Evaluation of benzyltetrahydroisoquinolines as ligands for neuronal nicotinic acetylcholine receptors

Article abstract of DOI:10.1038/sj.bjp.0706307

Effects of derivatives of coclaurine (C), which mimic the 'eastern' or the nonquaternary halves of the alkaloids tetrandrine or d-tubocurarine, respectively, both of which are inhibitors of nicotinic acetylcholine receptors (nACh), were examined on recomb

Full text of DOI:10.1038/sj.bjp.0706307

British Journal of Pharmacology (2005) 146, 15–24
&
2005 Nature Publishing Group All rights reserved 0007–1188/05
$30.00
www.nature.com/bjp
Evaluation of benzyltetrahydroisoquinolines as ligands for neuronal
nicotinic acetylcholine receptors
1Richard Exley, 2Patricio Iturriaga-Vasquez, 3Ronald J. Lukas, 4Emanuele Sher, 2Bruce K. Cassels
& *,1Isabel Bermudez
2
1School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP; Millennium Institute
for Advanced Studies in Cell Biology and Biotechnology and Department of Chemistry, Faculty of Sciences, University of Chile,
3
4
Santiago, Chile; Division of Neurobiology, Barrow Neurological Institute, Phoenix, AZ, U.S.A. and Eli Lilly and Co. Lilly
Research Centre, Erl Wood Manor, Windlesham, Surrey
1
Effects of derivatives of coclaurine (C), which mimic the ‘eastern’ or the nonquaternary halves of
the alkaloids tetrandrine or d-tubocurarine, respectively, both of which are inhibitors of nicotinic
acetylcholine receptors (nACh), were examined on recombinant, human a7, a4b2 and a4b4 nACh
receptors expressed in Xenopus oocytes and clonal cell lines using two-electrode voltage clamping and
radioligand binding techniques.
2
In this limited series, Cs have higher affinity and are most potent at a4 subunit-containing-nACh
receptors and least potent at homomeric a7 receptors, and this trend is very marked for the
N-unsubstituted C and its O,O0-bisbenzyl derivative.
3
7-O-Benzyl-N-methylcoclaurine (BBCM) and its 12-O-methyl derivative showed the highest
affinities and potencies at all three receptor subtypes, and this suggests that lipophilicity at C7 and/or
C12 increases potency.
4
Laudanosine and armepavine (A) were noncompetitive and voltage-dependent inhibitors of a7,
a4b2 or a4b4 receptors, but the bulkier C7-benzylated 7BNMC (7-O-benzyl-N-methylcoclaurine) and
7B12MNMC (7-O-benzyl-N,12-O-dimethyl coclaurine) were voltage-independent, noncompetitive
inhibitors of nACh receptors. Voltage-dependence was also lost on going from A to its N-ethyl
analogue.
5
These studies suggest that C derivatives may be useful tools for studies characterising the
antagonist and ion channel sites on human a7, a4b2 or a4b4 nACh receptors and for revealing
structure–function relationships for nACh receptor antagonists.
British Journal of Pharmacology (2005) 146, 15–24. doi:10.1038/sj.bjp.0706307;
published online 27 June 2005
Keywords: Tetrandrine; nicotinic acetylcholine receptors; laudanosine; armepavine; Xenopus oocytes
Abbreviations: A, armepavine; ACh, acetylcholine; a-BgTx, a-bungarotoxin; BBC, 7,12-O,O0-dibenzylcoclaurine; BBCM, 7,12-
O,O0-dibenzyl N-methyl coclaurine; BBIQ, bis-benzylisoquinoline; 7B12MNMC, 7-O-benzyl-N,12-O-dimethyl
coclaurine; 7BNMC, 7-O-benzyl-N-methylcoclaurine; BTHIQ, 1-benzyl-1,2,3,4-tetrahydroisoquinoline; C,
coclaurine; EC50, concentration of agonist eliciting a half-maximal response; IC50, antagonist concentration
eliciting half-maximal inhibition; MC, N-methylcoclaurine; nACh, nicotinic acetylcholine; NEA, N-ethyl
norarmepavine; nHill, Hill coefficient; s.e.m., standard error of the mean
Introduction
Neuronal nicotinic acetylcholine (nACh) receptors are cur-
rently the focus of considerable pharmaceutical interest
because of their potential as therapeutic targets for a wide
variety of brain diseases such as nicotine addiction, memory
and learning disabilities, Parkinson’s disease, Tourette’s
syndrome and Alzheimer’s disease (Astles et al., 2002). d-
Tubocurarine, a monoquaternary, head-to-tail bis-tetrahydro-
isoquinoline alkaloid isolated from curare, is the prototype of
an extensive series of natural and synthetic neuromuscular
nAChR receptor blockers with activity also at neuronal nACh
receptors (Buck, 1987; Garland et al., 1998). However, d-
tubocurarine and its mono- or bisquaternary analogues have
awakened little attention as possible neuronal nACh receptor
ligands because of their inability to pass the blood–brain
barrier. On the other hand, tetrandrine, a nonquaternary head-
to-head bis-tetrahydroisoquinoline alkaloid (Figure 1a) that is
the principal antihypertensive and muscle relaxant component
of the Chinese cardiovascular drug han fang chi (Stephania
tetrandra root) (Wang & Liu, 1985) and that might be expected
to reach the brain in pharmacologically active concentrations
has also been found quite recently to be a noncompetitive
inhibitor of both muscle and neuronal nACh receptors at low
micromolar concentrations (Slater et al., 2002).
