Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery.

Abstract

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

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Effect of Novel Negative Allosteric Modulators of Neuronal
Nicotinic Receptors on Cells Expressing Native
and Recombinant Nicotinic Receptors: Implications
for Drug Discovery
Tatiana F. Gonza ´lez-Cestari, Brandon J. Henderson, Ryan E. Pavlovicz, Susan B. McKay,
Raed A. El-Hajj, Aravinda B. Pulipaka, Crina M. Orac, Damon D. Reed, R. Thomas Boyd,
Michael X. Zhu, Chenglong Li, Stephen C. Bergmeier, and Dennis B. McKay
Divisions of Pharmacology (T.F.G.-C., B.J.H., S.B.M., R.A.E., D.B.M.) and Medicinal Chemistry and Pharmacognosy
(R.E.P., C.L.), College of Pharmacy, The Ohio State University, Columbus, Ohio; Department of Neuroscience,
The Ohio State University, College of Medicine and Public Health, Columbus, Ohio (R.T.B., M.X.Z.);
and Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio (A.B.P., C.M.O., D.D.R., S.C.B.)
Received August 14, 2008; accepted October 7, 2008
ABSTRACT
Allosteric modulation of nAChRs is considered to be one of the
most promising approaches for drug design targeting nicotinic
acetylcholine receptors (nAChRs). We have reported previously
on the pharmacological activity of several compounds that
seem to act noncompetitively to inhibit the activation of ?3?4*
nAChRs. In this study, the effects of 51 structurally similar
molecules on native and recombinant ?3?4 nAChRs are char-
acterized. These 51 molecules inhibited adrenal neurosecretion
activated via stimulation of native ?3?4* nAChR, with IC50
values ranging from 0.4 to 13.0 ?M. Using cells expressing
recombinant ?3?4 nAChRs, these molecules inhibited calcium
accumulation (a more direct assay to establish nAChR activity),
with IC50values ranging from 0.7 to 38.2 ?M. Radiolabeled
nAChR binding studies to orthosteric sites showed no inhibitory
activity on either native or recombinant nAChRs. Correlation
analyses of the data from both functional assays suggested
additional, non-nAChR activity of the molecules. To test this
hypothesis, the effects of the drugs on neurosecretion stimu-
lated through non-nAChR mechanisms were investigated; in-
hibitory effects ranged from no inhibition to 95% inhibition at
concentrations of 10 ?M. Correlation analyses of the functional
data confirmed this hypothesis. Several of the molecules (24/
51) increased agonist binding to native nAChRs, supporting
allosteric interactions with nAChRs. Computational modeling
and blind docking identified a binding site for our negative
allosteric modulators near the orthosteric binding site of the
receptor. In summary, this study identified several molecules
for potential development as negative allosteric modulators and
documented the importance of multiple screening assays for
nAChR drug discovery.
Several physiological functions involve neuronal nicotinic
receptors (nAChRs), including memory and learning, atten-
tion, pain perception, and body temperature regulation. In
addition, nAChRs may be linked to several disease states,
including nicotine addiction, epilepsy, Parkinson’s disease,
and Alzheimer’s disease. Progress in this area is slowed by
the lack of pharmacological tools to investigate nAChR in-
volvement in these processes. Most drug discovery programs
target the orthosteric site of nAChRs where agonists and
competitive antagonists bind (e.g., Dwoskin and Crooks,
2001). Our laboratory is targeting negative allosteric (non-
competitive) sites as a novel approach for nAChR drug dis-
covery. A large number of chemically diverse drugs have been
classified as noncompetitive antagonists and are believed to
bind to specific sites on nAChRs (Lloyd and Williams, 2000;
Arias et al., 2006). Noncompetitive antagonists inhibit
nAChR function either by binding allosteric sites and chang-
ing conformational states of the receptor (negative allosteric
modulators) or by directly blocking the receptor-associated
ion channel (ion channel blockers). Several noncompetitive
antagonist binding sites have been proposed (Lloyd and Wil-
liams, 2000; Arias et al., 2006). One of these sites is located
within the nAChR ion channel, in which ligands cause steric
This work was supported in part by National Institutes of Health [Grant
DA12707].
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.108.144576.
ABBREVIATIONS: nAChR, neuronal nicotinic acetylcholine receptor; MLA, methyllycaconitine; NAM, negative allosteric modulator; AM, ace-
toxymethyl ester; HEK, human embryonic kidney; LBD, ligand binding domain; MD, molecular dynamics.
0022-3565/09/3282-504–515$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
JPET 328:504–515, 2009
Vol. 328, No. 2
144576/3426311
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blockade. Allosteric sites have been described in the receptor
vestibule, in the ?M1 domain, or simply as locations at the
lipid-protein interface.
Most drug discovery programs focus on the development of
agonists and competitive antagonists that target the nAChR
orthosteric site. One potential difficulty in this approach is
the physiochemical resemblance of orthosteric sites among
subtypes of nAChRs. Another approach that could poten-
tially be a powerful tool for therapeutic intervention is the
design of drugs that act as allosteric modulators of receptors.
Allosteric modulators exist for a variety of receptors (Wess,
2005). Allosteric modulation of nAChRs is considered to be
one of the most promising approaches in ligand design for
nAChR research and therapeutics (Cassels et al., 2005; Iorga
et al., 2006), and the characterization and localization of
these sites are beginning to be elucidated (Costa et al., 2003;
Iorga et al., 2006; McKay et al., 2007).
Our laboratory has reported previously the synthesis and
preliminary characterization of several noncompetitive an-
tagonists of nAChRs that are structurally related to methyl-
lycaconitine (MLA) (Bergmeier et al., 1999, 2004; Bryant et
al., 2002; Huang et al., 2008) and has recently described a
pharmacophore for the binding site of these molecules
(McKay et al., 2007). In the present study, a novel class of
negative allosteric modulators (NAMs) of nAChRs is identi-
fied and characterized. The following experiments character-
ize the nature of the inhibitory activities of our NAMs, de-
scribe their structure-activity relationships, and define a
potential ligand binding domain of these NAMs on ?3?4
nAChRs using homology modeling and blind docking
approaches.
