Investigating the interaction of McN-A-343 with the M1 muscarinic receptor using its nitrogen mustard derivative and ACh mustard

Article abstract of DOI:10.1111/j.1476-5381.2010.00810.x

Background and purpose: We investigated how McN-A-343 inhibited the alkylation of the M₁ muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically. Experimental approach: We incubated the M₁ muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [³H]N- methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model. Key results: The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [³H]N- methylscopolamine binding. Conclusions and implications: Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M₁ muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.

Full text of DOI:10.1111/j.1476-5381.2010.00810.x

British Journal of Pharmacology (2010), 160, 1534–1549
© 2010 The Authors
Journal compilation © 2010 The British Pharmacological Society All rights reserved 0007-1188/10
www.brjpharmacol.org
RESEARCH PAPER
Investigating the interaction of McN-A-343 with the
M1 muscarinic receptor using its nitrogen mustard
derivative and ACh mustardbph_810 1534..1549
KW Figueroa*, H Suga and FJ Ehlert
Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, CA, USA
Background and purpose: We investigated how McN-A-343 inhibited the alkylation of the M1 muscarinic receptor by its
nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically.
Experimental approach: We incubated the M1 muscarinic receptor expressed in Chinese hamster ovary cells with ACh
mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping
the reaction and removing unreacted ligands, unalkylated receptors were measured using [3H]N-methylscopolamine. Analo-
gous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for
allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model.
Key results: The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent
with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent
complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was
consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants
estimated from alkylation kinetics agreed with those measured by displacement of [3H]N-methylscopolamine binding.
Conclusions and implications: Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively
with ACh and N-methylscopolamine at the orthosteric site on the M1 muscarinic receptor. Measuring how drugs modulate the
kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric
modulators and competitive inhibitors.
British Journal of Pharmacology (2010) 160, 1534–1549; doi:10.1111/j.1476-5381.2010.00810.x
Keywords: M1 muscarinic receptor; ACh mustard; McN-A-343; nitrogen mustard derivative; allosterism; competitive inhibition;
irreversible binding
Abbreviations: AChM, ACh mustard; BR384, 4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate; CHO
hM1 cells, Chinese hamster ovary cells stably transfected with the human M1 muscarinic receptor; KRB,
Krebs–Ringer bicarbonate; McN-A-343, [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammo-
nium chloride; NMS, N-methylscopolamine; WIN 51708, 17-b-hydroxy-17-a-ethynyl-5-a-androstano[3,2-
b]pyrimido[1,2-a]benzimidazole
atropine, adrenergic antagonism and bilateral adrenalectomy
and sympathectomy, but not by hexamethonium. The pressor
response occurred rapidly upon arterial injection of McN-A-
343 close to the superior sympathetic ganglia, and it was later
shown that the compound caused a release of catecholeam-
ines in the rat (Martin, 1996). Thus, McN-A-343 behaves as a
novel muscarinic ganglionic stimulant. In contrast, most
non-selective muscarinic agonists decrease blood pressure by
activating the M3 muscarinic receptor (receptor nomenclature
follows Alexander et al., 2009) on the endothelium of periph-
eral blood vessels. The pressor response to McN-A-343 in vivo
is antagonized by the M1 selective antagonist pirenzepine
with high potency (Hammer and Giachetti 1982), and McN-
A-343 behaves as an M1/M4 selective muscarinic receptor
Introduction
The compound, [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-
butynyl] trimethylammonium chloride (McN-A-343), was
first shown by Roszkowski (1961) to stimulate sympathetic
ganglia. When administered to cats and dogs, it caused an
increase in blood pressure, and this action was blocked by
Correspondence: FJ Ehlert, Department of Pharmacology, School of Medicine,
University of California, Irvine, Irvine, CA 92697-4625, USA. E-mail: fjehlert@
uci.edu
*Present address: Johnson & Johnson Pharmaceutical, La Jolla, CA 92121, USA.
Received 6 August 2009; revised 10 December 2009; accepted 10 March 2010

M1 muscarinic receptors and McN-A-343
KW Figueroa et al
1535
Figure 1 Structures of AChM, BR384, their aziridinium ions and alcoholic hydrolysis products. Also shown for comparison are ACh and
McN-A-343.
agonist in signalling assays on cell lines expressing muscarinic
receptors (McKinney et al., 1991; Lazareno et al., 1993;
Figueroa et al., 2008).
powerful means of detecting allosteric interactions (Suga
et al., 2008). Because neither the test drug nor reactive ligand
is radioactive, they can be used over a range of concentrations
much wider than that typically used in experiments investi-
gating the modulation of radioligand binding by a putative
negative allosteric modulator, for example. When used with
site-directed mutagenesis, either to introduce or to remove
accessible reactive amino acids lining the binding pocket, this
approach enables the identification of the binding site for the
irreversible ligand and its competitive inhibitors.
In the present report, we have utilized both ACh mustard
(AChM) and a nitrogen mustard derivative of McN-A-343,
4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophe
nyl)carbamate (BR384, see Figure 1) (Ringdahl et al., 1990), to
explore the interactions of orthosteric and allosteric ligands
with the human M1 receptor. We find that McN-A-343, as well
as ACh and NMS, competitively protects the M1 receptor from
irreversible alkylation by both AChM and BR348, whereas
17-b-hydroxy-17-a-ethynyl-5-a-androstano[3,2-b]pyrimido[
1,2-a]benzimidazole (WIN 51708) has little effect, and gal-
lamine modulates alkylation through an allosteric mecha-
nism. Our approach has widespread application to the study
of allosterism at subtypes of the muscarinic receptor and
other receptors.
The selective action of McN-A-343 raises the question of
how it binds differentially with muscarinic receptor subtypes.
The interaction between the binding of McN-A-343 and the
orthosteric, muscarinic antagonist [3H]N-methylscopolamine
([3H]NMS) in the cerebral cortex and heart has been inter-
preted as competitive and allosteric, respectively (Birdsall
et al., 1983). Given the abundance of M1 and M4 receptors in
the cortex, and M2 receptors in the heart, this conclusion
suggests that McN-A-343 interacts competitively with
[3H]NMS at M1 and M4 receptors, and allosterically with
[3H]NMS at the M2 receptor. Site-directed mutagenesis studies
have led to speculation that McN-A-343 (May et al., 2007) and
desmethylclozapine (Sur et al., 2003) interact allosterically
with [3H]NMS at the M2 muscarinic receptor. More recently, it
has been suggested that the binding site for McN-A-343 on
the M2 receptor shares a common region with that of orthos-
teric ligands, indicating a competitive interaction of McN-A-
343 with [3H]NMS and ACh even though part of McN-A-343
may interact with an allosteric site (Valant et al., 2008). In a
study on the interaction between McN-A-343 and [3H]NMS at
the M2 receptor in the presence of d-tubocurarine, the data
were interpreted according to the postulate that McN-A-343
competitively inhibits the binding of [3H]NMS at relatively
low concentrations, and at higher concentrations it binds to
the allosteric site (Waelbroeck, 1994). How this mechanism
can account for the incomplete inhibition of [3H]NMS
binding by McN-A-343 is unclear, although it is interesting
that McN-A-343 causes complete inhibition of [3H]NMS
binding to the M2 receptor in the presence of d-tubocurarine
(Waelbroeck, 1994).