Both d-tubocurarine and tetrandrine belong to the large
bis-benzylisoquinoline (BBIQ) alkaloid family that includes
anti-inflammatory, antiarrhythmic, bactericidal and muscle
relaxant alkaloids (Buck, 1987). While d-tubocurarine is
regarded primarily as a depolarising skeletal muscle relaxant,
which may cause hypotension at high doses, the antihyperten-
sive and smooth muscle relaxant properties of tetrandrine are
*Author for correspondence; E-mail: p0054922@brookes.ac.uk

16
R. Exley et al
Isoquinoline inhibitors of nicotinic receptors
CH3
O
a
H3C
b
H3C
CH3
O
O
BTHIQ
R7
H
R12
RN
H
R11
O
N
CH3
R7
O
N
Coclaurine (C)
H
H
H
RN
H
O
H
H
N
N-Methylcoclaurine (MC)
Armepavine (A)
H
H
H
CH3
CH3
CH3CH2
CH3
H
H3C
O
R12
R11
CH3
H
O
O
H3C
tetrandrine
coclaurine
N-Ethyl Norarmepavine
CH3
H
H
CH3
O
(NEA)
Laudanosine (L)
CH3
CH3
O-CH3
H
N
CH3
O
H
HO
7,12-O,O’-Dibenzylcoclaurine
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
H
(BBC)
d-tubocurarine
7,12-O,O’-Dibenzyl N-methyl
coclaurine (BBCM)
CH3
CH3
CH3
H
H3C
H3C
H
O
N+
OH
7-O-benzyl-N-
methylcoclaurine (7BNMC)
H
O
CH3
7-O-benzyl-N,12-O-Dimethyl
CH3
H
coclaurine (7B12MNMC)
Figure 1 Structure of BTHIQ. (a) Structures of tetrandrine, C (RN ¼ R7 ¼ R11 ¼ R12 ¼ H) and derivatives, and d-tubocurarine.
(b) C and its congeners used in this study.
thought to be a consequence of its ability to inhibit L-type
Ca2 þ channels (King et al., 1988; Felix et al., 1992).
Tetrandrine binds the benzothiazepine site of L-type Ca2 þ
channels and produces the same allosteric coupling pattern as
diltiazem (King et al., 1988; Felix et al., 1992), but its nicotinic
effects might contribute to its antihypertensive properties
(Slater et al., 2002).
nonquaternary half of d-tubocurarine), is a valid approach to
investigate the structure–functional relationships of tetran-
drine analogues at L-type Ca2 þ channels or noradrenergic
receptors (Iturriaga-Vasquez et al., 2003). In this study, we
have used a similar approach to investigate the structural
features of tetrandrine that may confer affinity for neuronal
nACh receptors and evaluated the effects of C and O- and/or
N-substituted C derivatives on human a7, a4b2 and a4b4
nACh receptors.
The structure of d-tubocurarine incorporates two mono-
meric 1-benzyl-1,2,3,4-tetrahydroisoquinoline (BTHIQ) moi-
eties bonded together in a head-to-tail manner by two ether
linkages between the isoquinoline and the benzyl benzene rings
(Figure 1a). One of these halves contains a permanently
charged, quaternary nitrogen atom, and the other incorporates
a tertiary amine function, which may or may not be
protonated to create a second positive charge. In contrast,
the two halves of the tetrandrine molecule are joined in a head-
to-head/tail-to-tail fashion, containing one tertiary nitrogen
atom each (Figure 1a). In all BBIQ alkaloids, the two halves
may be viewed as derived from the monomeric BTHIQ,
coclaurine (C), each with a stereogenic centre. In the case of
tetrandrine, as it is usually represented, distinct ‘eastern’ and
‘western’ regions are apparent.
Methods
Chemistry
C and its O-benzylated and/or O-methylated derivatives
(Figure 1b; BTHIQs will be abbreviated as in Figure 1
henceforward) were prepared by the Bischler-Napieralski 3,4-
dihydroisoquinoline synthesis and subsequent reduction of the
intermediates with NaBH4. N-methylation was carried out on
the BTHIQs with aqueous formaldehyde and NaBH4 as
previously described (Iturriaga-Vasquez et al., 2003).
The structural features that confer nicotinic activity to
tetrandrine have not been determined as yet, partly because the
macrocyclic nature of this alkaloid, with very specific bonding
between the ‘eastern’ and ‘western’ regions of the molecule,
hinders the chemical synthesis of a large number of derivatives.
However, a recent study has shown that the use of derivatives
of C, the monomer that most clearly mimics the structure of
the ‘eastern’ part of tetrandrine (Figure 1a) (and also the
Ligand binding assays
Established cultures of the SH-SY5Y-ha7 clonal cell line
(Houlihan et al., 2001), which overexpress the human a7 nACh
receptor, were used for [125I]a-bungarotoxin (a-BgTx) binding
assays. SH-EP1-ha4b2 (Eaton et al., 2003) and SH-EP1-ha4b4
(Eaton et al., 2000) clonal cell lines that express human a4b2
British Journal of Pharmacology vol 146 (1)

R. Exley et al
Isoquinoline inhibitors of nicotinic receptors
17
and a4b4 nACh receptors, respectively, were used for
[3H]cytosine binding assays. Membrane homogenates for all
clonal cell lines were prepared and utilized in binding assays
using methods previously described (Houlihan et al., 2001) to
give a final protein concentration of 30–50 mg per assay tube.
