Depicted is one experiment at each temperature for which
the t1/2 values of dissociation for [3H]A-585539 binding were 70.1 min at
4°C and 5.7 min at 22°C. The average t1/2 values for dissociation were
64.2 Ϯ 6.1 min at 4°C (n ϭ 5) and 7.3 Ϯ 0.5 min at 22°C (n ϭ 4). B,
association of [3H]A-585539. Samples in quadruplicate were incubated
with 0.1 nM [3H]A-585539 at 4°C (closed symbols, solid line) or at 22°C
(open symbols, dotted line) for various time intervals as indicated. Asso-
ciation was terminated with rapid filtration onto 96-well glass fiber filter
plates. One experiment is shown at each temperature for which the t1/2
values of association for [3H]A-585539 binding were 9.3 min at 4°C and
9.0 min at 22°C. The average t1/2 values for association were 8.0 Ϯ 0.8 min
at 4°C (n ϭ 5) and 7.4 Ϯ 0.7 min at 22°C (n ϭ 4).
sections were incubated with [3H]A-585539 at the same concentra-
tions together with either nicotine tartrate (1 mM) or unlabeled
A-585539 (500 ␮M). Remaining series were incubated with [125I]␣-
bungarotoxin at 1 nM either alone or together with 300 ␮M nicotine
tartrate. The binding procedure was conducted as described previ-
ously (Tribollet et al., 2004). Sections incubated with [3H]A-585539
were apposed for 3 weeks to a tritium-sensitive storage phosphorim-
aging screen. Digital images were obtained with a phosphorimaging
system (Cyclone storage Phosphor System; PerkinElmer Life and
Analytical Sciences) and Optiquant (PerkinElmer Life and Analyti-
cal Sciences), a Windows-based software. Sections incubated with
[
␤
125I]␣-bungarotoxin were placed in x-ray cassettes in contact with
hyperfilms (Amersham, Buckinghamshire, UK) for 3 days.
max
Films were developed in Kodak D19 (Kodak SA, Lausanne, Switzer-
land) and photographed with a digital camera.
Fig. 4. Displacement of [3H]A-585539 binding. Concentration-inhibition
curves for the displacement of [3H]A-585539 binding to rat brain (A) and
human cortex (B) membranes by three ␣7 nAChR compounds: MLA, PNU
282987, and racemic AR-R17779. Data points are the mean data from
three independent experiments. The Ki values are summarized in Table
2. C, correlation plot for log Ki values of displacement of [3H]A-585539
binding from rat brain versus human frontal cortex. Excluding MLA,
there was good correlation with a slope of 0.94 and a coefficient of
correlation of 0.88. Data points are the mean of 3 to 10 independent
determinations.
Data Analysis. Dissociation constant (Kd) and maximal binding
(Bmax) values from saturation binding and observed association rate
(kob) and dissociation rate (koff) from kinetic experiments were de-
termined using GraphPad Prism (GraphPad Software, San Diego,
CA). The Kd was determined from the equation Kd ϭ koff/kon, where
k
on ϭ (kob Ϫ koff)/L, where L is the concentration of radioligand. The
IC50 values were determined by nonlinear regression in Microsoft

[3H]A-585539 Binding to ␣7 nAChRs
183
half-time (t1/2) for [3H]A-585539 binding at 4°C was 8.0 Ϯ 0.8
min, and the dissociation t1/2 was 64.2 Ϯ 6.1 min (n ϭ 5).
Calculation of the dissociation constant from kinetic rate
constants yielded a Kd of 0.021 Ϯ 0.003 nM, which is in
reasonable agreement with the Kd value obtained from sat-
uration binding isotherms. At 22°C, the association t1/2 (7.4 Ϯ
0.7 min) was comparable to that obtained at 4°C, whereas the
dissociation t1/2 was much faster (7.3 Ϯ 0.5 min, n ϭ 4). The
rapid koff rate at 22°C precludes the determination of mean-
ingful dissociation constant by typical filtration assays.
Displacement Profile of [3H]A-585539. The radioli-
gand binding pharmacology of [3H]A-585539 was assessed
by examining the displacement of specific binding by ago-
nists, antagonists, and positive allosteric modulators from
rat and human brain membranes (Figs. 4 and 5; Table 2).