Methods
Cell culture
Chinese hamster ovary (CHO) cells stably expressing the
human muscarinic M1 receptor (CHO hM1) were obtained
from Acadia Pharmaceuticals (San Diego, CA, USA). CHO hM1
cells were cultured in F-12 media Kaighn’s modification
supplemented with 10% fetal calf serum, penicillin–
Measuring how a test drug interferes with the covalent
binding of a reactive orthosteric ligand to a receptor is a
British Journal of Pharmacology (2010) 160 1534–1549

M1 muscarinic receptors and McN-A-343
1536
KW Figueroa et al
streptomycin (100 units·mL-1 and 100 mg·mL-1) and G418
(0.4 mg·mL-1) at 37°C with 5% CO2.
Animals
All animal care and experimental procedures were approved
by the Institutional Animal Care and Use Committee at the
University of California, Irvine. Four male Sprague Dawley
rats (200–250 g) were used as a source of cerebral cortex for
binding experiments. The animals were maintained in the
university vivarium on a 12 h light–dark cycle with free access
to food and water.
Scheme 1 Model for the interaction of the aziridinium ion (X) with
the M1 receptor (R) in the presence of an allosteric modulator (A). The
aziridinium ion first forms a reversible complex with the receptor
(XR), and then forms a covalent complex (X-R). K1 and K2 denote the
affinity constants of the reversible complexes of XR and RA, a denotes
the cooperativity factor for the allosteric interaction and k1 denotes
the rate constant for alkylation of the receptor.
Receptor alkylation experiments in intact cells and homogenates
The strategy for measuring the rate of inactivation of the M1
receptor by AChM and BR384 involved: (i) incubating the M1
receptor preparation with the mustard; (ii) stopping the reac-
tion with thiosulphate and a competitive inhibitor; (iii)
washing the preparation to remove the stopping reagents;
and (iv) measuring the specific binding of [3H]NMS to esti-
mate the residual, unalkylated receptors. Experiments on
intact cells were carried out as described previously (Suga
et al., 2008). CHO hM1 cells incubated with the aziridinium
ion of the mustard analogs at 37°C for various times in a final
volume of 0.6 mL of Krebs–Ringer bicarbonate buffer (KRB
buffer; 26 mM NaHCO3, 1.2 mM KH2PO4, 124 mM NaCl,
5 mM KCl, 1.8 mM CaCl2, 1.3 mM MgCl2, 10 mM glucose, pH
7.4). The reaction was stopped at the indicated times by the
addition of a mixture of thiosulphate (10 mM) and N-methyl-
amitriptyline (10 mM), which completely prevented receptor
alkylation by AChM (1 mM) and BR384 (0.1 mM). The stop-
ping solution was left on the cells for a minimum of 15 min.
The adherent cells were washed five times, and residual mus-
carinic receptors were estimated using an intact cell [3H]NMS
binding assay at room temperature.
In other experiments, the rate of inactivation of muscarinic
receptors by AChM and BR384 was measured in homogenates
of CHO hM1 cells as described previously (Suga et al., 2008).
Cellular homogenate was prepared in 20 mM Na/HEPES
buffer, pH 7.4, containing EDTA (10 mM) and NaCl
(100 mM). Homogenates were incubated at 37°C with AChM,
BR384 and other drugs in microfuge tubes for various periods
of time, and the incubations were stopped by the addition of
thiosulphate (1 mM) and scopolamine. The concentration of
scopolamine was 1 mM for BR384 (1–10 mM) and AChM
(1–100 mM). When higher concentrations of each mustard
were used (BR384, 0.1–0.3 mM; AChM, 1 mM), the concen-
tration of scopolamine was 10 mM. These reagents completely
prevented receptor alkylation by AChM (1 mM) and BR384
(0.3 mM). The homogenates were washed twice (1 mM scopo-
lamine) or three times (10 mM scopolamine) by centrifuga-
tion, and residual muscarinic receptors were measured using
[3H]NMS at 23°C.
Competitive [3H]NMS binding experiments at 0°C
The competitive binding experiments with the nitrogen
mustard analogs and their transformation product were
carried out at 0°C as described by Suga et al. (2008).
Data analysis
The competitive and saturation binding data for [3H]NMS
were analysed with GraphPad Prism using the variable slope
dose–response and one-site binding functions respectively.
We derived a mathematical model to describe the kinetics
of receptor alkylation by BR384 under conditions where the
concentration of BR384 decays with time. Our approach is
similar to that described previously (Ehlert and Jenden, 1985).
The model for the interaction of the aziridinium ion of BR384
(X) with the M1 muscarinic receptor (R) in the presence of an
allosteric modulator (A) is shown in Scheme 1. We assume
that the aziridinium ion of BR384 rapidly equilibrates with
the receptor to form a reversible complex (XR) that converts
to a covalent complex (X-R) at a slower rate. K1 and K2 denote
the affinity constants (inverse molar units; reciprocal of dis-
sociation constant) of the aziridinium ion of BR384 and the
allosteric modulator for the free receptor, respectively; a
denotes the cooperativity factor for their interaction; and k1
denotes the rate constant for alkylation of the receptor by the
aziridinium ion. If the rate constants describing the reversible
binding of X and A are fast relative to k1, then the rate of loss
of unalkylated receptors (y) can be described by the following
differential equation as described previously (Suga et al.,
2008):
dy
dt
Xy
(1)
= −
k1
1
X +
1+ AαK2
1+ AK2
K1
(
)
The concentration of the aziridinium ion is not constant
during the incubation, but decays from its peak concentration
according to a first-order decay process approximated by the
following equation:
Experiments investigating the alkylation of muscarinic
receptors by BR384 in rat brain homogenate were carried out
using the same procedures as that described previously in a
study on an alkylating derivative of oxotremorine-M (Ehlert
and Jenden, 1985).
τ
X = X0e−t
(2)
x
in which X0 denotes the concentration of the aziridinium ion
at the start of the incubation, and tx denotes the time con-
stant for the decay of the aziridinium ion from its peak con-
British Journal of Pharmacology (2010) 160 1534–1549

M1 muscarinic receptors and McN-A-343
KW Figueroa et al
1537
centration. The value of tx was estimated at 14.3 min by
regression analysis of the data reported by Ringdahl et al.