Materials and Methods
Materials. (?)-Nicotine hydrogen tartrate, ?-bungarotoxin, poly-
ethylenimine, and components of N2? media were obtained from the
Sigma-Aldrich (St. Louis, MO). Fluo-4-acetoxymethyl ester (AM),
probenecid, and Pluronic F-127 were obtained from Invitrogen
(Carlsbad, CA). Dulbecco’s modified Eagle’s medium, Dulbecco’s
modified Eagle’s medium/F-12 (used in N2? medium), minimum
essential medium, penicillin, streptomycin, and L-glutamine were
obtained from Invitrogen. (?)-[5,6-bicycloheptyl-3H]epibatidine (spe-
cific activity, 55.5 Ci/mmol) and DL-[7-3H(N)]norepinephrine hydro-
chloride (specific activity, 10.9 Ci/mmol) were purchased from
PerkinElmer Life and Analytical Sciences (Boston, MA). Bovine ad-
renal glands were purchased from the Herman Falter Packing Com-
pany (Columbus, OH). Whatman GF/B filters were purchased from
Brandel Inc. (Gaithersburg, MD). All other reagents were purchased
from Thermo Fisher Scientific (Waltham, MA). In general, molecules
(Fig. 1) were prepared by reaction of hydroxymethyl piperidine with
the appropriate alkyl halide to provide the N-alkyl hydroxymethyl
piperidine, as reported previously by our laboratory (Bergmeier et
al., 1999, Bergmeier et al., 2004; Huang et al., 2008). This molecule
was then coupled to the appropriate carboxylic acid to provide the
target molecule (Bergmeier et al., 2004). All molecules were ?98%
pure as shown by
spectrometry. Before pharmacological studies, all compounds were
converted to their hydrochloride or oxalate salts.
Neurosecretion Studies. Bovine adrenal chromaffin cells were
dissociated from intact glands and placed into culture, as described
previously by our laboratory (Wenger et al., 1997). A [3H]norepineph-
rine assay was used to monitor neurosecretion from cultured cells
(Wenger et al., 1997). Cells were pretreated for 15 min with the drug
before stimulation (10 ?M nicotine or 56 mM KCl) in the continued
presence of the drug. The concentration of nicotine used in this
1H NMR,
13C NMR, and high-resolution mass
neurosecretion assay (10 ?M) is the EC100value of nicotine (Wenger
et al., 1997). When KCl was used as a stimulant, the NaCl concen-
tration in the buffer was reduced accordingly to maintain isotonicity.
Cultured adrenal chromaffin cells were typically used 3 to 7 days
after isolation.
[3H]Epibatidine Binding to Native and Recombinant ?3?4
nAChRs. Membrane preparations from bovine adrenal medullary
tissues or HEK 293 cells stably expressing bovine ?3?4 nAChRs
(BM?3?4 cells) were prepared, and binding assays were performed,
as described previously by our laboratory (Free et al., 2003). Briefly,
membranes were incubated at room temperature for 60 min in buffer
containing 1 nM [3H]epibatidine. Nonspecific binding was deter-
mined in the presence of 300 ?M nicotine. Specific binding was
typically 1 to 2% of total binding for recombinant nAChRs and 5 to
10% for native nAChRs. For homologous competition binding exper-
iments on adrenal membranes, epibatidine was used at increasing
concentrations in the presence or absence of 10 ?M APB-8. Typically,
single concentrations of our molecules (10 ?M) were used, except in
concentration-response experiments.
Measurement of Intracellular Calcium Using HEK 293 Cells
Stably Expressing Recombinant nAChRs. For the calcium accu-
mulation assays, KX?3?4R2 cells (Xiao et al., 1998), which express
rat ?3?4 nAChRs, were used because functional responses produced
by BM?3?4 cells were not as robust as needed for concentration-
response studies. Cells were plated on clear poly-D-lysine-coated
96-well plates at a density of 1.2 to 1.5 ? 105cells per well and
incubated at 37°C in 5% CO2, using minimum essential medium
supplemented with 10% fetal bovine serum, 10 mM L-glutamine, 0.7
mg/ml Geneticin (G418), 100 units/ml penicillin, and 100 ?g/ml
streptomycin. Forty-eight hours after plating, the cells were washed
with HEPES-buffered Kreb’s saline (HBK) (155 mM NaCl, 4.6 mM
KCl, 1.2 mM MgSO4, 1.8 mM CaCl2, 6 mM glucose, and 20 mM
HEPES, pH 7.4) by flicking the plate and loaded for 1 h at 24°C
(protected from light) with 40 ?l of HBK containing 2 ?M fluo-4-AM
solution, 2.5 mM probenecid, and 0.05% Pluronic F-127. Fluo-4-AM
and Pluronic F-127 were dissolved in dimethyl sulfoxide [100 and
20% (w/v), respectively], resulting in a final dimethyl sulfoxide con-
centration of ?0.1%. At the end of the incubation period, the cells
were washed, and 80 ?l of the corresponding buffers was added to
each well. The fluorescence was measured at ?0.7-s intervals using
a fluid handling integrated fluorescence plate reader (Flex Station;
Molecular Devices, Sunnyvale, CA).
nAChR antagonism was assessed using the following protocol. For
the nicotine control group, HBK (40 ?l) was added, and the fluores-
cence was measured for 40 s. Subsequently, nicotine (40 ?l of a 400
?M solution) was added to achieve a final concentration of 100 ?M
(EC100; see Fig. 3A), and the fluorescence was measured for 60 s.
Treatment groups received the antagonist (40 ?l of a 3? solution), in
the first addition, and then the same nicotine solution (400 ?M) with
the desired concentration of the antagonist, in the second addition.
Sham-treated groups (nonstimulated groups) were treated only with
HBK both times. Probenecid (2.5 mM) was included in all of the
solutions once the cells were loaded to prevent the leakage of fluo-4
from cells. The fluo-4 fluorescence was read at excitation of 494 nm
and emission of 520 nm from the bottom of the plate. Responses were
quantified by first calculating the net fluorescence, that is, the dif-
ference between stimulated groups’ fluorescence values (nicotine
control and treatment groups) and sham-treated group fluorescence
values. Peak fluorescence values (after stimulation) were obtained.
Results were expressed as a percentage of peak values of nicotine-
stimulated controls.
To assess nAChR agonist activity, cells were treated with 80 ?l of
a 2? solution in HBK of the desired concentrations of nicotine or
epibatidine. Fluorescence was recorded for 20 s before the stimula-
tion, and 100 s after the stimulation. Sham-treated group (nonstimu-
lated group) received only HBK. Peak fluorescence values were de-
termined after subtraction of sham group’s fluorescence values. The
percentages of the effects of each concentration of nicotine or epiba-
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tidine were calculated in relationship with the effect of 100 ?M
nicotine or 1 ?M epibatidine, respectively.