A number of ␣7-selective nAChR compounds, including
unlabeled A-585539, MLA, PNU-282987, (Ϯ)-AR-R17779,
Fig. 5. Comparison of displacement of [3H]A-585539 and [3H]MLA bind-
ing from rat brain membranes by nAChR agonists and antagonists. Data
points are the log Ki values from 3 to 11 independent determinations of ␣-bungarotoxin, and A-582941 were found to displace
[3H]A-585539 and [3H]MLA binding with the similar rank
data taken from Table 2. The coefficient of correlation and slope values
are 0.96 and 0.95, respectively.
order of affinity. Other nAChR ligands, such as (Ϯ)-epiba-
tidine, (Ϫ)-nicotine, (Ϫ)-cytisine, (ϩ)-anatoxin-A, vareni-
cline, and A-85380 also exhibited a high degree of correla-
ten times the Kd. At 22°C, the Kd was 0.188 nM, and the Bmax
was 31.7 fmol/mg protein (n ϭ 5). In contrast, the binding of
[3H]methyllycaconitine to rat membranes at 22°C was found
tion (Fig. 5). In general, a shift in Ki values was observed
between [3H]A-585539 and [3H]MLA binding displacement
to have a Kd of 1.26 Ϯ 0.33 nM and a Bmax of 32.7 Ϯ 5.8
in rat brain membranes. Displacement of [3H]A-585539
fmol/mg protein (n ϭ 3). However, nonspecific binding of
[3H]MLA ranged from 20 to 50% of total binding, even with
binding to human cortex membranes yielded nearly iden-
tical Ki values as those for rat brain membranes (Fig. 4C).
An interesting exception was MLA, which showed consid-
erably lower apparent affinity for human cortex mem-
branes. Positive allosteric modulators of ␣7 nAChRs, in-
cluding 5-hydroxyindole and PNU-120596, neither
displaced nor enhanced [3H]A-585539 binding under the
present experimental conditions.
the addition of 0.1% BSA to the incubation.
The high specific activity and excellent affinity of [3H]A-
585539 made it possible to directly detect binding to human
brain (Fig. 2B; Table 1). Human frontal cortex membranes
bound [3H]A-585539 at 4°C with a Kd value of 0.066 Ϯ 0.006
nM, comparable to that of rat brain, and a Bmax of 5.8 Ϯ 0.5
fmol/mg protein (n ϭ 8), which is lower than that observed in
the rat brain. Nonspecific binding ranged from 15 to 50% of
total binding for range of concentrations of [3H]A-585539. In
contrast, specific binding of [3H]MLA to human frontal cortex
membranes was not detectable due to low counts and high
nonspecific binding levels.
To determine specificity of A-585539 for the ␣7 nAChR, the
compound was further evaluated in a radioligand binding
screen panel containing representatives of multiple G-pro-
tein coupled receptors, and ligand- and voltage-gated ion
channel binding sites at CEREP (receptor binding and en-
zyme profile; CEREP, Potiers, France). A-585539 at 10 ␮M
did not show significant displacement of binding of over 75
targets, with the exception of 5-HT3 receptors, where 89%
Kinetics of [3H]A-585539 Binding. [3H]A-585539 bind-
ing was temperature-dependent. Kinetic analysis demon-
strated temperature dependence for the dissociation rate but
not for the association rate (Fig. 3, A and B). The association inhibition of [3H]BRL 43694 (granisetron) binding was ob-
TABLE 2
Displacement of ͓3H͔A-585539 binding to rat and human brain: comparison with ͓3H͔MLA
͓3H͔A-585539
͓3H͔MLA
Rat
Rat
Human
95% CI
Compound
Ki,
95% CI
nH
n
KI
nH
n
Ki
95% CI
nH
n
nM
nM
nM
A-585539
0.071
0.19
0.36
2.7
0.06–0.09
0.16–0.23
0.33–0.38
2.1–3.4
5.8–7.2
6.2–19
0.8
1.1
0.9
1.0
0.9
1.1
2.1
1.2
1.2
1.1
1.0
1.1
1.5
4
10
3
ND
1.01
0.6–1.6
1.5–2.7
1.0
1.1
1.6
1.4
1.1
1.7
1.2
1.6
1.3
1.6
1.5
1.6
1.6
4
10
3
4
6
MLA
2.5
1.8–3.6
0.9
9
2
TC-5280
ND
1.1
3.2
16.7
17.7
9.7
15.6
17.6
40.5
159
153
1.2
21
46.4
88
17.4
122
144
299
1390
1450
1580
0.9–1.5
Epibatidine
PNU 282987
A-582941
8
0.7–1.7
2.2–4.5
13–22
8.1–39
7.1–13
12.0–20
6.4–48
27–60
0.8
0.9
1.1
0.9
1.1
0.9
0.9
1.0
1.0
1.7
3
6
6
2
4
6
2
6
6
3
13–33
6.5
7
33–66
10.8
9
59–130
11
3
␣-Bungarotoxin
Anatoxin-A
Varenicline
A-85380
(Ϯ)-AR-R17779
(Ϫ)-Nicotine
DMPP
12.2
19.8
33.4
33.6
9.4–16
3
14–21
16–24
5
97–150
6
23–49
6
100–210
150–610
1100–1760
850–2500
1200–2100
7
20–57
4
4
128
176
308
100–160
62–500
220–420
5
5
8
4
110–230
110–210
6
4
CI, 95% confidence interval; n ϭ number of determinations, each carried out in duplicate; DMPP, 1,1-dimethyl-4-phenylpiperazinium; ND, not determined.