(1990) for the stability of BR384 in aqueous solution at pH
7.4. We use a time constant to describe the first-order decay of
the aziridinium ion from its peak concentration in solution to
distinguish this process from the two microscopic rate con-
stants describing the formation and decay of the aziridinium
ion (k1 and k2 described by Ringdahl et al., 1990). Substituting
in Equation (3) for X in Equation (2) yields:
It is well known that highly efficacious agonists, like ACh,
exhibit competitive [3H]NMS binding curves with Hill slopes
of less than one (Birdsall et al., 1978). Consequently, for the
analysis of the effects of ACh on receptor alkylation by
BR384, we modified Equations (6) and (7) by adding a Hill
slope (n) to account for the complex binding properties of
ACh:
k τ
x
n
1+ AK2
1+
(
(
( )
⎧
⎡
⎣
⎤ ⎫ 1
⎦
τ
X0e−t K1 +
x
⎪
⎪
⎢
n ⎥ ⎪
y
(
AαK2
)
⎪
⎬
τ
dy
dt
X0e−t
y
1
x
=
(
1− b
)
×
+ b
(9)
⎨
⎪
n
y0
)
= −
k1
⎡ 1+ AK2
⎤
⎪
X0K1 +
τ
X0e−t
+
⎢
n ⎥
⎦
x
⎪
⎩
⎪
⎭
(3)
1+ AαK
)
⎣
2
1+ AαK2
1+ AK2
K1
(
)
k τ
1
x
n ⎤ ⎫
τ
X e−t K + 1+
(
IKi
)
x
⎧
⎪
⎪
⎩
⎡
⎣
y
0
1
⎪
⎦
=
(
1− b
)
×
+ b
(10)
⎨
⎬
n
)
Dividing both sides of the equation by y and integrating over
the time interval t = 0 to t yields:
y0
X K + 1+
(
IKi
⎡
⎤
⎦
⎪
⎭
0
1
⎣
Because the aziridinium ion of AChM is relatively stable at
37°C, we used simpler equations to describe the kinetics of
receptor alkylation by AChM in the absence [Equation (11)]
and presence of allosteric [Equation (12)] and competitive
[Equation (13)] inhibitors:
⎧
⎪
⎡
⎢
⎤
⎥
⎥
⎥
y
1
τ
x
ln
= k1τx⎨ln X0e−t
+
⎢
1+ AαK2
1+ AK2
y0
⎢
⎪
K1
(
)
⎢
⎥
⎦
⎩
⎣
(4)
⎡
⎢
⎤⎫
⎥⎪
1
− ln X0 +
⎛
⎞
⎢
⎥
⎥
⎥
⎬
⎪
1+ AαK2
⎜
⎜
⎜
⎝
⎟
⎟
⎟
⎠
X
⎢
−
k t
1
K1
1
(
)
⎢
⎣
X+
(11)
1+ AK2 ⎦⎭
y
K
1
=
(
1− b
)
× e
+ b
yo
In this equation, y0 denotes the total receptor density at the
start of the reaction, and y denotes the residual, unalkylated
receptors at time t. This equation reduces to:
⎡
⎢
⎢
⎢
⎤
⎥
⎥
⎥
X
−
k t
1
⎢
⎢
⎢
⎥
⎥
⎥
1
k τ
X+
1
x
(12)
1+AαK
1+AK
1+ AK2
⎛
K
1⎜
2 ⎞
2
⎡
⎢
⎢
⎢
τ
⎤
⎥
⎥
⎥
X0e−t K1 +
x
⎟
⎠
Y
t
⎝
⎢
⎣
⎥
⎦
(
)
=
(
1− b
)
× e
+ b
y
1+ AαK2
Yo
=
(5)
1+ AK2
y0
X0K1 +
(
)
⎢
⎥
⎦
⎣
1+ AαK2
⎡
⎢
⎤
⎥
⎥
⎥
X
1+IK
K
−
k t
1
⎢
⎢
(
)
i
X+
(13)
Y
We found that a small proportion of the M1 receptor popula-
tion labelled by [3H]NMS was not alkylated by the aziridinium
ion of BR384. Equation (5) was modified; therefore, by adding
a constant (b) denoting the proportion of non-alkylated
sites:
t
⎢
⎣
⎥
⎦
1
=
(
1− b
)
× e
+ b
Yo
These
equations
have
been
described
previously
(Suga et al., 2008) and are based on the assumption that the
aziridinium ion of AChM remains constant during the
incubation.
k τ
1
x
1+ AK2
1+ AαK2
⎡
⎢
⎢
⎢
τ
⎤
⎥
⎥
⎥
X0e−t K1 +
x
(
)
y
=
(
1− b
)
×
+ b
(6)
We also used a more empirical approach to analyse the
effects of non-labelled inhibitors on the kinetics of receptor
alkylation by AChM and BR384. The data for the loss of
[3H]NMS binding as a function of time were fitted to a single
exponential decay equation using Prism to estimate the
observed rate constant for decay in the absence (kobs) and
presence (kobs′) of inhibitors. With BR384, only the data
obtained over the first 8 min were used to avoid estimation
error caused by the decay in the aziridinium ion. The ratio (R)
of time constants for receptor alkylation in the presence of
the inhibitor (t′) divided by that measured in its absence (t) is
defined as:
1+ AK2
y0
X0K1 +
(
)
⎢
⎥
⎣
1+ AαK2
⎦
Equation (6) describes the kinetics of the loss of [3H]NMS
binding sites in the presence of BR384 and an allosteric modu-
lator. When the negative cooperativity is very great (i.e. log a
<< 0), then A behaves like a competitive inhibitor, and Equa-
tion (6) reduces to the following:
k τ
1
x
τ
x
y
X e−t K1 +
(
1+ IKi
)
⎡
⎤
0
=
(
1− b
)
×
+ b
(7)
⎢
⎣
⎥
⎦
y0
X0K1 + 1+ IKi
( )
in which A and K2 have been replaced with I and Ki to denote
the concentration of the competitive inhibitor and its affinity
constant respectively. In the absence of an allosteric modula-
tor, Equation (6) reduces to:
kobs
τ′
τ
R =
=
(14)
′
kobs
The effect of allosteric [Equation (15)] and competitive [Equa-
tion (16)] inhibitors on the relative ratio of time constants (R)
for receptor alkylation is described by:
k τ
1
x
τ
X e−t K +1
x
y
⎛
⎞
0
1
=
(
1− b
)
×
+ b
(8)
⎜
⎝
⎟
⎠
y0
X0K1 +1
British Journal of Pharmacology (2010) 160 1534–1549

M1 muscarinic receptors and McN-A-343
1538
KW Figueroa et al
1+ AK2
1+ AαK2
1+ XK1
⎛
⎜
⎜
⎞
⎟
⎟
XK1 +
Log
(
R
)
= Log
(15)
(16)
⎜
⎝
⎟
⎠
1+ XK1 + IK1
1+ XK1
⎛
⎝
⎞
⎠
Log
(
R
)
= Log
The theoretical basis of Equations (15) and (16) has been
described previously (Suga et al., 2008).