Homology Modeling and Docking. A homology model of the rat
?3?4 ligand binding domain (LBD) was constructed to computation-
ally probe for potential noncompetitive antagonist binding sites.
Crystal structures for three molluskan species of homopentameric
acetylcholine binding proteins (Celie et al., 2004, 2005; Hansen et al.,
2005), along with the mouse ?1 LBD monomer (Dellisanti et al.,
2007), were used as templates for the modeling process. Models were
built in an iterative manner with MODELLER9v1 (Andrej Sali, San
Francisco, CA) in which successive rounds focused on refining par-
ticular loop regions of the LBD while incorporating the lowest energy
model from the previous iteration as an additional template. Model
energies were evaluated in the AMBER suite of programs (Univer-
sity of California, San Francisco, CA) in which after explicit water
energy minimization, a generalized Born method was used to esti-
mate the free energy of solvation for the 200 models built in each
iteration. The internal energy and solvation free energy of each
model were summed, and the model with the lowest total energy was
chosen as the conformation to which the antagonists were docked.
The relaxed complex method (Lin et al., 2003) coupled with a blind
docking approach was used to search for noncompetitive antagonist
sites on the final ?3?4 LBD model. In the relaxed complex method,
docking to different protein conformations as extracted from a mo-
lecular dynamics (MD) simulation enhances the probability of repro-
ducing a correct binding mode by taking into consideration the
dynamic movements of the receptor. An explicit water MD simula-
tion of the ?3?4 model was carried out in AMBER 9 (University of
California, San Francisco, CA), and trajectory snapshots were ex-
tracted at 200-ps intervals for docking. Blind docking grids were
93.75 ? 93.75 ? 71.25 Å, with a grid point spacing of 0.375 Å. This
encompassed the entire LBD except for the loops that would nor-
mally come into contact with the membrane. Following grid con-
struction, the two stereoisomers of COB-3 were docked with Auto-
Dock 4 (The Scripps Research Institute, Jupiter, FL). In all docking
runs, compounds were docked 100 times with 100 million energy
evaluations, allowing all bonds to rotate freely. Antagonists were
docked to a ternary complex in which epibatidine was present in both
agonist binding sites to search for noncompetitive binding sites.
After docking the two COB-3 stereoisomers, the results for each
compound were clustered according to their centroid, with a toler-
ance of 4 Å. The top four clusters of the each compound at each
snapshot were then clustered together to identify consensus docking
positions.
Calculations and Statistics. Results were calculated from the
number of observations (n) performed in duplicate, triplicate, or
quadruplicate. Curve fitting was performed by Prism software
(GraphPad Software Inc., San Diego, CA) using the following equa-
tion for a single-site sigmoidal dose-response curve with a variable
slope: Y ? 100/{1 ? 10[(log IC50? X) ? HS]}, where Y is the
percentage of the maximal effect at a given concentration (X), and
HS is the Hill coefficient. IC50values and Hill coefficients were
obtained by averaging values generated from each individual con-
centration-response curve. For a few molecules, the IC50values were
extrapolated since 50% inhibition was not achieved at the highest
concentration used (10 ?M); higher concentrations could not be
tested due to solubility problems. For these molecules, extrapolated
IC50values, as well as the corresponding Hill coefficients, were taken
from the mean curve. Results were expressed as arithmetic means ?
S.E.M. (n ? 3) or S.D. (n ? 2), except for IC50and EC50values, which
were expressed as geometric means (95% confidence limits).
Results
In this study, a variety of techniques and approaches were
used to identify the sites and mechanisms of action of the
51 molecules. The chemical structures of these molecules are
found in Fig. 1. Initially, the effects of the drugs on nicotine-
stimulated adrenal neurosecretion were investigated. As il-
lustrated in Fig. 2 and Table 1, the drugs inhibited adrenal
neurosecretion stimulated using 10 ?M nicotine, with IC50
values ranging from 0.4 to 13.0 ?M. Hill coefficients varied
from ?1.2 to ?5.1. None of the molecules showed agonist
activity (data not shown).
The site of action of these molecules for their inhibitory
effects on nAChR-stimulated neurosecretion cannot be estab-
lished using this functional paradigm since multiple steps
are involved in the stimulus-secretion coupling pathway. To
investigate whether these molecules act via direct interac-
tion with nAChRs, calcium accumulation assays were per-
formed using cells expressing recombinant nAChRs. For this
assay, the functional responses (i.e., increases in intracellu-
lar calcium) are directly linked to nAChR activation (Staud-
erman et al., 1998; Chavez-Noriega et al., 2000). For these
studies, an HEK 293 cell line stably expressing rat ?3?4
nAChRs (KX?3?4R2) was used (Xiao et al., 1998). Table 2
established the feasibility of using this cell line. The relative
potencies of nicotine, epibatidine, and one of our own antag-
onists, COB-2, were identical to values reported in the liter-
ature for native bovine adrenal nAChRs (McKay and Trent-
Sanchez, 1990; McKay and Burkman, 1993; Wenger et al.,
1997; McKay et al., 2007); native nAChRs showed slightly
higher apparent affinities for the agonists. The inhibitory
effects of the competitive antagonist tubocurarine and the
noncompetitive antagonists mecamylamine and tetracaine
were also compared (Table 2); no major differences were seen
between native bovine and recombinant rat nAChRs. The
functional responses of KX?3?4R2 cells were also compared
with those of BM?3?4 cells (Fig. 3). For these studies, the
calcium concentration in the buffer for BM?3?4 cells was
increased to 20 mM to obtain a more robust response
(Chavez-Noriega et al., 2000; Pacheco et al., 2001). The con-
centration-dependent effects of nicotine, tubocurarine, and
tetracaine were identical (Fig. 3, A, C, and E), and recombi-
nant bovine nAChRs showed slightly lower apparent affini-
ties for epibatidine, mecamylamine, and COB-2 (Fig. 3, B, D,
and F).
The concentration-response effects of the 51 molecules
(Fig. 1) on nAChR-stimulated calcium accumulation in
KX?3?4R2 cells are found in Fig. 4. All 51 molecules pro-
duced concentration-dependent inhibition of nicotine-stimu-
lated increases in [Ca2?]i, with IC50values ranging from 0.7
to 38.2 ?M (Fig. 4). These data documented direct antagonist
activity of the molecules on nAChRs. Hill coefficients ranged
from ?0.6 to ?2.0; these values were not significantly differ-
ent from ?1.0 (unpaired t test, p values ?0.05). None of these
molecules showed any agonist activity (data not shown).