Competition binding studies were performed in a final volume
of 250 ml of binding saline (in mM: 140NaCl, 1 EGTA, 10
Hepes, pH 7.4 for [125I]a-BgTx binding and 120NaCl, 5 KCl,
1 MgCl2, 2.5 CaCl2, 50Tris, pH 7.0for [ 3H]cytisine binding).
For [125I]a-BgTx binding assays, preparations were incubated
for 90min at room temperature (21 1C) and the concentration
of radiolabelled toxin was 1 nM. In [3H]cytisine binding studies,
the concentration of radiolabelled cytisine was 1 nM and
incubations were carried out at 41C for 75 min. For both
binding assays, 10 mM nicotine was used to define nonspecific
binding. Bound and free fractions were separated by rapid
vacuum filtration through Whatman GF/C filters presoaked
in binding saline supplemented with 0.1% polyethyleneimine.
Radioactivity was determined with a g counter or by liquid
scintillation, as appropriate.
an EC50 ACh concentration and increasing concentrations of
compound were normalised to the responses elicited by an
EC50 concentration of ACh alone. ACh EC50 concentrations at
a7 and a4b4 nACh receptors were 100 and 30 mM, respectively
(Houlihan et al., 2001; Figures 3b, 6b). The concentration–
response curve of ACh at a4b2 nACh receptors is biphasic
comprising a high-affinity (EC50 1 mM) and a low-affinity
component (EC50 100 mM) (Figure 5b; see also, Zwart &
Vijverberg, 1998; Buisson
& Bertrand, 2001; Houlihan
et al., 2001); the human a4b2 receptor studies reported here
were carried out using the low-affinity ACh EC50 concen-
tration (100 mM) due to the predominance of this com-
ponent (approximately 85% of the overall ACh response;
Figure 5b). Constant responses to ACh were obtained before
the coapplication of ACh and compound. In these studies,
oocytes were preincubated with compounds for 3 min prior to
the coapplication procedure to ensure equilibration between
receptors and compound. To maintain ongoing measurements
of the control response to ACh throughout the experiment,
each coapplication was bracketed by an application of EC50 of
agonist alone.
nACh receptor expression in Xenopus oocytes
Data analyses
Stage V and VI Xenopus oocytes were prepared as previously
described (Houlihan et al., 2001) and injected nuclearly with
PcDNa3.1-ha7 or combinations of PcDNA3.1-ha4 plus
PcDNA3.1hb2 or PcDNA3.1-hb4 (1 : 1 molar ratio) containing
the indicated, human nACh receptor subunit cDNA using a
Nanoject Automatic Oocyte Injector (Drummond, Broomall,
PA, U.S.A.). Approximately 1 ng of each plasmid was injected
in a total injection volume of 18.4 nl oocyteÀ1. After injection,
the oocytes were incubated at 191C in modified Barth’s
solution containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3,
0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 15 mM
HEPES and 50 mg mlÀ1 neomycin (pH 7.6 with NaOH).
Experiments were performed on oocytes after 2–6 days of
incubation (Houlihan et al., 2001).
Concentration–effect data for agonists and antagonists were
fitted by nonlinear regression (Prism 3.01, GraphPad, U.S.A.)
to the equations:
nHill
ðaÞ i ¼ imax=½1 þ ðEC50=xÞ
ꢀ or
nHill
ðbÞ i ¼ imax=½1 þ ðIC50=xÞ
ꢀ
wherein imax ¼ maximal normalised current response (in the
absence of antagonist for inhibitory currents), x ¼ agonist or
antagonist concentration, EC50 ¼ concentration of agonist
eliciting a half-maximal response, IC50 ¼ antagonist concen-
tration eliciting half-maximal inhibition and nHill ¼ Hill
coefficient. ACh concentration–response data from a4b2
receptors were fitted using a two-component Hill equation
(Houlihan et al., 2001). The magnitudes of the responses to the
agonist concentrations greater than 1 mM decreased in a
concentration-dependent manner due to receptor desensitisa-
tion and/or agonist-induced open channel block and were
excluded from the analysis of the data. Results are presented as
mean7standard error of the mean (s.e.m.) of at least four
separate experiments from at least two different batches
of oocytes. Where appropriate, one-way Anova or Student’s
t-tests for paired or unpaired data were used, and values
of Po0.05 were regarded as significant.
Electrophysiological recordings
Whole-cell currents were measured by two-electrode voltage
clamp (GeneClamp 500, Axon Instruments, U.S.A.) using
agarose-cushioned electrodes containing 3 M KCl. Oocytes
were continually supplied with fresh Ringer solution (in mM:
115 NaCl, 2.5 KCl, 1.8 CaCl2, 10HEPES, pH 7.2) in a 60 ml
bath, using a gravity-driven perfusion system at a rate of
5 ml minÀ1. Modified Ringer solution (CaCl2 replaced by
BaCl2) was used when recording from oocytes expressing
human a7 nACh receptors. Compounds were applied by
gravity perfusion using a manually activated valve. The
agonist acetylcholine (ACh) was applied for a period sufficient
(approx. 10–15 s) to obtain a stable plateau response (at low
concentrations) or the beginning of a sag after a peak (at
higher concentrations). Between each successive ACh and/or
compound application, the cell was perfused with Ringer
solution for 3 min to allow drug clearance and prevent receptor
desensitisation. Concentration–response curves for ACh were
constructed by normalising to the maximal response to ACh
and used to generate EC50 (concentration of agonist eliciting a
half-maximal response) and nHill (Hill coefficient) estimates
(Houlihan et al., 2001). To construct antagonist concentra-
tion–effect curves, the responses elicited by coapplication of
The binding parameters (KD and Bmax) of [3H]cytisine
binding were determined from saturation binding isotherm
data using the equation y ¼ (Bmax ꢁ x)/(KD þ x), wherein
Bmax ¼ maximal binding, KD ¼ apparent equilibrium dissocia-
tion binding constant, x ¼ concentration of ligand and
y ¼ specific binding.