Figure 2 Effects of AChM and BR384 on the binding of [3H]NMS to
homogenates of CHO cells expressing the M1 receptor. Cellular
homogenate was incubated with the aziridinium ion of BR384 (1 mM)
or AChM (10 mM) for 8 min at 37°C. These concentrations refer to
the starting concentration of the nitrogen mustard before transfor-
mation to the aziridinium ion. Sodium thiosulphate (1 mM) was
added subsequently, and the homogenates were washed by centrifu-
gation and suspension in fresh buffer. Control homogenate was
treated in the same manner except for exposure to the mustards. The
data represent the mean specific binding values Ϯ SEM of two
experiments, each in triplicate. The mean Ϯ SEM denotes the range
of the two measurements, because N = 2.
When fitting Equations (6)–(13) and (15) and (16) to the
binding data by nonlinear regression analysis, the parameter
K1 was constrained as a constant. The log molar values of this
constant for the aziridinium ions of AChM and BR384 were
4.62 and 5.1, respectively, as determined from the data shown
in Figure 4.
To test whether the various allosteric models [Equations (6),
(9), (12) and (15)] provided a better fit to the data than the
corresponding competitive models [Equations (7), (10), (13)
and (16)], we compared the residual estimate of the variance
for both models using an F distribution as described previ-
ously (Suga et al., 2008).
Results
Effects of AChM and BR384 on the specific binding of [3H]NMS
To examine the influence of AChM and BR384 on the binding
of [3H]NMS, CHO hM1 cells were incubated with the corre-
sponding aziridinium ions at 37°C and then assayed for
[3H]NMS binding at room temperature. In control CHO hM1
cells, [3H]NMS bound in a saturable fashion characterized by
an observed log affinity constant (KA) of 9.41 Ϯ 0.05; mean Ϯ
SEM (Figure 2). Treatment of CHO hM1 cells with the aziri-
dinium ions of AChM (10 mM) and BR384 (1 mM) for 8 min at
37°C followed by washing caused a subsequent reduction in
the binding capacity of [3H]NMS to 64 Ϯ 1.8 and 44 Ϯ 1.8%,
respectively, without having a significant effect on KA (9.44 Ϯ
0.04 and 9.52 Ϯ 0.06 respectively). The data show that the
aziridinium ions of AChM and BR384 bind irreversibly to the
M1 muscarinic receptor.
Formation of the aziridinium ions of BR384 and AChM
BR384 was incubated at 37°C for 5 min in a mixture of 80%
50 mM phosphate buffer (pH 7.4) and 20% acetone to form its
aziridinium ion (see Figure 1 for the transformation products
of BR384 in aqueous solution at neutral pH). The small flask
(10 mL) containing cyclized BR384 was attached to a rotary
evaporator suspended in air at room temperature, and the
acetone was quickly removed in vacuo (5 min). The solution
quickly cooled to about 10°C because of the loss of the heat of
vaporization. After removal of the acetone, the solution was
placed on ice and used as soon as possible. At 37°C, the
aziridinium ion of BR384 decays from its peak concentration
of 54% (relative to the parent mustard) achieved after 5 min
of incubation to 5.3% 30 min later (Ringdahl et al., 1990).
AChM was cyclized to its aziridinium ion by incubation at
37°C for 15 min as described previously (Suga et al., 2008).
The solution was placed on ice and used as soon as possible.
The aziridinium ion of AChM is relatively stable at 37°C and
only declines to 86% of its peak concentration in 60 min. All
concentrations of the transformation products are given in
the text as the starting concentration of the parent mustard.
Competitive binding experiments with AChM, BR384 and their
transformation products
We determined the extent to which the aziridinium ion of
AChM alkylates the M1 muscarinic receptor at 0°C. CHO hM1
cells were incubated with four concentrations of the aziri-
dinium ion of AChM for 60 min, and then washed prior to
measuring specific [3H]NMS binding. The results in Figure 3A
show negligible alkylation of the M1 receptor by the aziri-
dinium ion of AChM at 0°C, indicating that it should be
possible to estimate its dissociation constant for the M1 recep-
tor in a competitive binding assay at 0°C. Figure 3B shows the
competitive effects of ACh, AChM and its transformation
products on the binding of [3H]NMS to intact CHO hM1 cells
at 0°C. Both the parent mustard and the alcoholic hydrolysis
product of AChM were without effect on the binding of
[3H]NMS, whereas ACh and the aziridinium ion of AChM
behaved as competitive inhibitors. Their IC50 values were cor-
rected for the competitive effect of [3H]NMS to yield log affin-
ity constants of 4.77 Ϯ 0.12 and 4.24 Ϯ 0.13 respectively.
Materials
Drugs and chemicals were obtained from the following
sources: [3H]NMS (PerkinElmer Life and Analytical Sciences,
Boston, MA, USA); F-12 media Kaighn’s modification, fetal
calf serum, trypsin–EDTA, G418 (Invitrogen, San Diego, CA,
USA); ACh, NMS, gallamine, scopolamine, Na2S2O3, WIN
51708 (Sigma-Aldrich, St Louis, MO, USA). AChM and McN-
A-343 were synthesized as described previously (Suga et al.,
2008) using modifications of procedures described by Jackson
and Hirst (1972) and Mellin et al. (1989) respectively.
N-methylamitriptyline was synthesized as described by Suga
et al. (2008), and BR384 was synthesized as described by
Ringdahl et al. (1990).
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M1 muscarinic receptors and McN-A-343
KW Figueroa et al
1539
also inhibited binding, although the data are insufficient to
resolve its reversible binding properties from its covalent
interaction with the receptor. The data were analysed none-
theless using a competitive model to yield an ‘apparent’ log
affinity constant of 4.93 Ϯ 0.14.
Kinetics of hM1 receptor alkylation
We incubated M1 receptors with different concentrations of
the aziridinium ions of the mustard analogs for various times,
and then used [3H]NMS to measure the residual muscarinic
receptor population after stopping the reaction and washing
the cells (Figure 4A,C). The aziridinium ions of both AChM
and BR384 caused a time- and concentration-dependent
decrease in specific [3H]NMS binding. The kinetic data for
AChM were fitted simultaneously to Equation (11) by nonlin-
ear regression analysis to estimate the affinity constant of the
aziridinium ion (K1) and the rate constant for alkylation (k1).
A similar analysis was done for BR384 using Equation (8),
which accounts for the decay in the aziridinium ion. These
fits yielded K1 and k1 parameter estimates for AChM of 4.62 Ϯ
0.07 and 0.10 Ϯ 0.01 min-1, and for BR384 of 5.14 Ϯ 0.03 and
0.55 Ϯ 0.03 min-1 respectively. The estimates of the percent-
age of receptors not alkylated by AChM and BR384 were 8.1
and 4.5% respectively.
We also estimated the observed rate constant for alkylation
(kobs) at each concentration of the mustards using an expo-
nential decay equation. Only the data obtained during the
first 8 min of incubation with BR384 were used to avoid error
in the estimate of kobs due to decay of the aziridinium ion. A
one-site equation was fitted to these estimates of kobs to yield
the log affinity constant (K1) and the maximal rate constant
for alkylation (k1) for the aziridinium ions of AChM (-4.08 Ϯ
0.24 and 0.14 Ϯ 0.015 min-1) and BR384 (-4.82 Ϯ 0.09 and
0.61 Ϯ 0.017 min-1) (Figure 4B,D). The latter values are similar
to those estimated by global nonlinear regression analysis of
the data according to Equations (8) and (11) as described
above. The log affinity constant of the aziridinium ion of
AChM, determined by the kinetic analysis at 37°C (4.62 Ϯ
0.01), is also approximately similar to that estimated from the
competitive binding assay at 0°C (4.24 Ϯ 0.10; see Figure 3).