When the effects of the inhibitors on native and recombi-
nant nAChRs were directly compared via Pearson analysis,
no correlation was demonstrated (Pearson r ? ?0.176, p ?
0.217) (Fig. 5). One possibility involves the neurosecretion
assay; the inhibitors may have additional effects on more
distal steps (downstream of nAChR activation) of the stimu-
lus-secretion coupling pathway. To address this hypothesis,
the effects of the drugs on neurosecretion stimulated using
depolarizing concentrations of KCl (56 mM) were investi-
gated. This method of stimulation bypasses activation of
nAChRs and directly depolarizes adrenal membranes, result-
ing in neurosecretion. This approach has been used to local-
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ize the activity of drugs to nAChRs (e.g., McKay and Schnei-
der, 1984). Inhibition of KCl-simulated neurosecretion will
document additional inhibitory actions by the molecules (i.e.,
nonreceptor mechanisms). As seen in Fig. 6, the effects of the
drugs at 10 ?M concentrations varied from little (?25%) or
no inhibitory activity to significant (?75%) inhibitory activ-
ity. These observations suggested that most of these drugs
tested have at least two inhibitory effects: one effect medi-
ated through nAChR mechanisms (seen with the calcium
accumulation assays) and one effect mediated through non-
nAChR mechanisms (seen with the KCl-neurosecretion as-
say). These data also suggested that the lack of correlation
observed in Fig. 5 was due to these dual inhibitory activities.
In support of this, a significant correlation using molecules
with little or no non-nAChR effects was found when we
directly compared IC50values obtained from the nAChR-
neurosecretion assays and the calcium accumulation assays
(r ? 0.93, p ? 0.0001) (Fig. 7A). For drugs with large non-
nAChR effects, no significant correlation was observed (r ?
0.36, p ? 0.31) (Fig. 7B).
In the next series of experiments, radiolabeled agonist
binding studies were performed to investigate direct interac-
tions of our molecules with the orthosteric site (i.e., compet-
itive inhibition). The drugs (10 ?M) showed little (?25%) or
no inhibitory effects on [3H]epibatidine binding to recombi-
nant ?3?4 nAChRs (Table 1). The effects of these drugs on
Fig. 1. Chemical structures.
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binding to native ?3?4* nAChRs were somewhat more di-
verse than their effects on recombinant nAChRs, ranging
from little (?20%) or no effects to large increases in binding
(Table 1). At a concentration of 10 ?M, approximately half
(24/51) of our drugs increased (?1.2-fold) [3H]epibatidine
binding; 16 of these molecules increased binding by 1.4- to
Fig. 2. Effects of drugs on adrenal neurosecretion stimulated via activation of native nAChRs. Cultured bovine adrenal chromaffin cells expressing
?3?4* nAChRs were treated for 15 min with the drug before their stimulation with 10 ?M nicotine in the continued presence of the drug.
Concentration-response curves shown in A and B correspond to group A in Fig. 1. The concentration-response curves in C, D, and E correspond to
groups B, C, and D, respectively. The concentration-response curves in F correspond to groups E and F. Values represent means ? S.E.M. (n ? 3–8).
Results are expressed as a percentage of control, nicotine-stimulated neurosecretion.
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2.3-fold (Table 1). The concentration dependencies of the
binding effects for several molecules are seen in Fig. 8. When
homologous competition binding experiments were per-
formed in the absence or presence of 10 ?M APB-8, the IC50
values of epibatidine significantly (p ? 0.01) decreased from
1.51 (1.45–1.58) nM in the absence to 0.97 (0.90–1.05) nM in
the presence of APB-8 (Fig. 9). APB-8 also increased total
[3H]epibatidine binding (Fig. 9, inset), but it did not affect
nonspecific binding (data not shown). The data suggested
that the observed increase in radiolabeled ligand binding in
the presence of the drugs was at least partially due to an
increase in apparent affinity of the radiolabeled ligand for its
binding site.
In this final series of experiments, computational homol-
ogy modeling and blind docking approaches were used to
identify the potential binding site of our negative allosteric
TABLE 1
Functional and binding effects of the nAChR negative allosteric modulators
Drug
Functional Studies