184
Anderson et al.
served with 10 ␮M A-585539. The 5-HT3 antagonist, MDL ent Kd of 0.801 Ϯ 0.118 nM and a Bmax of 10,500 Ϯ 1000
72222, does not significantly inhibit [3H]A-585539 binding to fmol/mg protein (n ϭ 4). The pharmacology of [3H]A-585539
rat brain at concentrations up to 10 ␮M. In contrast, MDL binding was found to be consistent with an interaction with
72222 inhibited the binding of the 5-HT3 antagonist ␣7 nAChRs. A number of ␣7 nAChR-selective compounds,
[3H]GR65630 to human 5-HT3 serotonin receptors with a Ki including MLA, TC-5280, PNU-282987, ␣-bungarotoxin, and
of 12 nM (95% CL 9–15 nM, n ϭ 3), whereas A-585539 had a SSR180711 were found to displace [3H]A-585539 binding
Ki of 1.4 ␮M (95% CL 1.2–1.7 ␮M, n ϭ 3).
with the same rank order of affinity as that seen in rat brain.
Localization of [3H]A-585539 Binding. To assess the Non-␣7 nAChR-selective nAChR compounds, such as (Ϯ)-
regions on ␣7 nAChRs responsible for binding interactions, epibatidine, (Ϫ)-nicotine, (ϩ)-anatoxin-A, 1,1-dimethyl-4-
studies were conducted using a ␣7/5-HT3 chimera where the phenylpiperazinium, varenicline, and A-85380, had a high
N-terminal domain of ␣7 nAChR was fused with the trans- degree of correlation for displacement between ␣7/5-HT3 chi-
membrane/pore-forming region of the 5-HT3 receptor. [3H]A- mera-expressing cells and rat brain (Fig. 6B). Overall, there
585539 showed no detectable binding to 5-HT3 receptors was a coefficient of correlation of 0.94 and a slope of 1.0.
(data not shown), whereas specific and saturable binding of
Regional Distribution of [3H]A-585539 Binding. Auto-
[3H]A-585539 was detected in cells expressing the ␣7/5-HT3 radiography studies using [3H]A-585539 in rat brain showed
chimera (Fig. 6A). Binding was rapid and represented Ͼ95% a distribution of binding comparable with that of [125I]␣-
of total binding over the concentration range (0.05 to 5 nM) bungarotoxin, with a high level of binding in hippocampus
examined. Saturation binding isotherms revealed an appar- and the superficial gray layer of the superior colliculus
(Fig. 7A). Binding was fully displaced by unlabeled A-585539
(Fig. 7C). Nicotine also effectively displaced binding, al-
though some residual labeling was seen in the CA3 area of
the hippocampus (Fig. 7B). In contrast, the intense labeling
yielded by [125I]␣-bungarotoxin, in particular in the hip-
pocampus and in the superior colliculus, (Fig. 7D), was fully
displaced (Fig. 7E).