Figure 3 Effect of AChM, BR384, their transformation products,
ACh and McN-A-343 on the binding of [3H]NMS to intact CHO hM1
cells at 0°C. (A) CHO hM1 cells were incubated with various concen-
trations of the aziridinium ions of AChM and BR384 for 1 h at 0°C,
washed and then assayed for [3H]NMS binding at 0°C. (B) The
competitive inhibition of the binding of [3H]NMS by various concen-
trations of ACh, AChM and its transformation products was measured
at 0°C. (C) The binding of [3H]NMS in the presence of various
concentrations of McN-A343, BR384 and its transformation products
was measured at 0°C. The data represent the mean binding values Ϯ
SEM of three to four experiments, each in triplicate. The concentra-
tion of [3H]NMS was 1.0 nM, and its log affinity constant was esti-
mated to be -9.53 in separate experiments at 0°C. The concentration
of aziridinium ion is expressed as the total concentration of the parent
mustard from which it was derived.
Effects of competitive inhibitors and allosteric modulators on
receptor alkylation by AChM and BR384
To determine the site of action of the mustard analogs on the
M1 receptor, we investigated how some known competitive
inhibitors and allosteric modulators affect the kinetics of
receptor alkylation by the mustard analogs. The rate of recep-
tor alkylation by the aziridinium ion of AChM at concentra-
tions of 0.1 mM (Figure 5A) and 1 mM (Figure 5B) was
investigated in intact CHO hM1 cells in the presence of dif-
ferent concentrations of NMS. NMS caused a concentration-
dependent slowing and ultimately stopped receptor
alkylation by AChM at both the low and high concentrations
of the mustard. Similar behaviour was observed in experi-
ments with the aziridinium ion of BR384 at concentrations of
0.01 mM (Figure 5C) and 0.1 mM (Figure 5D). The data with
both mustards are generally consistent with a competitive
mechanism. To test this postulate, the competitive model
In contrast, the aziridinium ion of BR384 caused a moderate
alkylation of the hM1 receptor after a 60 min incubation at
0°C as shown by the reduction in [3H]NMS binding measured
after washing the reactive ligand from CHO hM1 cells
(Figure 3A). We also examined McN-A-343, BR384 and its
transformation products in competitive binding assays with
[3H]NMS at 0°C (Figure 3C). Both the parent mustard and the
alcoholic hydrolysis product had little effect on [3H]NMS
binding, whereas McN-A-343 inhibited binding with a log
affinity constant of 4.35 Ϯ 0.14. The aziridinium ion of BR384
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KW Figueroa et al
Figure 4 The kinetics of alkylation of M1 receptors by AChM (A, B) and BR384 (C, D). Intact CHO hM1 cells were incubated with various
concentrations of cyclized AChM (A) or BR384 (C) at 37°C, washed and then assayed for [3H]NMS binding. The theoretical curves represent
the global least square fit of Equations (12) and (9) to the data for AChM and BR384 respectively. The observed rate constant for alkylation
of M1 receptors was estimated for each concentration of AChM and BR384 in (A) and (C), and plotted against the mustard concentration in
(B) and (D) respectively. A one-site model was fitted to each plot of the observed rate constants. The data represent the mean values Ϯ SEM
of two experiments, each in triplicate. The mean Ϯ SEM denotes the range of the two measurements, because N = 2. The concentration of
[3H]NMS was 1.0 nM. The concentration of cyclized nitrogen mustard is expressed as its initial concentration before transformation into the
aziridinium ion.
Figure 5 Effects of NMS on the kinetics of receptor alkylation by AChM (A, B) and BR384 (C, D). Intact CHO hM1 cells were incubated at 37°C
with cyclized AChM at concentrations of 0.1 mM (A) or 1.0 mM (B), and in the presence of the indicated concentrations of NMS. Similar
experiments were run with cyclized BR384 at concentrations of 0.01 mM (C) and 0.1 mM (D). Following incubation with the mustards, the
cells were washed and assayed of [3H]NMS binding at a concentration of 1.0 nM. The data represent the mean binding values Ϯ SEM of two
(A), four (B), five (C) and five (D) experiments, each in triplicate. The concentration of cyclized nitrogen mustard is expressed as its initial
concentration before transformation into the aziridinium ion.
[Equation (13)] was fitted to the data obtained at both con-
centrations of AChM by global nonlinear regression analysis
sharing the estimate of the affinity constant of NMS between
the data and setting the affinity constant of the aziridinium
ion of AChM at a constant value (log K1 = 4.62) equivalent to
that estimated from the data in Figure 3. A similar approach
was used in the analysis of the data with BR384, except that
Equation (10) was used to allow for the decay in the aziri-
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Figure 6 Effects of gallamine on the kinetics of receptor alkylation by AChM (A, B) and BR384 (C, D). Intact CHO hM1 cells were incubated
at 37°C with cyclized AChM at concentrations of 0.1 mM (A) or 1.0 mM (B), and in the presence of the indicated concentrations of gallamine.
Similar experiments were run with cyclized BR384 at concentrations of 0.01 mM (C) and 0.1 mM (D). Following incubation with the mustards,
the cells were washed and assayed of [3H]NMS binding at a concentration of 1.0 nM. The data represent the mean binding values Ϯ SEM of
three (A), four (B), four (C) and two (D) experiments, each in triplicate. The concentration of cyclized nitrogen mustard is expressed as its initial
concentration before transformation into the aziridinium ion.
dinium ion of BR384. The estimates of the affinity constants
of NMS in experiments with AChM and BR384 were 10.04 Ϯ
0.07 and 9.97 Ϯ 0.07 respectively. There was no significant
improvement in residual error when the allosteric models
[Equation (12) for AChM and Equation (9) for BR384] were
fitted to the data.
Analogous experiments were carried out with the allosteric
modulator gallamine in intact CHO hM1 cells (Figure 6).
When the concentration of the aziridinium ion of AChM was
low (0.1 mM), gallamine caused a concentration-dependent
slowing in the rate of receptor alkylation (Figure 6A). When
Figure 7 Effect of WIN 51708 on kinetics of receptor alkylation by
BR384. Intact CHO hM1 cells were incubated at 37°C with cyclized
BR384 at a concentration of 3.0 mM (initial concentration before
the concentration of the aziridinium ion of AChM was
increased to 1 mM, however, the inhibitory effect of gal-
lamine reached a plateau at high concentrations, consistent
with an allosteric mechanism (Figure 6B). There was a sub-
stantial improvement in residual error when the data were
fitted to the allosteric model [Equation (12)] as compared to
the competitive model [Equation (13)] (F1,204 = 107.5; P = 1.7 ¥
10-20). The estimate of the log affinity constant of gallamine
(K2) and its log cooperativity factor (a) for its interaction with
the aziridinium ion of AChM were 5.06 Ϯ 0.09 and -2.04 Ϯ
0.07 respectively. We also observed similar behaviour in
experiments with gallamine and BR384 at low (0.01 mM;
Figure 6C) and high (0.1 mM; Figure 6D) concentrations.