Native nAChRs
(Nicotine-Stimulated Neurosecretion)
Recombinant nAChRs
(Nicotine-Stimulated ?Ca2??i)
Binding Studies
(nAChR Specific Bindinga,d)
IC50c
nHd
IC50c
nHd
Native nAChRsRecombinant nAChRs
?M
?M % Control
APB-2
APB-5
APB-6
APB-7
APB-8
APB-9
APB-10
APB-12
APB-20
APB-21
COB-1
COB-2
COB-3
DDR-1
DDR-13
DDR-14
DDR-15
DDR-17
DDR 18
DDR-19
IB-2
IB-4
IB-10
JHB-1
JHB-2
JHB-7
KAB-8
KAB-9
KAB-10
KAB-11
KAB-14
KAB-16
KAB-18
KAB-23
KAB-24
KAB-25
KAB-28
KAB-32
KAB-34
KAB-35
KAB-36
KAB-37
KAB-38
PPB-2
PPB-5
PPB-6
PPB-7
PPB-9
PPB-10
PPB-11
PPB-12
2.8 (2.5–3.1)e
7.1 (5.8–8.7)e
6.6 (6.1–7.2)e
6.7 (4.4–10.4)
2.8 (2.1–3.9)e
4.0 (3.2–4.8)e
8.2 (6.9–9.8)e
13.0 (10.8–16)e
2.4 (2.3–2.6)
1.6 (1.3–1.9)
3.5 (3.1–3.9)e
1.2 (1.2–1.3)e
3.0 (2.5–3.7)e
1.7 (1.4–2.0)e
2.2 (1.8–2.7)
1.8 (1.4–2.1)
1.4 (1.3–1.5)
0.4 (0.4–0.4)
2.6 (2.3–3.0)
2.4 (2.2–2.6)
3.3 (3.2–3.3)
3.5 (2.7–4.6)
1.3 (1.1–1.4)e
1.3 (1.1–1.5)e
0.7 (0.6–0.8)
11.9 (9.7–14.6)
5.4 (5.0–5.8)e
6.9 (6.5–7.3)e
3.5 (3.5–3.6)e
2.8 (2.4–3.1)e
2.1 (1.9–2.4)e
2.5 (2.2–2.8)e
1.7 (1.6–1.8)e
3.2 (3.1–3.4)f
3.2 (3.1–3.2)f
5.2 (4.8–5.6)f
1.8 (1.6–2.1)
6.7 (6.0–7.5)
6.2 (5.3–7.2)e
3.3 (2.8–3.9)e
2.0 (1.8–2.2)e
1.9 (1.6–2.2)e
3.5 (3.2–3.8)e
0.9 (0.8–1.0)
1.2 (1.1–1.3)e
1.6 (1.6–1.7)e
1.2 (1.1–1.3)e
5.7 (5.0–6.5)e
4.1 (3.8–4.5)e
2.6 (2.2–3.1)e
1.1 (0.9–1.3)
?1.3 ? 0.2
?2.8 ? 1.0
?2.1 ? 0.5
?1.7 ? 0.3
?2.1 ? 0.2
?2.3 ? 0.4
?1.4 ? 0.2
?1.6 ? 0.2
?2.1 ? 0.2
?3.2 ? 1.2
?2.8 ? 1.3
?2.0 ? 0.2
?1.6 ? 0.5
?5.1 ? 2.3
?1.3 ? 0.3
?1.7 ? 0.5
?2.6 ? 0.4
?2.4 ? 0.5
?1.2 ? 0.2
?1.7 ? 0.5
?3.9 ? 1.2
?2.0 ? 0.2
?2.4 ? 0.3
?3.9 ? 1.5
?3.9 ? 1.2
?4.0 ? 2.5
?2.1 ? 0.2
?1.8 ? 0.2
?4.1 ? 0.5
?4.2 ? 0.6
?1.9 ? 0.2
?2.3 ? 0.1
?2.2 ? 0.1
?2.3 ? 0.1
?1.9 ? 0.3
?2.4 ? 0.2
?1.9 ? 0.4
?1.7 ? 0.6
?1.6 ? 0.7
?1.7 ? 0.3
?1.9 ? 0.1
?1.8 ? 0.2
?4.4 ? 1.9
?3.2 ? 0.1
?2.7 ? 0.5
?2.4 ? 0.3
?4.1 ? 2.2
?2.9 ? 0.4
?3.8 ? 1.1
?2.9 ? 0.4
?3.6 ? 1.2
15.7 (11.9–20.8)b
38.2 (13.1–111.8)b
3.7 (3.5–3.8)
3.8 (3.2–4.4)
10.0 (9.5–10.6)
4.2 (3.9–4.5)
2.6 (2.3–3.0)
6.6 (6.0–7.2)
10.9 (9.1–13.2)b
19.0 (12.8–28.3)b
1.2 (1.0–1.4)e
1.1 (1.0–1.1)e
0.7 (0.6–0.9)e
30.0 (23.5–38.3)b,e
15.0 (12.1–18.3)b
20.3 (13.4–30.6)b
2.4 (2.2–2.6)
7.7 (5.8–10.1)
20.4 (14.1–29.6)b
2.0 (1.5–2.7)
1.5 (1.3–1.9)
4.8 (3.7–6.1)
7.6 (6.8–8.6)e
4.5 (4.0–5.1)
4.6 (4.1–5.2)
5.6 (5.2–6.0)
5.2 (4.8–5.6)
2.8 (2.5–3.1)
4.5 (3.9–5.3)
7.2 (6.3–8.3)
6.8 (5.8–7.9)
6.1 (5.1–7.3)
10.2 (7.9–13.3)
1.9 (1.7–2.1)
2.7 (2.2–3.3)
2.6 (2.2–3.1)
12.2 (10.4–14.4)b
1.6 (1.4–1.9)
3.1 (2.8–3.5)e
6.2 (5.6–6.8)
20.3 (19.4–21.2)b
9.5 (9.1–9.8)
11.9 (10.8–13.2)b
17.2 (12.4–23.9)b
13.4 (10.3–17.3)b,e
1.8 (1.5–2.0)
4.6 (4.0–5.3)
3.1 (2.3–4.2)
2.6 (2.1–3.2)
8.0 (5.9–10.8)e
21.0 (15.4–28.4)b
?2.1 ? 1.1
?1.1 ? 0.9
?1.3 ? 0.1
?1.1 ? 0.1
?1.4 ? 0.5
?1.1 ? 0.1
?0.9 ? 0.0
?1.1 ? 0.2
?1.3 ? 0.4
?1.3 ? 0.6
?0.7 ? 0.1
?0.8 ? 0.0
?0.9 ? 0.1
?0.7 ? 0.1
?1.9 ? 0.7
?1.5 ? 0.7
?0.9 ? 0.1
?1.3 ? 0.3
?1.6 ? 0.6
?1.0 ? 0.1
?1.1 ? 0.1
?0.8 ? 0.1
?1.4 ? 0.1
?1.0 ? 0.2
?0.7 ? 0.1
?1.3 ? 0.1
?1.6 ? 0.1
?0.9 ? 0.2
?1.0 ? 0.1
?1.4 ? 0.2
?1.1 ? 0.3
?1.2 ? 0.2
?1.1 ? 0.2
?0.8 ? 0.1
?0.7 ? 0.1
?1.2 ? 0.0
?1.5 ? 0.5
?1.1 ? 0.1
?1.1 ? 0.1
?1.4 ? 0.1
?1.0 ? 0.3
?1.2 ? 0.2
?1.4 ? 0.7
?0.6 ? 0.3
?0.7 ? 0.3
?0.6 ? 0.1
?1.0 ? 0.1
?0.7 ? 0.1
?1.0 ? 0.1
?1.3 ? 0.4
?0.8 ? 0.3
118.6 ? 14.6
108.2 ? 11.2
95.5 ? 8.1
103.4 ? 8.1
174.4 ? 14.8
107.3 ? 7.7
98.7 ? 4.7
98.7 ? 4.4
112.8 ? 17.7
130.3 ? 17.4
113.5 ? 4.8e
125.8 ? 3.8e
106.0 ? 2.5e
118.7 ? 13.9e
224.3 ? 12.1
216.6 ? 6.0
201.6 ? 6.3
215.0 ? 8.8
190.6 ? 8.0
169.5 ? 5.6
102.2 ? 6.5
107.0 ? 16.1
151.3 ? 15.7e
102.0 ? 13.5
112.9 ? 12.2
99.5 ? 3.6
137.4 ? 2.8
115.2 ? 3.0
146.9 ? 1.9
136.7 ? 16.9
111.8 ? 6.0
108.4 ? 3.4
110.1 ? 14.1
135.3 ? 12.8
173.5 ? 16.1
145.2 ? 11.4
127.5 ? 4.7
97.9 ? 7.9
83.2 ? 1.4e
68.7 ? 3.7
121.6 ? 11.5
138.9 ? 5.3
139.9 ? 6.0
147.7 ? 7.7
152.8 ? 7.6e
123.8 ? 7.6
144.3 ? 9.4
112.7 ? 2.1
118.8 ? 4.4
118.4 ? 12.5e
117.9 ? 6.7
75.6 ? 9.6
75.5 ? 6.1
98.5 ? 2.4
100.7 ? 2.7
71.2 ? 3.9
96.9 ? 1.6
99.8 ? 1.8
120.7 ? 9.3
75.0 ? 5.8
80.6 ? 3.4
87.3 ? 2.6
88.7 ? 1.5
68.8 ? 5.6
66.3 ? 4.8
113.0 ? 7.3g
92.8 ? 6.1g
103.1 ? 9.8g
75.3 ? 8.1g
91.2 ? 10.1g
91.6 ? 10.0g
78.4 ? 6.7
95.5 ? 7.5
96.7 ? 7.0
87.2 ? 5.8
100.1 ? 5.5
74.3 ? 4.6
98.0 ? 3.8
95.1 ? 1.8
92.6 ? 4.4
89.1 ? 1.6
79.8 ? 6.9
82.8 ? 4.2
69.9 ? 3.8
77.6 ? 2.6
72.1 ? 1.8
70.1 ? 3.7
71.8 ? 3.7
108.6 ? 15.5
79.9 ? 11.4
71.0 ? 29.1g
95.6 ? 28.1
92.1 ? 7.0
93.5 ? 10.4
97.9 ? 2.6
90.3 ? 1.1
101.8 ? 1.5
89.1 ? 3.7
96.6 ? 5.1
92.7 ? 3.2
104.0 ? 16.4
83.3 ? 16.1
nH, Hill coefficient.