Results
The BTHIQ analogues listed in Figure 1b were designed to
evaluate the importance of N-alkylation and O-benzylation
or methylation with regard to affinity and selectivity for
recombinant, human a7, a4b2 or a4b4 nACh receptors
British Journal of Pharmacology vol 146 (1)

18
R. Exley et al
Isoquinoline inhibitors of nicotinic receptors
expressed heterologously in Xenopus oocytes or in transfected
clonal cell lines. Each BTHIQ inhibited a7, a4b2 and a4b4
nACh receptor radioligand binding and/or function with a
potency and profile that was influenced by the receptor
subtype (Tables 1 and 2). None of the ligands tested displayed
agonist or potentiating effects (not shown), even at the highest
concentrations tested (100–300 mM).
most potent inhibitory BTHIQs at concentrations close to their
IC50 concentration on the saturation binding of 125I-BgTx
showed that they are noncompetitive inhibitors of human a7
nACh receptors: A, L, NEA, 7BNMC and 7B12MNMC
significantly decreased Bmax from 1.55 pmol mg proteinÀ1 to
about 0.8–0.9 pmol mg proteinÀ1 (Po0.05) without significantly
changing KD values. Control KD was 0.98 nM and in the presence
of BTHIQ increased to no more than 1.2 nM (Figure 2b).
EC50 ACh-mediated currents through a7 nACh receptors
expressed heterologously in Xenopus oocytes were fully
inhibited by 7BNMC, 7B12MNMC, A, L and NEA, with
IC50 (antagonist concentration eliciting half-maximal inhibi-
tion) values of 1.4 and 2.8 mM for 7BNMC and 7B12MNNC
and 26 and 22 mM for A and L (Figure 3a; Table 2). Inhibition
occurred at concentrations similar to or lower than those
producing inhibition of radiotoxin binding. NEA, the N-
ethylated analogue of A, was significantly less potent
a7 nACh receptors
Binding of 125I-BgTx to SH-SY5Y-ha7 cell membrane homo-
genates was inhibited by 7BNMC (7-O-benzyl-N-methylco-
claurine), 7B12MNMC (7-O-benzyl-N,12-O-dimethyl coclaurine),
laudanosine (L), armepavine (A) and N-ethyl norarmepavine
(NEA) (Figure 2a). In contrast, N-methylcoclaurine (MC) and
C did not inhibit binding, and BBC (7,12-O,O0-dibenzylco-
claurine) or BBCM (7,12-O,O0-dibenzyl N-methyl coclaurine)
caused less than 10% inhibition at the highest concentration
tested (300 mM) (Figure 2a). Estimated IC50 values are
summarised in Table 1. The rank order of potency of
inhibition of 125I-BgTx binding to SH-SY5Y-ha7 cell a7
nACh receptors was 7BNMC47B12MNMC4L4A4
NEAbBBCEBBCM. Characterisation of the effects of the
a
C
NEA
100
A
L
80
BBC
7BNMC
60
7B12MNMC
MC
40
Table 1 Ligand binding affinities (IC50 (mM); mean
(95% CI); 4–5 independent experiments) for BTHIQ
compounds at human a4b2 or a4b4 nACh receptors
expressed in SH-EP1 cells or a7 nACh receptors
expressed in SH-SY5Y cells
BBCM
20
0
-7
-6
-5
-4
-3
log[BTHIQ] M
IC50 (95% CI) (mM)
1.5
1.0
0.5
0.0
a7
a4b2
a4b4
b
control
BTHIQ
C
MC
[
125I]a-BgTx
b500
b500
28 (13–59)
44 (38–92)
21 (11–41)
4300
[3H]cytisine
4200
[3H]cytisine
132 (125–140)
23 (19–27)
14 (9–24)
47 (37–60)
12 (10–16)
+ 44 µM NEA
+30 µM A
+ 20 µM L
27 (18–38)
24 (13–47)
59 (38–92)
16 (13–19)
13 (8–21)
15 (11–22)
1.4 (1.2–1.7)
8.1 (7.0–9.5)
A
NEA
L
BBC
7.8 (6.0–10)
7.3 (6.1–21.7)
0.37 (0.26–0.52)
2.6 (2.1–3.1)
BBCM
7BNMC
7B12MNMC
4300
11 (9–13)
14 (11–18)
0
1
2
3
4
5
[
125I-α-BgTx] nM
In competition studies using a7-, a4b2 or a4b4 nACh
receptors, the radiolabelled ligand concentration was 1 nM.
1.5
1.0
0.5
0.0
control
+ 10 µM
7BNMC
Table 2 Functional affinities (IC50 (mM); mean (95%
CI); 6–10independent experiments) for BTHIQ
compounds at human a7, a4b2 or a4b4 nACh
receptors expressed heterologously in Xenopus oocytes
+ 15 µM
7B12MNMC
IC50 (95% CI) (mM)
0
1
2
3
4
5
a7
a4b2
a4b4
[
125I-α-BgTx] nM
BTHIQ
ACh EC50
100 mM
4200
25 (19–36)
43 (34–54)
22 (16–29)
4200
ACh EC50
100 mM
49 (23–100)
ACh EC50 30 mM
Figure 2 Effects of BTHIQs on binding of [125I]a-BgTx to SH-
SY5Y-ha7 membrane homogenates. (a) Displacement of [125I]a-
BgTx binding to SH-SY5Y-ha7 cells by BTHIQ. SH-SY5Y-ha7-
membrane homogenates were incubated with 1 nM [125I]a-BgTx for
90min at room temperature in the presence of various concentra-
tions of BTHIQ. Data are the mean7s.e.m. of 4–5 experiments.