There was a highly significant improvement in residual error
when the data were fitted to the allosteric model with [Equa-
tion (6)] as compared to the analogous competitive model
[Equation (7)] (F1,110 = 135.4; P = 7.9 ¥ 10-21). The estimates of
the log affinity constant of gallamine for the unoccupied M1
receptor (K2) and the log of the cooperativity constant (a) for
its interaction with the aziridinium ion of BR384 were 5.02 Ϯ
0.08 and –1.73 Ϯ 0.07 respectively.
cyclization) and in the presence of the indicated concentrations of
WIN 51708. Following incubation with BR384, the cells were washed
and assayed of [3H]NMS binding at a concentration of 1.0 nM. The
data represent the mean binding values Ϯ SEM of two experiments,
each in triplicate. The mean Ϯ SEM denotes the range of the two
measurements, because N = 2.
of strychnine and gallamine (Lazareno et al., 2002), so we
investigated its potential effects on M1 receptor alkylation by
the aziridinium ion of BR384 in intact CHO hM1 cells
(Figure 7). BR384 was used at a low concentration of 3 mM to
optimize conditions for detecting a small allosteric effect. No
influence on receptor alkylation was observed with WIN
51708 when used at concentrations of 3 and 10 mM. At a
concentration (30 mM) approaching its limiting solubility,
WIN 51708 reduced the observed rate constant for alkylation
to 57% of the control value.
The effects of McN-A-343 on receptor alkylation by the
aziridinium ion of AChM at low (0.1 mM) and high (1.0 mM)
concentrations are shown in Figure 8A and B respectively.
These experiments used homogenates of CHO hM1 cells
because the rate of alkylation was faster in homogenates com-
pared to intact cells. This provided a greater range over which
The compound WIN 51708 is thought to bind to an allos-
teric site on the M1–M5 muscarinic receptors distinct from that
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Figure 8 Effects of McN-A-343 on the kinetics of receptor alkylation by AChM (A, B) and BR384 (C, D). Homogenates of CHO hM1 cells were
incubated at 37°C with cyclized AChM at concentrations of 0.1 mM (A) or 1.0 mM (B), and in the presence of the indicated concentrations
of McN-A-343. Similar experiments were run with cyclized BR384 at concentrations of 0.01 mM (C) and 0.1 mM (D). Following incubation
with the mustards, the cellular homogenates were washed and assayed of [3H]NMS binding at a concentration of 1.0 nM. The data represent
the mean binding values Ϯ SEM of two experiments, each in triplicate. The mean Ϯ SEM denotes the range of the two measurements, because
N = 2. The concentration of cyclized nitrogen mustard is expressed as its initial concentration before transformation into the aziridinium ion.
a slowing in the kinetics could be measured, which is useful
for discriminating a large negatively cooperative effect from
competitive inhibition. In homogenates, the average values
of the observed rate constants of AChM (1.0 mM) and BR384
(0.1 mM) were 0.75 and 1.02 min-1, respectively, whereas the
estimates of k1 in intact cells were 0.11 and 0.45 min-1 respec-
tively. We have previously suggested that this difference may
be related to recycling of receptors after the alkylation step
(Suga et al., 2008). McN-A-343 caused
a concentration-
dependent slowing in the rate of receptor alkylation by AChM
at both the high and low concentrations. Similar behaviour
was noted in experiments investigating the influence of McN-
A-343 on the alkylation of the M1 receptor by the aziridinium
ion of BR384 (Figure 8C,D). Using the competitive model
[Equation (13)], we estimated the affinity constant of McN-A-
343 to be 5.23 Ϯ 0.05 in experiments with AChM. The value
estimated in experiments with BR384 was 5.21 Ϯ 0.05 [using
Equation (10)]. There was no significant improvement in
residual error when the data were fitted to the allosteric
models [Equation (12) for AChM and Equation (9) for BR384]
as compared to the competitive models.
The influence of ACh on the rate of M1 receptor alkylation
by the aziridinium ion of BR384 is shown in Figure 9. These
experiments used homogenates of CHO hM1 cells using
BR384 at concentrations of 0.01 mM (Figure 9A) and 0.1 mM
(Figure 9B). The effect of ACh on receptor alkylation
resembled that of a competitive inhibitor, with no evidence of
a plateau in its inhibitory effect at high concentrations. The
competitive model [Equation (10)] was fitted to the data to
yield an estimate of the affinity constant of ACh (4.77 Ϯ
0.08). No significant improvement in residual error occurred
when the allosteric model [Equation (9)] was fitted to the
data.
Figure 9 Effects of ACh on the kinetics of receptor alkylation by
BR384. Homogenates of CHO hM1 cells were incubated at 37°C with
cyclized BR384 at concentrations of 0.01 mM (A) or 0.1 mM (B), and
in the presence of the indicated concentrations of ACh. Following
incubation, cellular homogenate was washed and assayed of
[3H]NMS binding at a concentration of 1.0 nM. The data represent
the mean binding values Ϯ SEM of two experiments, each in tripli-
cate. The mean Ϯ SEM denotes the range of the two measurements,
because N = 2. The concentration of cyclized nitrogen mustard is
expressed as its initial concentration before transformation into the
aziridinium ion.
We also investigated the influence of NMS (Figure 10A) and
gallamine (Figure 10B) on the rate of inactivation of muscar-
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KW Figueroa et al
1543
Figure 10 Effects of NMS (A) and gallamine (B) on the kinetics of
receptor alkylation by BR384 in rat cortex. Tissue homogenates were
incubated at 37°C with cyclized BR384 (0.1 mM, initial concentration
before cyclization to the aziridinium ion) and in the presence of the
indicated concentrations of NMS or gallamine. Following incubation
with BR384, brain homogenate was washed and assayed of [3H]NMS
binding at a concentration of 1.0 nM. The data represent the mean
binding values Ϯ SEM of two experiments, each in triplicate. The
theoretical curves represent a best fit of an exponential decay equa-
tion to the data.
inic receptors in homogenates of rat cerebral cortex by the
aziridinium ion of BR384 (0.1 mM). This brain region
expresses mainly M1 and M4 muscarinic receptors, but also M2,
M3 and M5 (Yasuda et al., 1993). NMS slowed the rate of
receptor alkylation in a manner generally consistent with
competition, whereas the inhibitory effect of gallamine
reached a clear plateau at 170 mM and did not increase with a
further increase in concentration. We did not analyse the data
in detail because of the heterogeneity of the receptor
population.