aThe effects of the analogs were determined at a fixed concentration of 10 ?M.
bExtrapolated IC50values taken from mean curves.
cValues represent geometric means (confidence limits), n ? 3 to 8.
dValues represent arithmetic means ? S.E.M. (n ? 3–12).
eValues from McKay et al. (2007).
fValues from Bergmeier et al. (2004).
gValues represent means ? S.D. (n ? 2).
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modulators. We hypothesized that since these molecules are
structurally similar to MLA (Bergmeier et al., 1999, 2004;
Bryant et al., 2002; McKay et al., 2007) and MLA is a com-
petitive antagonist for ?3?4 nAChRs (Free et al., 2003), the
binding site of the negative allosteric modulators would be
near the orthosteric binding site. Two stereoisomers of
COB-3 were docked to 72 receptor conformations taken at
200-ps intervals over the duration of an MD simulation of the
agonist-bound model. COB-3 was chosen because of its rela-
tively high potency. The most frequently occurring docking
cluster for COB-3 is shown in Fig. 10. This position on the
receptor was the consensus docking site for both COB-3 ste-
reoisomers. After docking to receptor conformations that rep-
resent 15 ns of simulation, one of the top four docking clus-
ters for the two COB-3 compounds occurred at the most
prominent site in 58.3% of the receptor conformations that
were docked to, whereas the second most prominent site had
an occurrence rate of 42.4%. The site characterized in Fig. 10
was located within the pore of an ?/? interface, ?7 Å from the
bound epibatidine in the agonist binding site. This docking
site remained the most prominent site over the duration of
the simulation (20 and 72 snapshots), supporting consistency
of the data and lack of stereoselectivity at the site. Key
interactions included a hydrogen bonding potential between
the ester linkage of COB-3 with Asn108 and a stabilizing
electrostatic interaction between the positively piperidine
moiety of the ligand and Asp88. All stereoisomers of three
additional compounds (PPB-9, APB-10, and APB-12), repre-
sentative of the various chemical scaffolds (Fig. 1), were also
blind-docked to the homology models in an identical manner
as described for COB-3; all of these compounds were docked
to the same corresponding site (data not shown).
Discussion
Our laboratory has identified a series of molecules that
inhibit nAChR-mediated neurosecretion (Bergmeier et al.,
1999, 2004; Bryant et al., 2002; McKay et al., 2007). In the
studies reported here using cells expressing recombinant
nAChRs, we confirm that these drugs act at the level of the
nAChR to noncompetitively inhibit activation of nAChRs.
The calcium accumulation assay is directly linked to nAChR
activation (Stauderman et al., 1998; Chavez-Noriega et al.,
2000). The IC50values of our molecules ranged from 0.7 to
38.2 ?M. Their potencies are similar to other inhibitors of
adrenal neurosecretion, including d-tubocurarine, hexametho-
nium, decamethonium, tetracaine, pentolinium, and mecamyl-
amine (IC50values ranging from 17 to 0.1 ?M) (McKay and
Trent-Sanchez, 1990; McKay and Burkman, 1993).
Our compounds (51/51) showed an average increase in
agonist binding of 1.3-fold, with approximately 50% (24/51) of
these drugs (10 ?M) increasing [3H]epibatidine binding to
the orthosteric site by an average of 1.6-fold. This drug-
induced increase in binding to the agonist binding site is at
least partially due to an increase in affinity of epibatidine to
this site (Fig. 9). Many noncompetitive antagonists have
TABLE 2
Comparison of effects of agonists and antagonists on functional
responses from native and recombinant ?3?4 nAChRs
Recombinant Rat ?3?4
nAChRsa
(?Ca2??i)
EC50or IC50
Native Bovine ?3?4*
nAChRs
(Neurosecretion)
EC50or IC50
?M
Nicotine
Epibatidine
d-Tubocurarine
Mecamylamine
Tetracaine
COB-2
18.0 (15.9–20.4)
0.06 (0.05–0.07)
6.3 (5.0–8.1)
0.6 (0.4–0.8)
2.5 (2.4–2.6)
1.1 (1.0–1.1)e
3.6 (2.9–4.5)b
0.009 (0.006–0.014)b
2.2 (1.4–3.6)c
0.1 (0.1–0.7)d
4.0 (2.2–7.4)d
1.2 (1.2–1.3)e
aData are means (confidence limits) of three to four different experiments per-
formed in triplicate or quadruplicate.
bEC50value from Wenger et al. (1997).
cIC50value from McKay and Burkman (1993).
dIC50value from McKay and Trent-Sanchez (1990).
eIC50value from McKay et al. (2007) and Table 1.