(b) Saturation analysis of the specific binding of [125I]a-BgTx to
SH-SY5Y-ha7-membrane homogenates in the absence and presence
of IC50 concentrations of BTHIQ. Data points are the means of
triplicate samples7s.e.m. of eight experiments.
C
A
NEA
L
BBC
7BNMC
7B12MNMC
18 (15–23)
4.8 (3.9–5.8)
17 (14–19)
3.3 (2.0–5.5)
2.2 (1.5–3.1)
13 (9–18)
21 (15–28)
8.6 (5.8–12.8)
9.7 (6.9–13.6)
0.97 (0.72–1.31) 0.24 (0.23–0.26)
4.0(3.4–4.7) 1.2 (0.8–1.7)
1.4 (1.2–1.5)
2.8 (2.3–3.3)
Data represent 6–10independent experiments.
British Journal of Pharmacology vol 146 (1)

R. Exley et al
Isoquinoline inhibitors of nicotinic receptors
19
1.2
1.0
0.8
0.6
0.4
0.2
0.0
a
C
A
NEA
L
BBC
7BNMC
7B12MNMC
-7
-6
-5
-4
-3
log[BTHIQ] M
c
0.6
b
ACh
1.0
0.8
0.6
0.4
0.2
0.0
NEA
A
0.5
0.4
0.3
0.2
L
+ 40 µM NEA
+ 25 µM A
+ 20 µM L
-6
-5
-4
-3
-120
-90
-60
-30
log[ACh] M
Membrane Potential (mV)
0.60
0.55
0.50
0.45
0.40
ACh
1.0
0.8
0.6
0.4
0.2
0.0
7B12MNMC
7BNMC
+ 1 µM 7BNMC
+ 3 µM 7B12MNMC
-6
-5
-4
-3
-120
-90
-60
-30
log[ACh] M
Membrane Potential (mV)
Figure 3 Functional effects of BTHIQs on human a7 nACh receptors. (a) Concentration–response curve for antagonist effects of
the indicated BTHIQs on function of human a7 nACh receptors. The data were normalised to the responses elicited by 100 mM ACh
(approx. EC50 of ACh at a7 nACh receptors) and then fitted to a single site Hill equation. Data points represent the mean7s.e.m. of
6–10experiments. Where no error bars are shown, they are smaller than the symbols. (b) Concentration–response curve for ACh
responses in the absence or presence of IC50 concentrations of BTHIQ. Oocytes were first exposed to ACh to obtain control
responses, then to BTHIQ for 2 min, and finally to both ACh and BTHIQ. Data were normalised to responses elicited by 1 mM ACh
(maximal ACh response) and represent the mean7s.e.m. of 8–10experiments. (c) Data show the inhibition of a7 receptor function
by concentrations of BTHIQ close to their respective IC50 values at a range of membrane potentials. Inhibition by NEA, 7BNMC
and 7B12MNMC was equivalent at all potentials, but inhibition by L or A was dependent on holding potential.
(IC50 ¼ 43 mM; Po0.05) than any of the N-methylated BTHIQs
tested (MC and BBCM were not tested). N-unsubstituted
BTHIQ (C and BBC) had weaker effects on the function of a7
nACh receptors. C, at 300 mM, inhibited less than 10% of the
ACh EC50 response, while 100 mM BBC inhibited approxi-
mately 30% of the ACh EC50 response (Figure 3a). The rank
order of potency of inhibition of human a7 receptors by
BTHIQs is 7BNMC47B12MNMC4LIA4NEAbBBC4C.
The most potent inhibitors of a7 receptor function
(7BNMC, 7B12MNMC, L, A, NEA) were selected to
investigate how BTHIQs affect the concentration–response
curve of ACh at a7 receptors. These studies were carried out
using concentrations of BTHIQs close to their functional IC50
concentrations. Figure 3b shows that all the BTHIQ com-
pounds tested significantly lowered ACh efficacy throughout
the agonist dose range by about 40–50% (Po0.05) without
substantial increases in the ACh EC50. In a further series of
experiments, EC50 concentrations of ACh for a7 (100 mM) were
used to determine whether the antagonism by 7BNMC,
7B12MNMC, L, A or NEA was voltage-dependent. For these
experiments, oocytes were stepped at 20mV intervals between
À100 and À40mV and the response to ACh was determined at
each potential in the presence and absence of antagonist. The
percentage of inhibition for NEA, 7BNMC and 7B12MNMC
was not significantly different at each holding potential
(Figure 3c). In contrast, the effects of A and L were
significantly more pronounced at voltages lower than
À80mV (Figure 3c; ( Po0.05). These data indicate that
BTHIQs inhibited a7 nACh receptors noncompetitively and
confirm the findings of Chiodini et al. (2001) that L blocks a7
nACh receptors in a voltage-dependent manner.
a4b2 and a4b4 nACh receptors
Binding of [3H]cytisine to both human a4b2 and a4b4 nACh
receptors was inhibited by all BTHIQs shown on Figure 1
(Figure 4a). At both receptor subtypes, the most potent
inhibitors were 7BNMC and 7B12MNMC, which inhibited
British Journal of Pharmacology vol 146 (1)

20
R. Exley et al
Isoquinoline inhibitors of nicotinic receptors
binding at low micromolar levels (Table 1). MC fully inhibited
binding of [3H]cytisine to a4b2 or a4b4 nACh receptors with
potencies significantly higher than that displayed by C at either
receptor subtype (Po0.05; Table 1). The potency of BBCM at
a4b2 or a4b4 nACh receptors was not significantly different
from that of BBC. Inhibition of [3H]cytisine binding to both
receptor subtypes occurred at concentrations similar to or
higher than those producing functional inhibition, and they all
decreased maximal binding from about 5 pmol mg proteinÀ1 to
about 3–3.5 pmol mg proteinÀ1 without any significant increase
in KD values (Figure 4b), indicating that BTHIQs inhibited
binding of [3H]cytisine to human a4b2 or a4b4 nACh receptors
by a noncompetitive mechanism.