Figure 11 The effects of NMS, gallamine, McN-A-343 and ACh on
the observed time constant for alkylation (tobs) of the M1 muscarinic
receptor by AChM (A) and BR384 (B and C). The log ratio (R) of time
constant for alkylation in the presence of the inhibitor divided by that
measured in its absence is plotted against the log concentration of
the inhibitor. The theoretical curves represent the best fit of Equa-
tions (16) (gallamine) and 17 (NMS, ACh and McN-A-343) to the
data. The data obtained for a given test ligand at the two concen-
trations of AChM (0.1 and 1.0 mM) or BR384 (0.01 and 0.1 mM)
were fitted simultaneously. Open symbols represent data obtained
with 0.1 mM AChM or 0.01 mM BR384, whereas closed symbols
denote data obtained with 1.0 mM AChM or 0.1 mM BR384. The
concentration of cyclized nitrogen mustard is expressed as its initial
concentration before transformation into the aziridinium ion. The R
values were calculated from the data shown in Figures 5, 6, 8 and 9.
The estimates of the parameters Ϯ SEM in experiments with AChM
are: NMS, pKi = 9.96 Ϯ 0.12; gallamine, pK2 = 5.20 Ϯ 0.11, log a =
-2.08 Ϯ 0.10; McN-A-343, pKi = 5.85 Ϯ 0.05. The corresponding
estimates for the experiments with BR384 are: gallamine, pK2 = 5.10
Ϯ 0.17, log a = -1.77 Ϯ 0.14; McN-A-343, pKi = 5.21 Ϯ 0.06; ACh,
pKi = 4.58 Ϯ 0.04. With regard to NMS, the pK1 values at 0.01 and
0.1 mM BR384 were 9.47 Ϯ 0.08 and 9.86 Ϯ 0.13, respectively.
Effects of ACh, McN-A-343, NMS and gallamine on the time
constant for receptor alkylation by AChM and BR384
A more intuitive way to analyse the kinetic data in Figures 5,
6, 8 and 9 is to plot the ratio of the time constant for alkyla-
tion in the presence of the inhibitor divided by that measured
in its absence against the inhibitor concentration (Figure 11).
Figure 11A shows the data obtained for the aziridinium ion of
AChM at low (0.1 mM) and high (1.0 mM) concentrations.
Both NMS and McN-A-343 increased the time constant for
receptor alkylation in a manner that was proportional to their
concentration. This effect occurred with both concentrations
of AchM, and is indicative of a competitive mechanism.
Although gallamine caused
a
concentration-dependent
increase in the time constant at the low concentration of
AChM, its inhibitory effect clearly reached a limit at the
higher concentration of AChM. Similar behaviour is seen in
the corresponding plot of the data for the aziridinium ion of
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KW Figueroa et al
BR384. The competitive model was fitted to the data for NMS,
ACh and McN-A-343 (Figure 11B,C), whereas the allosteric
model was fitted to the data for gallamine (Figure 11B). The
results of this analysis were similar to those described above in
connection with Figures 5, 6, 8 and 9, and are described in the
legend to Figure 11. There was a tendency for the potency of
NMS to be greater when antagonizing receptor alkylation by
the higher concentration of BR384 (0.1 mM). As discussed
later, this phenomenon may be attributed to the slow disso-
ciation of NMS from the M1 receptor.
When plotting the relative time constant for alkylation
against the inhibitor concentration as shown in Figure 11, it
is difficult to discriminate between R values when the rate of
receptor alkylation is very slow. Consequently, in Figure 11,
we have omitted the R value for AChM (0.1 mM) in the
presence of 10 mM McN-A343 as calculated from the experi-
ments shown in Figure 8A. The rate of alkylation in the pres-
ence of 1 and 10 mM McN-A-343 appears similar, which
might suggest allosteric behaviour. Nonetheless, it is possible
to resolve this issue by repeating these measurements using a
higher concentration of AChM (1 mM) as shown in Figure 8B.
Under this conditions, a clear distinction between the rate of
alkylation in the presence of 1 and 10 mM McN-A-343 is
possible, which illustrates a consistency with a competitive
interaction as shown in Figure 11. In Figure 11, we also
omitted the R value measured at 1 mM NMS in the presence of
0.1 mM BR384 as calculated from the experiments shown in
Figure 5D. In this case, the rates of receptor alkylation by
BR384 in the presence of 0.1 and 1.0 mM NMS are both
approximately zero. This situation represents a limitation in
our ability to resolve competitive inhibition from high nega-
tive cooperativity because it is difficult to use much higher
concentrations of the aziridinium ion of BR384.
Because M1 receptor alkylation by the aziridinium ions of
AChM or BR384 was prevented by NMS, ACh and McN-A-343
in a manner proportional to their concentration (Figures 5, 8
and 9), it seems likely that the nitrogen mustards interact
competitively with the former ligands at the orthosteric
binding site. In contrast, increasing concentrations of the
allosteric ligand, gallamine, did not cause a proportional
increase in the protection of the M1 receptor from alkylation
by high concentrations of the nitrogen mustards (Figure 6).
This behaviour is consistent with an allosteric interaction
with the M1 receptor. Our estimate of the log affinity constant
of gallamine for the allosteric site on the M1 receptor, based
on its ability to interfere with the kinetics of alkylation (5.02),
agreed with that measured in equilibrium binding experi-
ments with [3H]NMS (5.21) (Matsui et al., 1995).
Our estimate of the negative cooperativity between the
binding of gallamine and the aziridinium ions of the mustard
analogs is based on the assumption that the microscopic rate
constants for alkylation of the free and the gallamine-
occupied receptors are the same (see Scheme 1). The estimate
of the cooperativity between AChM and gallamine (log a =
-2.01) is approximately the same as that measured between
gallamine and ACh in binding experiments on rat cerebral
cortex (-1.82) (Stockton et al., 1983), which is rich in M1
muscarinic receptors (Yasuda et al., 1993). This agreement
suggests that gallamine has little or no effect on the rate
constant for alkylation of M1 receptors by AChM. It seems
likely that the same is true for BR384 because there is no
evidence that gallamine alters the equilibrium between
ground and active states of the receptor. It often affects ligand
affinity only, perhaps through allosteric modulation of a
peripheral docking site (Ehlert and Griffin, 2008).
Although our experiments showed that the interaction
between BR384 and ACh was consistent with competitive
inhibition, we cannot rule out the possibility that there is
very high negative cooperativity between the two. The
minimum (log) estimate of this negative cooperativity was
-3.33 (a = 0.00046), which is defined as that required to cause
a significant increase in residual error, assuming an allosteric
mechanism. For the interaction between McN-A-343 and
AChM, the minimum logarithm of the estimate was approxi-
mately -3.01 (a = 0.001). Similarly, we cannot rule out the
possibility that there is very high negative cooperativity
between the binding of NMS and BR384.