Discussion
Selective modulation of ␣7 nAChRs is thought to regulate
cellular processes impaired in schizophrenia, Alzheimer’s
disease, and other dementias. Our laboratory and others
have previously reported on the synthesis of novel analogs
derived from various diamines exemplified by SSR180711
(Biton et al., 2007), A-582941 (Bitner et al., 2007), and quinu-
clidine derivatives, such as PNU-282987 (Bodnar et al., 2005;
Hajos et al., 2005), PHA-543613 (Wishka et al., 2006), and
AR-R17779 (Levin et al., 1999; Van Kampen et al., 2004), as
␣7 nAChR agonists. Despite the heightened interest in this
molecular target for a number of central nervous system
indications, developments in high-affinity radioligands, espe-
cially agonists with which to study this important subtype,
have been less forthcoming. In the course of our discovery
efforts to develop novel selective ␣7 nAChR agents from a
series of 2.2.1 diamines, we found the diazabicycloheptane
analog A-585539 to have greater than 1000-fold selectivity
for ␣7 nAChRs over ␣4␤2 nAChRs in radioligand binding.
Initial experiments in X. laevis oocytes expressing rat ␣7
nAChRs showed that A-585539 activated ionic currents with
an EC50 of 0.61 ␮M (95% CI 0.29–1.2 ␮M) and efficacy of
94.0 Ϯ 6.4% (n ϭ 3). No agonist activity was detected at
concentrations up to 100 ␮M in IMR-32 (ATCC, Manassas, VA)
Fig. 6. Binding of [3H]A-585539 to ␣7/5-HT3 chimera expressed in HEK-
neuroblastomas or in transfected cell lines containing ␣3␤2,
␣3␤4, ␣4␤2, or ␣4␤4 nAChRs using fluorometric imaging plate
293 cells. A, saturation binding of [3H]A-585539 to ␣7/5-HT3 cell mem-
branes. One of four independent experiments is represented. Specific
binding was determined in the presence of [3H]A-585539 (0.05–10 nM;
90-min incubation at 4°C). Nonspecific binding was determined in the
presence of 30 ␮M MLA. Data were analyzed by nonlinear regression, and
the Kd and Bmax values were determined for each experiment. The
mean Ϯ S.E.M. estimates for Kd were 0.80 Ϯ 0.12 nM and 10,500 Ϯ 1000
fmol/mg protein for Bmax. B, comparison of displacement of [3H]A-585539
binding from rat brain membranes and ␣7/5-HT3 chimera expressing cell
membranes by nAChR ligands. Data points are log Ki values of 3 to 10
independent determinations for rat brain and three to four determina-
tions for ␣7/5-HT3 cell membranes. The coefficient of correlation and
slope values are 0.94 and 1.02, respectively.
reader-based calcium flux assays (J.-H. Gronlein, J. Malysz,
and M. Gopalakrishnan, unpublished observations). Given that
A-585539 could be modified using a tritium methylation, it was
chosen as a suitable candidate for radiolabeling. Here, we de-
scribe the characterization of [3H]A-585539, one of the first
selective high-affinity ␣7 agonist radioligand with rapid kinet-
ics, low nonspecific binding, and high specific activity.
[3H]A-585539 bound to rat brain and to human frontal
cortex membranes with high affinity and with a pharmaco-

[3H]A-585539 Binding to ␣7 nAChRs
185
high affinity and high specific activity (63 Ci/mmol) of [3H]A-
585539 allowed for reliable pharmacological analysis of the
low density of receptors in the human brain. This is a distinct
advantage over [3H]MLA, which with an affinity of 2 nM does
not generate measurable labeling of ␣7 nAChRs in human
membranes under standard experimental conditions. An ad-
ditional attribute of [3H]A-585539 is its relatively low non-
specific binding. At concentrations near the Kd, the nonspe-
cific binding accounts for only 5% of total binding in rat brain
and approximately 20% in human brain. In contrast, with
[3H]MLA binding, substantially higher levels of nonspecific
binding (ϳ35% at Kd) were observed in rat brain, even with
the addition of 0.1% BSA in the incubation. Another limita-
tion with [3H]MLA binding is the existence of displaceable
binding from glass fiber filters, which can be reduced to some
extent by presoaking the filter plates with 2% BSA. Such
limitations were not encountered with [3H]A-585539.
The kinetics of binding of [3H]A-585539 at 4°C were rapid
for association (t1/2 ϭ 8.0 min) and slower for dissociation
(t1/2 ϭ 64 min), which allows for vacuum filtration assays.
The Kd value determined from kinetic experiments (21 pM) is
in reasonable agreement with values obtained from equilib-
rium binding experiments. At 22°C, the association rate was
similar (t1/2 ϭ 7.4 min), although the dissociation rate was
more rapid (t1/2 ϭ 7.3 min) compared to that observed at 4°C.