(Figures 5a, 6a) with IC50 values ranging from low to moderate
micromolar concentrations (Table 2). BTHIQs were more
potent at inhibiting the function of a4b4 than that of a4b2
nACh receptors, but in both receptor subtypes the rank
order of potency of functional inhibition was 7BNMC4
7B12MNMC4BBCIL4A4NEAIC.
To study further the mechanism whereby 7BNMC,
7B12MNMC, BBC, L, A, NEA and C inhibit human a4b2
or a4b4 nACh receptors, we analysed the effects of IC50
concentrations on membrane currents elicited by different
ACh concentrations applied to human a4b2 or a4b4 nACh
receptors. Figures 5b and 6b show that all the BTHIQ
compounds tested significantly reduced the responses to ACh
equieffectively for both a4b2 or a4b4 nACh (Po0.05), without
significant changes in the ACh EC50. 7BNMC, 7B12MNMC,
NEA, C and BBC blocked the response to ACh at human a4b2
or a4b4 nACh receptors in a voltage-independent manner
(Figures 5c, 6c). However, human a4b2 or a4b4 nACh
receptors, as human a7 nACh receptors, were inhibited in a
voltage-dependent manner by A and L (Figures 5c and 6c).
The function of human a4b2 and a4b4 nACh receptors was
fully inhibited by 7BNMC, 7B12MNMC, BBC, L, A, NEA
and C (MC and BBCM were not tested), generally with IC50
values significantly lower than those observed at a7 nACh
receptors (Po0.05; Table 2). Thus, C and BBC, which had
little effect on the function of a7 nACh receptors, fully
inhibited the ACh responses of a4b2 and a4b4 nACh receptors
a
α4β2
α4β4
C
100
100
80
60
40
20
0
A
C
A
L
NEA
BBC
NEA
L
80
BBC
7BNMC
60
7B12MNMC
MC
BBCM
7BNMC
7B12MNMC
40
MC
20
BBCM
0
-8
-7
-6
-5
-4
-3
-9 -8 -7 -6 -5 -4 -3
log[BTHIQ] M
log[BTHIQ] M
b
α4β2
α4β4
6
5
4
3
2
1
0
ACh
5.0
control
+140 µM C
+47 µM NEA
+15 µM A
+ 60 µM NEA
+24 µM A
+12 µM L
2.5
+16 µM L
0.0
0
1
2
3
4
0
1
2
3
4
5
[3H]cytisine] M
[3H]cytisine] M
6
5
4
3
2
1
0
ACh
5.0
2.5
0.0
+7 µM BBC
control
+1 µM 7BNMC
+2 µM 7B12MNMC
+ 12 µM BBC
+2 µM
7BNMC
+8 µM
7B12MNMC
0
1
2
3
4
0
1
2
3
4
5
[3H]cytisine] M
[3H]cytisine] M
Figure 4 Effects of BTHIQ on [3H]cytisine binding to human a4b2 and a4b4 nACh receptors. (a) Displacement of [3H]cytisine
binding to SHEP-ha4b2 or SHEP-ha4b4 membrane homogenates by BTHIQ. SH-EP1-ha4b2 or SHEP-ha4b4 membrane
homogenates were incubated with 1 nM [3H]cytisine for 75 min at 41C in the presence of various concentrations of BTHIQ. Data are
the mean7s.e.m. of 10experiments. (b) Saturation analysis of the specific binding of [ 3H]cytisine to SH-EP1-ha4b2 or SHEP-ha4b4
membrane homogenates in the absence and presence of IC50 concentrations of BTHIQ. Data points are the means of triplicate
samples7s.e.m. of 10( a4b2) or 4–5 (a4b4) experiments.
British Journal of Pharmacology vol 146 (1)

R. Exley et al
Isoquinoline inhibitors of nicotinic receptors
21
a
1.2
1.0
0.8
0.6
0.4
0.2
0.0
C
A
NEA
L
BBC
7BNMC
7B12MNMC
-9 -8 -7 -6 -5 -4 -3 -2
log[BTHIQ] M
c
0.6
1.2
1.0
0.8
0.6
0.4
0.2
0.0
b
+ C
+ L
ACh
+
A
+ 20 µM NEA
+ 12 µM A
+ 8 µM L
NEA
0.4
0.2
0.6
-120
-80
-40
-8
-7
-6
-5
-4
-3
-2
log[ACh] M
Membrane Potential (mV)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
ACh
+ BBC
10 µM BBC
+ 7BNMC
1 µM 7BNMC
0.4
0.2
+ 7B12MNMC
4 µM 7B12MNMC
-120
-80
-40
-8
-7
-6
-5
-4
-3
-2
log[ACh] M
Membrane Potential (mV)
Figure 5 Functional effects of BTHIQ on human a4b2 nACh receptors. (a) Concentration–response curve for the antagonist
effects of the test BTHIQ on human a4b2 nACh receptors. The data were normalised to the responses elicited by 100 mM ACh
(approximate low affinity EC50 of ACh at a4b2 nACh receptors) and then fitted to a two-component Hill equation. Data points
represent the mean7s.e.m. of 8–10independent experiments. Where no error bars are shown, they are smaller than the symbols. (b)
Concentration–response curve for ACh responses in the absence or presence of IC50 concentrations of BTHIQ. After control EC50
ACh responses were elicited, oocytes were first superfused with BTHIQ alone for 2 min and then with EC50 ACh and BTHIQ. Data
were normalised to responses elicited by 1 mM acetylcholine (maximal ACh response) and represent the mean7s.e.m. of 10
experiments. (c) Inhibition of a4b2 receptor function by IC50 concentrations of BTHIQ at a range of holding potentials. Inhibition
by 7BNMC, 7B12MNMC, BBC, NEA and C was equivalent at all potentials, but inhibition by L or A was dependent on holding
potential (n ¼ 10, Anova test).