Fig. 3. Comparison of effects of agonists and antagonists on HEK 293
cells expressing either rat or bovine recombinant nAChRs. KX?3?4R2
(?) and BM?3?4 (f) cells were loaded with fluo-4-AM for 60 min. For
BM?3?4 cells, experiments were performed in buffer containing 20 mM
calcium. Fluorescence was measured after stimulation with increasing
concentrations of nicotine (A) or epibatidine (B). Effects of d-tubocurarine
(C), mecamylamine (D), tetracaine (E), and COB-2 (F) on nicotine (100
?M)-stimulated increases in calcium accumulation were also determined.
Results are expressed as either the percentage of the effect of 100 ?M
nicotine (A), the percentage of the effect of 1 ?M epibatidine (B), or the
percentage of control, nicotine-stimulated peak fluorescence levels (C–F).
Values represent means ? S.E.M. (n ? 3–4).
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shown to enhance agonist binding to the AChR orthosteric
site (Kato and Changeux, 1976; Herz et al., 1987), including
NAMs (Herz et al., 1987), and these increases in agonist
binding to the orthosteric site have been linked to desensiti-
zation of nAChRs (Kato and Changeux, 1976). In addition,
similar increases in agonist binding affinity have been re-
ported for noncompetitive antagonists; chlorpromazine and
vinblastine decrease IC50values from 4.3 to 1.6 ?M (Carp et
al., 1983) and from ?0.5 to 0.1 ?M (McKay et al., 1985),
respectively, and dibucaine and histrionicotoxin decreased
IC50values from 20.7 to 8.6 ?M and 21.0 to 2.6 ?M, respec-
tively (Sine and Taylor, 1982). The binding of NAMs to allo-
steric sites may shift the receptors from the resting to the
desensitized state or may stabilize the desensitized state; in
either case, these desensitized receptors are not functional
(Monod et al., 1965; Quick and Lester, 2002). The ability of
our molecules to increase agonist binding to orthosteric sites
of native nAChRs supports their classification as NAMs and
suggests that their inhibitory actions involve receptor desen-
sitization. No increases in agonist binding to recombinant
receptors were observed, though. The reasons for these find-
ings are unknown but may be due to potential differences in
the subunit composition of native and recombinant ?3?4
nAChRs; it has been reported that native ?3?4* nAChRs
may contain ?5 or ?7 subunits (Campos-Caro et al., 1997;
Gerzanich et al., 1998; El-Hajj et al., 2007).
The data indicate that many of our allosteric modulators
have additional, non-nAChR inhibitory activity. From a drug
development perspective, it is important to identify drugs
that have non-nAChR activity to reduce potentially problem-
Fig. 4. Effects of drugs on intracellu-
lar calcium levels stimulated via acti-
vation of recombinant nAChRs. Cul-
tured KX?3?4R2 cells were loaded
with fluo-4-AM for 60 min and treated
with the drug 40 s before their stimu-
lation with 100 ?M nicotine in the
continued presence of the drug. Con-
centration-response curves shown in
A and B correspond to group A in Fig.
1. The concentration-response curves
in C to E correspond to groups B, C,
and D, respectively. The concentra-
tion-response curves in F correspond
to groups E and F. Values represent
means ? S.E.M. (n ? 3–4). Results
are expressed as percentage of con-
trol, nicotine-stimulated peak fluores-
cence levels.
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atic side effects. Comparisons of Hill coefficients of inhibition
curves in the two functional assays (native versus recombi-
nant nAChRs) support differences in mechanisms of inhibi-
tion of the antagonists. The Hill coefficients of antagonists in
the neurosecretion assay have a mean value of ?2.53 ? 0.13,
suggesting positive cooperativity. In contrast, the Hill coeffi-
cients of the antagonists in the calcium accumulation assay
have a mean value of ?1.11 ? 0.05, suggesting lack of coop-
erativity. These results indicate that the inhibitory mecha-
nisms of the antagonists in the calcium accumulation assay
probably involve a single site of action. In addition, approx-
Fig. 6. Effects of nAChR Antagonists on KCl-stimulated Neurosecretion. KCl-stimulated neurosecretion studies were performed, as described under
Materials and Methods, using a single concentration (10 ?M) of each compound. Results are expressed as percentage of control, KCl-stimulated
neurosecretion. Values represent means ? S.E.M. (n ? 4–9).
Fig. 5. Comparison of effects of drugs on recombinant and native
nAChRs. Linear regression analysis of IC50values of all our drugs on
nicotine-stimulated calcium accumulation (recombinant nAChRs) and
adrenal neurosecretion (native nAChRs) was performed. Actual IC50val-
ues for recombinant nAChRs are found in Table 1. Values for native
nAChRs are from McKay et al. (2007) and Bergmeier et al. (2004).
Fig. 7. Regression analyses comparing the effects of antagonists on
recombinant and native nAChRs. Linear regression analysis of IC50val-
ues obtained using nAChR antagonists with little or no non-nAChR
effects (A: COB-3, APB-10, APB-6, APB-12, COB-1, PPB-9, APB-7,
JHB-7, PPB-6, and COB-2) or nAChR antagonists with large non-nAChR
actions (B: KAB-28, KAB-14, DDR-18, JHB-2, IB-10, DDR-1, DDR-17,
KAB-38, JHB-1, and KAB-18) on nicotine-stimulated increase in intra-
cellular calcium (recombinant nAChRs) and adrenal neurosecretion (na-
tive nAChRs). Actual IC50values are found in Table 1. Dotted lines
represent linear regression analyses when the data are forced through
x ? 0, y ? 0.
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imately 80% of our molecules (40/51) inhibit KCl-stimulated
neurosecretion by 25% and more, establishing that these
drugs interact with steps in the stimulus-secretion process
distal to membrane depolarization (i.e., nonreceptor mecha-
nisms). The KCl-neurosecretion assay was found to be a
useful secondary assay for the screening of NAMs for addi-
tional, non-nAChR inhibitory activity. The correlation data
in Fig. 7 support this approach. Drugs that inhibit neurose-
cretion stimulated via direct depolarization of cells must act
on a site distal to nAChR activation. Additional sites of action
may include other cationic channels (Na?, K?, and/or Ca2?
channels). Several drugs have been identified that target
both nAChRs and ion channels. Galantamine, an nAChR
allosteric potentiator, blocks K?channels in chromaffin cells
(Ale ´s et al., 2006) and hippocampal neurons (Pan et al.,
2003). Nicardipine and other dihydropyridines, which are
voltage-gated calcium channel blockers, inhibit nAChR acti-
vation in rat superior cervical ganglion neurons (Wheeler et
al., 2006), in skeletal muscle (Adam and Henderson, 1990),
and adrenal chromaffin cells (Lo ´pez et al., 1993).