From saturation binding isotherms, the estimated Kd was
188 pM, which is some 8-fold lower than the value measured
at 4°C. Under similar conditions, the Kd for [3H]MLA binding
did not show temperature dependence (D. J. Anderson, un-
published observations). The lower affinity of binding ob-
served with [3H]A-585539 at increased temperatures is
characteristic of thermodynamic discrimination of binding
kinetics in which enthalpy-driven processes produce a tem-
perature-dependent shift in apparent affinity, unlike entro-
py-driven processes that do not exhibit temperature-depen-
dent shifts in affinity (Borea et al., 1998). These observations
are consistent with thermodynamic analysis with [3H]cy-
tisine binding that predominantly labels high-affinity ␣4␤2
nAChRs, where it was shown that agonist binding was both
enthalpy- and entropy-driven, whereas antagonist binding
was totally entropy-driven (Borea et al., 2000).
Fig. 7. Comparison of labeling obtained with [3H]A-585539 and [125I]␣-
bungarotoxin in the rat brain. A to C, taken from three adjacent coronal
sections cut through the diencephalon. Section A was incubated with 25
nM [3H]A-585539; sections B and C with 25 nM [3H]A-585539 together
with 1 mM nicotine tartrate (B) or 500 ␮M unlabeled A-585539 (C). D and
E are from two adjacent sections cut from another rat brain and incu-
bated with 1 nM 125I-␣-bungarotoxin either alone (D) or together with 300
␮M nicotine tartrate (E). Note that [3H]A-585539 and [125I]␣-bungaro-
toxin yielded a similar labeling of field CA3 of the hippocampus (CA3) and
of the superficial gray layer of the superior colliculus (SuG). Note also
that [3H]A-585539 binding in CA3 (A and B) is only partially displaced by
nicotine tartrate (B), whereas it is not detectable in the presence of
unlabeled A-585539 in excess (D). Bar ϭ 5 mm.
Displacement of [3H]A-585539 binding by structurally di-
verse nAChR ligands, including agonists and antagonists,
indicated a rank order of selectivity characteristic of ␣7-
selective binding (Table 2; Fig. 4), with A-588539 showing
highest affinity. In general, a good correlation between log Ki
values for inhibition of [3H]A-585539 binding from both rat
brain and human cortex membranes was observed. A notable
exception was MLA, which interestingly had a 10-fold
greater affinity for rat brain than for human cortex. This may
be an additional reason why [3H]MLA binding was not mea-
surable in human cortex in our hands. When displacement of
logical profile akin to that of [3H]MLA, a well characterized [3H]A-585539 binding in rat brain membranes was compared
␣7 nAChR antagonist (Ward et al., 1990; Davies et al., 1999; to displacement of [3H]MLA binding in the same tissue (Ta-
Whiteaker et al., 1999). Saturation binding isotherms re- ble 2; Fig. 5), an excellent correlation was noted. However,
vealed comparable binding affinities in rat (63 pM) and hu- compounds showed some 5 to 10-fold higher affinity in dis-
man (66 pM), whereas analysis of receptor density demon- placement of [3H]A-585539 binding relative to [3H]MLA,
strated that human cortex expressed some 6-fold lower which may be related to the temperature-dependent shift in
density of binding sites compared to the rat. A similar ratio affinity with [3H]A-585539.
and density were as previously observed with [125I]␣-Bgt in
To further investigate the regions responsible for high-
rat P2 membranes (35 fmol/mg protein) and human cortex (5 affinity interactions of [3H]A-585539 binding, we assessed its
fmol/mg protein) (Davies et al., 1999; Falk et al., 2003). The interaction with a chimeric ␣7 nAChR, the ␣7/5-HT3 chi-

186
Anderson et al.
mera, where the N-terminal domain of the human 5-HT3 it ideal for filtration assays. It provides an advantage to be
receptor was replaced with the N-terminal domain of the able to study agonist-agonist interactions at the ␣7 nAChR
human ␣7 nAChR. Figure 6 shows [3H]A-585539 binding to without having to interpret agonist displacement of tena-
the ␣7/5-HT3 chimera where high levels of expression of the cious antagonists. [3H]A-585539 binding also avoids the pit-
chimeric receptor could be detected. The log Ki values for the falls of working with the pseudo-irreversible [125I]␣-Bgt that
displacement by a range of ligands at the ␣7/5-HT3 chimera requires 37°C incubation and the reversible nonspecific bind-
were highly correlated to the log Ki values obtained in rat brain ing of [3H]MLA. A key advantage of [3H]A-585539 is that it
membranes. In general, incorporation of the N-terminal do- enables the measurement of ␣7 nAChRs in a variety of tis-
main of ␣7 nAChR alone was sufficient to faithfully repro- sues, especially human brain, unlike MLA, and should aid in
duce the pharmacology of [3H]A-585539 binding. Interest- the further study of ligand interactions with the ␣7 nAChR
ingly, even though the transmembrane/pore-forming region subtype under physiological and pathological conditions.