Discussion
d-tubocurarine, BTHIQs only exhibited noncompetitive
(voltage-dependent or -independent) inhibitory effects. This
suggests, as has been previously shown for muscle nACh
receptors (Codding & James, 1973), that two appropriately
spaced positively charged nitrogen atoms borne on a rigid
hydrocarbon scaffolding fulfil the basic requirement for
curariform competitive antagonism at neuronal nACh recep-
tors.
The results presented here show that C and its congeners listed
in Figure 1 inhibit human a7, a4b2 and a4b4 nACh receptors
in a noncompetitive manner and with different strengths. The
BTHIQs tested in this study are structural mimics of the
‘eastern’ moiety of the hypotensive alkaloid tetrandrine, which
inhibits muscle and neuronal nAChR with low micromolar
affinity (Slater et al., 2002). Substitutions at C7, C12 or N of
the basic BTHIQ structure (Figure 1) produced derivatives
that were more potent than C and in some cases (e.g. 7BNMC)
more potent than tetrandrine.
In comparing functional IC50 values to radioligand binding
inhibition IC50 values for these noncompetitive interactions
acting at a specific nACh receptor subtype, BBC was four-fold
functionally less potent, A, L and NEA were approximately
equipotent, and 7BNMC and 7B12MNMC were 5-8-fold
functionally more potent when acting at a7 nACh receptors,
suggesting possible ability of these agents to discriminate sites
for radiotoxin binding from functionally relevant agonist
binding sites. However, all BTHIQs were slightly more potent
in functional than in radioagonist binding competition assays
when acting at a4b2 (1.5–2.8-fold) and a4b4 (1.5–3.8-fold
C derivatives also mimic the nonquaternary half of the
d-tubocurarine molecule. The latter has a broad range of
pharmacological effects on neuronal nACh receptors, includ-
ing competitive inhibition (Lipscombe & Rang, 1987; Bertrand
et al., 1990; Chavez-Noriega et al., 1997), partial agonism
(Nooney et al., 1992; Cachelin & Rust, 1994) and competitive
potentiation (Cachelin
& Rust, 1994). However, unlike
British Journal of Pharmacology vol 146 (1)

22
R. Exley et al
Isoquinoline inhibitors of nicotinic receptors
a
1.2
1.0
0.8
0.6
0.4
0.2
0.0
C
A
L
NEA
BBC
7BNMC
7B12MNMC
-9
-8
-7
-6
-5
-4
-3
-2
log[BTHI] M
c
0.7
0.6
0.5
0.4
0.3
1.2
1.0
0.8
0.6
0.4
0.2
0.0
b
ACh
+C
+ NEA
+A
+18 µM C
+16 µM NEA
+5 µM A
+ L
+3 µM L
-120
-90
-60
-30
-7.5
-5.0
-2.5
log[ACh] M
Membrane Potential (mV)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.7
0.6
0.5
0.4
0.3
ACh
+2µM BBC
+BBC
+1µM 7BNMC
+7BNMC
+2µM
7B12MNMC
+7B12MNMC
-120
-90
-60
-30
-7.5
-5.0
-2.5
log[ACh] M
Membrane Potential (mV)
Figure 6 Functional effects of BTHIQ on human a4b4 nACh receptors. (a) Concentration–response curve for the antagonist
effects of the test BTHIQ on human a4b4 nACh ceptors. The data were normalised to the responses elicited by 30 mM ACh
(approximate EC50 of the ACh response at a4b4 nACh receptors) and then fitted to a single component Hill equation. Data points
represent the mean7s.e.m. of 3–4 experiments. Error bars are not shown when they are smaller than the symbols. (b)
Concentration–response curve for ACh responses in the absence or presence of IC50 concentrations of BTHIQ. After control EC50
ACh responses were elicited, oocytes were first superfused with BTHIQ alone for 2 min and then with EC50 ACh and BTHIQ. Data
were normalised to responses elicited by 1 mM ACh (maximal ACh response) and represent the mean7s.e.m. of 6–10experiments.
(c) Inhibition of a4b4 receptor function by concentrations of BTHIQ close to their respective IC50 concentrations at a range of
holding potentials. Inhibition by 7BNMC, 7B12MNMC, BBC, C and NEA was equivalent at all potentials at which ACh responses
could be elicited, but inhibition by L or A was dependent on holding potential (n ¼ 10, Anova test).
excluding C) nACh receptors, possibly suggesting a systematic
difference in affinity determinations based on the two assays
probing effects on agonist binding domains. In absolute terms,
each BTHIQ was most potent at a4b4 nAChRs and least
potent (except functionally for 7B12MNMC and in binding
assays for NEA) at a7 nAChRs.
What are the key structural features of C and its congeners
that influence potency in antagonism of nACh receptors?