Analyses of the structure-activity relationship studies
were performed to investigate structural determinants of our
drugs that affect their interactions with nAChR and non-
nAChR sites. We focused on both potency of our drugs, ob-
tained from their direct actions on nAChRs (calcium accumu-
lation), and their non-nAChR related actions (KCl-mediated
neurosecretion). The general structure of most compounds is
that of a piperidine ring linked to a substituted benzoyl group
via a hydroxymethyl group on the piperidine ring. One of the
most drastic and important findings is that the replacement
of the phenylpropyl group in KAB-18 [IC50value ? 10.2
(7.9–13.3) ?M and ?90% non-nAChR actions] with a small
group both increases potency and eliminates non-nAChR-
related effects. For example, COB-3 showed a 14-fold in-
crease in potency and an elimination of non-nAChR-related
actions. Similar differences were found with COB-1 and
COB-2. Clearly, small alkyl groups on the piperidine nitro-
gen can provide potent drugs when coupled with the biphenyl
ester. However, the biphenyl ester is not sufficient to en-
hance potency of large alkyl groups on the piperidine nitro-
gen (e.g., DDR-18). In addition to these biphenyl esters, com-
pounds PPB-6 and PPB-9 provide similar pharmacological
enhancements. Both of these compounds have a small alkyl
group (N-iPr for PPB-6 and N-Et for PPB-9) on the piperidine
nitrogen. Unlike compounds COB-1, -2, and -3, these com-
pounds have a benzyl-substituted succinimide on the benzoyl
group. This combination of a relatively large benzoyl group
(biphenyl or benzyl-substituted succinimide) and a small al-
kyl group on the piperidine provide an interesting divergence
from observations reported previously by our laboratory
(Bryant et al., 2002). We should note that the replacement of
the small alkyl group of PPB-6 with the larger 3-phenylpro-
pyl group (IB-10) provides compounds essentially similar in
both potency and non-nAChR-related effects to KAB-18.
Another structural set of compounds that have improved
pharmacological properties are those exemplified by ABP-6,
APB-7, APB-9, and APB-10. This set of compounds showed
improved potency (?3–4-fold) and an elimination of non-
nAChR-related actions. Structurally, these compounds con-
tain a large alkyl group on the piperidine nitrogen. Unlike all
of the other compounds examined, the benzoyl group has
been replaced with a heterocyclic group (a chromone or cou-
marin). It is interesting to note that both 2- and 3-substituted
chromones have roughly similar activities. In addition, sub-
stitution on the terminal end of the piperidine alkyl substitu-
ent (4-methoxyphenyl in APB-10 compared with the 4-chlo-
rophenyl of APB-6) again showed only minor differences. A
final compound that showed improved pharmacological prop-
erties is APB-12. This compound is almost identical to IB-2;
the only difference is the change of the piperidine ring of IB-2
to a pyrrolidine ring and a change in the position of the
hydroxymethyl linker to the benzoyl group.
The structure-activity relationship studies reported here
have allowed us identify three general structural classes of
molecules with improved pharmacological profiles relative to
initially identified compounds. One group has a small alkyl
group on the piperidine nitrogen and a large benzoyl ester.
The other group has a large alkyl group on the piperidine
nitrogen and a heterocyclic ester. The final group of one
Fig. 8. Effects of nAChR antagonists on [3H]epibatidine binding to native
nAChRs. Competition binding experiments were performed on bovine
adrenal medulla membrane homogenates using indicated concentrations
of the molecules. Data are expressed as a percentage of control specific
binding. Values represent means ? S.E.M. (n ? 4).
Fig. 9. Effects of APB-8 on [3H]epibatidine binding. [3H]epibatidine ho-
mologous competition binding experiments were performed on adrenal
medulla membranes in the absence (F) and presence of 10 ?M APB-8 (E).
Data are expressed as percentage specific [3H]epibatidine binding or as
specific [3H]epibatidine binding in femtomoles per milligram of protein
(inset). Triangles represent [3H]epibatidine binding in the absence of
epibatidine (inset). Values represent means ? S.E.M. (n ? 4).
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contains both a large piperidine alkyl substituent and a large
benzoyl group on a pyrrolidine ring. Molecules that fall out-
side these general structural requirements while potent tend
to exhibit significant non-nAChR activity.
Using homology modeling and blind docking approaches,
we identified a potential binding site for our NAMs, ?7 Å
from the agonist binding site. This binding site, as we hy-
pothesized, is adjacent to the binding site of MLA (Hansen et
al., 2005). Interestingly, the docking site identified for COB-3
is similar in position to that of the nAChR allosteric poten-
tiator galantamine, which was also identified via blind dock-
ing (Iorga et al., 2006).
In summary, we have shown that our primary assay (cal-
cium accumulation) and our secondary assay (KCl-neurose-
cretion) are important in nAChR drug development. We have
identified several molecules showing promise as NAMs of
nAChRs. Although our paradigms involve either native or
recombinant ?3?4 nAChRs, these drugs show limited nAChR
subtype selectivity (data not shown); however, computer-
assisted drug design is currently being investigated to im-
prove selectivity. To date, we have discovered a small group
of drugs that 1) inhibit adrenal nAChR-activated neurosecre-
tion, 2) inhibit nAChR-activated increases in intracellular
calcium from cells expressing recombinant nAChRs, 3) exert
little or no effects on non-nAChR-mediated neurosecretion,
and 4) exert little or no inhibitory effects on binding to native
and recombinant adrenal nAChRs. Correlation of functional
data (Fig. 7A) supports a single site of action of this group of
drugs on nAChRs. The structure-activity relationship study
emphasizes that small substitutions to the piperidine ring or
to the benzoyl ester also improve potency and avoid non-
nAChR actions. Computational modeling and blind docking
identify a binding site for our NAMs near the orthosteric
binding site of the receptor. The localization and character-
ization of the binding site for these novel drugs should pro-
vide insight into synthetic alterations that will produce more
potent and more selective antagonists for specific subtypes of
nAChRs. The discovery of novel nAChR negative allosteric
modulators will provide new experimental approaches to in-
vestigate nAChR involvement in physiological functions and
pathophysiological conditions and, possibly, novel therapeu-
tic strategies to treat disease.
Acknowledgments
The KX?3?4R2 cells were kindly provided by Drs. Xiao and Kellar
(Georgetown University). We thank the Ohio Supercomputer Center
for a grant of computational resources.
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