of 5-HT3 receptor has a similar protein structure and a rel-
atively high degree of homology with the ␣7 nAChR, the Kd
of 0.8 nM represents approximately 10-fold decrement in the
affinity of [3H]A-585539 binding. This relatively lower
affinity was also translated across the range of nAChR com-
pounds tested, as reflected in the leftward shift of the cor-
relation curve when comparing log Ki values. This im-
plies that regions beyond the N-terminal domain of the ␣7
nAChR may also be involved in the formation of the binding
pocket responsible for high-affinity binding interactions of
[3H]A-585539.
The conventional target for ␣7 nAChR drug discovery has
focused on the “agonist”-binding site of nAChRs, with the
emergence of several agonist and competitive antagonists.
More recently, ligands that are positive allosteric modulators
of ␣7 nAChRs have been identified (Hurst et al., 2005; Gron-
lein et al., 2007; reviewed in Faghih et al., 2007). The positive
allosteric modulators, 5-hydroxyindole and PNU-120596,
failed to displace or increase [3H]A-585539 or [3H]MLA bind-
ing to brain membranes. This is consistent with the notion
that, unlike agonists, positive allosteric modulators do not
directly activate or desensitize receptors by interacting with
orthosteric binding sites, but instead they enhance the sen-
sitivity and/or efficacy of the receptor during agonist activa-
tion (Bertrand and Gopalakrishnan, 2007).
Acknowledgments
We thank Steven Cassar for generating the chimera construct and
Kirsten Thorin-Hagene for generating the HEK-293 cell line.
References
Berg DK and Conroy WG (2002) Nicotinic alpha 7 receptors: synaptic options and
downstream signaling in neurons. J Neurobiol 53:512–523.
Bertrand D and Gopalakrishnan M (2007) Allosteric modulation of nicotinic acetyl-
choline receptors. Biochem Pharmacol 74:1155–1163.
Bitner RS, Bunnelle WH, Anderson DJ, Buccafusco J, Curzon P, Decker MW, Frost
JM, Gronlien JH, Gubbins E, Li J, et al. (2007) Broad-spectrum efficacy across
cognitive domains by ␣7 nicotinic acetylcholine receptor agonism correlates with
activation of ERK1/2 and CREB phosphorylation pathways. J Neurosci 27:10578–
10587.
Biton B, Bergis OE, Galli F, Nedelec A, Lochead AW, Jegham S, Godet D, Lanneau
C, Santamaria R, Chesney F, et al. (2007) SSR180711, a novel selective alpha7
nicotinic receptor partial agonist: (1) binding and functional profile. Neuropsycho-
pharmacology 32:1–16.
Bodnar AL, Cortes-Burgos LA, Cook KK, Dinh DM, Groppi VE, Hajos M, Higdon NR,
Hoffmann WE, Hurst RS, Myers JK, et al. (2005) Discovery and structure-activity
relationship of quinuclidine benzamides as agonists of alpha7 nicotinic acetylcho-
line receptors. J Med Chem 48:905–908.
Borea PA, Varani K, Gessi S, Gilli P, and Gilli G (1998) Binding thermodynamics at
the human neuronal nicotine receptor. Biochem Pharmacol 55:1189–1197.
Borea PA, Dalpiaz A, Varani K, Gilli P, and Gilli G (2000) Can thermodynamic
measurements of receptor binding yield information on drug affinity and efficacy?
Biochem Pharmacol 60:1549–1556.
Burghaus L, Schutz U, Krempel U, de Vos RA, Jansen Steur EN, Wevers A,
Lindstrom J, and Schroder H (2000) Quantitative assessment of nicotinic acetyl-
choline receptor proteins in the cerebral cortex of Alzheimer patients. Brain Res
Mol Brain Res 76:385–388.
Dajas-Bailador F and Wonnacott S (2004) Nicotinic acetylcholine receptors and the
regulation of neuronal signalling. Trends Pharmacol Sci 25:317–324.