From the data shown in Tables 1 and 2 it is clear that 7BNMC
and 7B12MNMC are the most potent ligands at human a7,
a4b2 and a4b4 nACh receptors. These compounds differ
from C7-hydroxyl, C12-hydroxyl, N-unsubstituted C in that
they are N-methylated and contain a bulky benzyloxy group
at C7 and a phenolic hydroxyl (7BNMC) or methoxyl
(7B12MNMC) group at C12. Simpler N-methylated Cs
contain a hydroxyl (MC) or methoxyl (A, NEA, L) group at
C7 and either a hydroxyl (MC, A, NEA) or methoxyl (L)
group at C12.
A large, lipophilic substituent at C7 of BTHIQs, which
corresponds to part of the ‘western’ tetrahydroisoquinoline
moiety of tetrandrine, is an important element for activity at
nACh receptors. Lipophilic substituents at C7 may enhance
binding of the ligand to a lipophilic region at or around the
BTHIQ binding domain, which may contribute favourable
hydrophobic interactions to the free energy of BTHIQ binding
to the receptors. Nevertheless, the overall bulkiness in the
region also is important. A large lipophilic substituent at C7
such as a benzyloxy group favours interaction with a7, a4b2
and a4b4 nACh receptors more than a small group such as a
methoxy group (e.g., 7BNMC is more potent than A), and
compounds with a C7-methoxyl group also are generally more
potent than hydroxyl analogues (e.g., A is more potent than
MC except at a4b2-nACh receptors). However, the presence of
bulky benzyloxy substituents at both C12 and C7 (i.e., BBC
and BBCM) decreases potency relative to the potency
displayed by 7BNMC or 7B12MNMC, and C12-hydroxylated
British Journal of Pharmacology vol 146 (1)

R. Exley et al
Isoquinoline inhibitors of nicotinic receptors
23
7BNMC has higher potency than C12-methoxylated
7B12MNMC for compounds already carrying C7-benzyloxy
groups. Such a decrease in potency does not occur in L, which
is methoxylated at both C12 and C7, when compared to A.
Thus, although lipophilicity on the ‘western’ side of BTHIQs
increases potency, excessive bulkiness may distort the folding
of the BTHIQs and weaken their interaction with nACh
receptors.
substituents at C7 and C12 interact to influence BTHIQs’
activity at a4b2 and a4b4 nACh receptors.
Inhibition of nACh receptors by A or L was voltage
dependent. Surprisingly, NEA, which is an N-ethylated analog
of A, blocked nACh receptors in a voltage-independent
manner. This loss of voltage-dependence may be related to
steric hindrance of a key interaction between the nitrogen
atom and a site at or within the ion channel. Voltage-
dependence is also lost with increasing bulk: benzylated
N-methylated BTHIQs (7BNMC and 7B12MNMC) inhibited
nACh receptors in a voltage-independent manner, which
suggests that in addition to accessibility to the substituted
nitrogen atom, bulk and/or lipophilicity at C7 also influence
the ability of BTHIQ to interfere with voltage sensing by
nACh receptors.
Effects of N-alkylation of BTHIQs on affinity for nACh
receptors are influenced by the type of alkyl substituent and
crucially by receptor subtype. N-unsubstituted BTHIQ (i.e., C
and BBC) are poor functional antagonists (IC50 values in
millimolar range) of a7 nACh receptors, but the N-methylated
A, L, 7BNMC and 7B12MNMC inhibit function and binding
with micromolar potency. NEA, which is the N-ethylated
analogue of A, is slightly less potent than A, but it is still
significantly more potent than C and BBC. On the other
hand, N-methylation of C to MC or of BBC to BBCM does
not improve or diminishes ability of the compounds to inhibit
125I-BgTx binding to a7 nACh receptors. Thus, BTHIQ
activity at a7 nAChR seems to be mostly influenced by the
type of substituents at C7 and C12: a bulky lipophilic group at
C7 conferring highest potency.
Our results show that subtle chemical modifications to the
basic BTHIQ structure bring about significant changes in both
nACh receptor affinity and mode of inhibition. The impact of
the structural changes upon affinity is significantly influenced
by receptor subunit composition, which further highlights the
potential of nicotinic antagonists in the development of high
affinity, receptor subtype-specific probes as tools to enhance
the study of the roles of nACh receptors in both normal brain
functions and in disease. Moreover, BTHIQs have potential
for revealing structure–function relationships for nACh
receptor antagonists.
In contrast, although BTHIQ activity at a4b2 and a4b4
nACh receptors does not require N-alkylation, this structural
modification increases affinity assessed using binding assays
when imposed on C. NEA, however, is the least potent of the
N-alkylated BTHIQs, and its interactions with a4b2 and a4b4
nACh receptors (and with a7 receptors) may be weakened
(compared to A) by the larger bulk of the N-ethyl group.
Moreover, the effect of N-methylation on potency when acting
at a4b2 and a4b4 nACh receptors is diminished when other
structural requirements such as appropriate bulk on the
‘western’ side of the BTHIQs are met: BBCM and BBC have
comparable affinities for [3H]cytisine binding to a4b2 and a4b4
nACh receptors. Thus, N-methylation and the overall bulk of
This work was funded in part by a Wellcome Trust CRIG and ICM
Grant No. P99-031-F. B.K.C. acknowledges a generous gift of
equipment from the Alexander von Humboldt Foundation (Germany).
R. Exley was funded by a BBSRC-Eli Lilly Case PhD studentship.
Funding from National Institutes of Health grants NS40417 and
DA015389, from Arizona Disease Control Research Commission
grants 9730and 9615, and by endowment and/or capitalization funds
from the Men’s and Women’s Boards of the Barrow Neurological
Foundation also is acknowledged (R.J.L.).
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(Received February 1, 2005
Revised March 24, 2005
Accepted May 16, 2005
Published online 27 June 2005)
British Journal of Pharmacology vol 146 (1)