Dani JA and Bertrand D (2007) Nicotinic acetylcholine receptors and nicotinic
cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol
Toxicol 47:699–729.
Davies AR, Hardick DJ, Blagbrough IS, Potter BV, Wolstenholme AJ, and Wonna-
cott S (1999) Characterisation of the binding of [3H]methyllycaconitine: a new
radioligand for labelling alpha 7-type neuronal nicotinic acetylcholine receptors.
Neuropharmacology 38:679–690.
Dineley KT, Xia X, Bui D, Sweatt JD, and Zheng H (2002) Accelerated plaque
accumulation, associative learning deficits, and up-regulation of alpha 7 nicotinic
receptor protein in transgenic mice co-expressing mutant human presenilin 1 and
amyloid precursor proteins. J Biol Chem 277:22768–22780.
Faghih R, Gfesser G, and Gopalakrishnan M (2007) Advances in the discovery of
novel positive allosteric modulators of the ␣7 nicotinic acetylcholine receptor.
Recent Patents on CNS Drug Discovery 2:99–106.
Falk L, Nordberg A, Seiger A, Kjaeldgaard A, and Hellstrom-Lindahl E (2003)
Higher expression of alpha7 nicotinic acetylcholine receptors in human fetal com-
pared to adult brain. Brain Res Dev Brain Res 142:151–160.
Felix R and Levin ED (1997) Nicotinic antagonist administration into the ventral
hippocampus and spatial working memory in rats. Neuroscience 81:1009–1017.
Freedman R, Hall M, Adler LE, and Leonard S (1995) Evidence in postmortem brain
tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia.
Biol Psychiatry 38:22–33.
Freedman R, Coon H, Myles-Worsley M, Orr-Urtreger A, Olincy A, Davis A, Poly-
meropoulos M, Holik J, Hopkins J, Hoff M, et al. (1997) Linkage of a neurophys-
iological deficit in schizophrenia to a chromosome 15 locus. Proc Natl Acad Sci
U S A 94:587–592.
Gronlein J-H, Håkerud M, Ween H, Thorin-Hagene K, Briggs CA, Gopalakrishnan
M, and Malysz J (2007) Distinct profiles of ␣7 nAChR positive allosteric modula-
tion revealed by structurally diverse chemotypes. Mol Pharmacol 72:715–724.
Hajos M, Hurst RS, Hoffmann WE, Krause M, Wall TM, Higdon NR, and Groppi VE
(2005) The selective alpha7 nicotinic acetylcholine receptor agonist PNU-282987
[N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] enhances
GABAergic synaptic activity in brain slices and restores auditory gating deficits in
anesthetized rats. J Pharmacol Exp Ther 312:1213–1222.
Hurst RS, Hajos M, Raggenbass M, Wall TM, Higdon NR, Lawson JA, Rutherford-
Root KL, Berkenpas MB, Hoffmann WE, Piotrowski DW, et al. (2005) A novel
positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine recep-
tor: in vitro and in vivo characterization. J Neurosci 25:4396–4405.
In autoradiography studies, [3H]A-585539 displayed a re-
gional binding pattern similar to that observed with 125I-␣-
bungarotoxin (Fig. 7). Both ligands labeled comparable re-
gions of the rat brain with highest density of binding in the
CA3 area of the hippocampus (CA3) and the superficial gray
layer of the superior colliculus. Whereas unlabeled A-585539
fully displaced [3H]A-585539 binding, 1 mM nicotine did not
completely displace the [3H]A-585539 label in the CA3 region
of the hippocampus, an area that was intensely labeled by
[3H]A-585539. It is possible that [3H]A-585539 labeled a
small population of nAChRs in this discrete area that are
resistant to displacement by nicotine. However, because the
concentration of [3H]A-585539 used was 25 ␮M, which was
ϳ400-fold above the Kd, it is also possible that small amounts
of a localized non-nAChR receptor were labeled. Absolute
comparison of the two radioligands is difficult because of the
difference in attainable resolution between tritium and 125I.
Phosphorimaging of tritium is inferior to film imaging of 125I,
both in terms of visualization of structures and duration of
exposure. Nonetheless, the obtained images show clearly
that the highest levels of [3H]A-585539 binding are observed
in regions known to have high density of ␣7 nAChRs.
In summary, [3H]A-585539 demonstrates high-affinity
binding consistent with ␣7 nAChR pharmacology, a rapid
association rate, and a relatively slow dissociation rate—the
latter attributes, coupled with low nonspecific binding, make

[3H]A-585539 Binding to ␣7 nAChRs
187
Leonard S, Gault J, Hopkins J, Logel J, Vianzon R, Short M, Drebing C, Berger R,
Venn D, Sirota P, et al. (2002) Association of promoter variants in the alpha7
nicotinic acetylcholine receptor subunit gene with an inhibitory deficit found in
schizophrenia. Arch Gen Psychiatry 59:1085–1096.
Levin ED, Bettegowda C, Blosser J, and Gordon J (1999) AR-R17779, and alpha7
nicotinic agonist, improves learning and memory in rats. Behav Pharmacol 10:
675–680.
Levin ED and Rezvani AH (2002) Nicotinic treatment for cognitive dysfunction. Curr
Drug Targets CNS Neurol Disord 1:423–431.
Luntz-Leybman V, Bickford PC, and Freedman R (1992) Cholinergic gating of
response to auditory stimuli in rat hippocampus. Brain Res 587:130–136.
Martin LF, Kem WR, and Freedman R (2004) Alpha-7 nicotinic receptor agonists:
potential new candidates for the treatment of schizophrenia. Psychopharmacology
(Berl) 174:54–64.
Rezvani AH and Levin ED (2001) Cognitive effects of nicotine. Biol Psychiatry
49:258–267.
Role LW and Berg DK (1996) Nicotinic receptors in the development and modulation
of CNS synapses. Neuron 16:1077–1085.
Schrimpf MR, Sippy KB, Anderson DJ, Brunnelle WH, and Nersesian DL (2007)
inventors; Abbott Laboratories, assignee. Amino-aza-adamantane derivatives and
methods of use. U.S. patent application US2007/0072892 A1. 2007 Mar 29.
Stevens KE, Kem WR, Mahnir VM, and Freedman R (1998) Selective alpha7-
nicotinic agonists normalize inhibition of auditory response in DBA mice. Psycho-
pharmacology (Berl) 136:320–327.
Tribollet E, Bertrand D, Marguerat A, and Raggenbass M (2004) Comparative
distribution of nicotinic receptor subtypes during development, adulthood and
aging: an autoradiographic study in the rat brain. Neuroscience 124:405–420.
Van Kampen M, Selbach K, Schneider R, Schiegel E, Boess F, and Schreiber R (2004)
AR-R 17779 improves social recognition in rats by activation of nicotinic alpha7
receptors. Psychopharmacology (Berl) 172:375–383.
Ward JM, Cockcroft VB, Lunt GG, Smillie FS, and Wonnacott S (1990) Methyllyca-
conitine: a selective probe for neuronal alpha-bungarotoxin binding sites. FEBS
Lett 270:45–48.
Wehner JM, Keller JJ, Keller AB, Picciotto MR, Paylor R, Booker TK, Beaudet A,
Heinemann SF, and Balogh SA (2004) Role of neuronal nicotinic receptors in the
effects of nicotine and ethanol on contextual fear conditioning. Neuroscience 129:
11–24.
Whiteaker P, Davies AR, Marks MJ, Blagbrough IS, Potter BV, Wolstenholme AJ,
Collins AC, and Wonnacott S (1999) An autoradiographic study of the distribution
of binding sites for the novel alpha7-selective nicotinic radioligand [3H]-
methyllycaconitine in the mouse brain. Eur J Neurosci 11:2689–2696.
Wishka DG, Walker DP, Yates KM, Reitz SC, Jia S, Myers JK, Olson KL, Jacobsen
EJ, Wolfe ML, Groppi VE, et al. (2006) Discovery of N-[(3R)-1-azabicyclo[2.2.2]oct-
3-yl]furo[2,3-c]pyridine-5-carboxamide, an agonist of the alpha7 nicotinic acetyl-
choline receptor, for the potential treatment of cognitive deficits in schizophrenia:
synthesis and structure-activity relationship. J Med Chem 49:4425–4436.
Sullivan JP and Anderson DJ (1998) Characterization of neuronal nicotinic acetyl-
choline receptors. In: Current Protocols in Pharmacology (Enna SJ, Williams M,
Ferkany JW, Porsolt RD, Kenakin T, and Sullivan JP, eds.) pp 1:1.8.1–1.8.9,
Wiley, New York.
Address correspondence to: David J. Anderson, R-47W, AP9A, Abbott
Laboratories, Abbott Park, IL 60064-6125. E-mail: david.j.anderson@
abbott.com