The dissociation constant of WIN 51708 for its allosteric
site on the M1 receptor has been estimated to be 1.6 mM
(Lazareno et al., 2002). If BR384 were binding to the allosteric
site for WIN 51708, then a significant inhibition in the rate of
receptor alkylation should have been detected with WIN
51708 at concentrations of 3 and 10 mM. In addition, if WIN
51708 exhibits a significant cooperative interaction, then this
effect should have been observed at the low concentration of
BR384 used (3 mM). Our data show that BR384 does not alky-
late the allosteric site for WIN 51708 on the M1 receptor
(Figure 7).
Discussion
Both AChM and BR384 bind irreversibly to the M1 muscarinic
receptor because prior treatment with the aziridinium ions of
the mustards followed by washing caused a decrease in the
binding capacity of [3H]NMS with no change in affinity
(Figure 2). The mechanism presumably involves the direct
covalent binding of the aziridinium ion to the orthosteric
binding site. Spalding et al. (1994) have shown that
[3H]AChM labels aspartic acid 105 in the M1 muscarinic recep-
tor, which presumably explains the actions of AChM
described here.
Because ACh and McN-A-343 equilibrate rapidly with mus-
carinic receptors, it is likely that the aziridinium ions of
AChM and BR384 do as well, and that their rate of alkylation
of muscarinic receptors should be proportional to receptor
occupancy. This appears to be the case because the affinity
constant of the aziridinium ion of AChM estimated in a
competitive binding assay with [3H]NMS (Figure 3) generally
agreed with that measured from the kinetics of receptor alky-
lation (Figure 4), although the two assays were at different
temperatures (0 and 37°C respectively). In addition, there was
general agreement between the affinities of various ligands
measured in equilibrium binding assays with [3H]NMS and
those estimated from their inhibition of the kinetics of the
alkylation by AChM or BR384.
We cannot rule out the possibility that BR384 binds
covalently to a third allosteric site, distinct from that for
gallamine and WIN 51708, and exhibits very high negative
cooperativity with ACh and [3H]NMS. Alternatively, it might
be possible that BR384 alkylates the allosteric site for gal-
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KW Figueroa et al
1545
(i.e. KB = 0.1), then k1-obs ª 0.1k1. The relatively high rate
constant of BR384 compared to AChM (see below) argues
against Scheme 2. A ligand with a higher KB value would
alkylate the orthosteric site with a faster rate, yet, by defini-
tion, such a ligand would be more orthosteric.
The second argument against Scheme 2 is that, under con-
ditions where the aziridinium ion of BR384 does not bind
covalently (i.e. low temperature), its maximal inhibition of
orthosteric ligand binding would be incomplete and only
reach a non-zero plateau of a/(1 + KB) times control binding
when the radioligand concentration is low [see Appendix,
Equation (A18)]. Both McN-A-343 and BR384 are capable of
fully displacing [3H]NMS binding from the M1 receptor,
however, as shown in Figure 3. If the negative cooperativity is
very great (i.e. a << 1), however, then near complete displace-
ment of binding could occur. This seems unlikely because the
requisite negative cooperativity would need to be very high
(–log a > 3).
Scheme 2 Model for the initial reversible interaction of the aziri-
dinium ion (X) with an allosteric site on the M1 receptor followed by
its subsequent irreversible binding to the orthosteric site in the pres-
ence of a competitive orthosteric ligand (I). The aziridinium ion first
equilibrates with the receptor in the form of reversible complexes (XR
and RX) as determined by the unimolecular constant KB. The RX
complex converts to a covalent complex (R-X) as determined by the
rate constant k1. The orthosteric ligand (I) only interacts reversibly
with the orthosteric site. KX and KI denote the affinity constants of the
reversible complexes of RX and IR, and a denotes the cooperativity
between the binding of X and I.
lamine at low concentrations, but then at high concentra-
tions, alkylates the orthosteric site. The converse situation is
also possible. Investigating how the alkylation of the receptor
is affected by mutagenesis of nucleophilic residues in the
binding pocket (e.g. D105) should settle these questions. The
simplest model to explain the data so far is a competitive
interaction between BR384 and orthosteric ligands.
A third reason against Scheme 2 is that, according to this
model, allosteric ligands should competitively inhibit the
covalent binding of BR384 to R2, yet gallamine and WIN
51708 do not (Figures 6 and 7).
Finally, the consequences of Scheme 2 predict that a large
increase in the concentration of BR384 should be unable to
overcome the inhibitory effect of orthosteric ligands on recep-
tor alkylation [see Appendix, Equation (A12)], yet our data
with ACh show a reciprocal competitive interaction (i.e. an
increase in the concentration of one ligand can overcome the
effect of an increase in the concentration of the other). In the
case of NMS, it exhibited moderately greater potency at inhib-
iting receptor alkylation at the higher concentration of
BR384, suggesting some deviation from a reciprocal competi-
tive interaction. In these experiments, NMS was allowed to
incubate with the receptor to reach equilibrium first, and then
the aziridinium ion of BR384 was added to start the reaction
at time zero. After this starting condition, it seems unlikely
that the reversible binding interaction between NMS and the
aziridinium ion is at equilibrium because NMS dissociates
slowly from the M1 receptor. As a result, the estimate of the
affinity constant of NMS is overestimated, particularly when
it is used at high concentrations (Suga et al., 2008). The data
with NMS illustrate a limitation in our analysis, because the
latter is based on the assumption that the reversible ligand
interactions equilibrate quickly compared to the rate of recep-
tor alkylation. We expect that high-affinity ligands (i.e. log K1
> 8.0) would not satisfy this criterion.
The rate constant for alkylation of the M1 receptor by the
aziridinium ion of AChM in intact cells was only one-fifth
that of BR384, but approximately equal to that of a nitrogen
mustard analog of oxotremorine-M (BM123) for alkylating
muscarinic receptors in rat cortex (Ehlert and Jenden, 1985).
The aziridinium ion of the latter compound alkylates
superhigh- and high-affinity agonist binding sites in rat cere-
bral cortical homogenate at much slower rates (0.014 and
0.022 min-1) compared to that of the more abundant low-
affinity agonist site (0.26 min-1). Because a greater proportion
of superhigh- and high-affinity agonist–receptor complexes is
likely to be in the active state, it has been suggested that
BM123 alkylates the ground state of the receptor at a faster
rate than the active state (Ehlert and Jenden, 1985). Spalding
It is often assumed that, because of high reactivity, an
electrophilic ligand is capable of alkylating a nucleophilic
residue residing beyond its binding pocket. Thus, it might be
argued that BR384 binds reversibly to an allosteric site, but
reacts covalently with the nearby nucleophilic D105 at the
orthosteric site of the M1 receptor. This hypothesis could
explain why orthosteric ligands, like ACh and NMS, protect
the M1 receptor from alkylation by BR384, even if the latter
bound reversibly to an allosteric site. This mechanism is
described in Scheme 2 where X denotes the aziridinium ion of
BR384, RX the reversible complex of X with the allosteric site,
XR the reversible complex of X with the orthosteric site, X-R
the covalent complex of X with the orthosteric site, KA the
affinity constant of X for the allosteric site and KB the unimo-
lecular constant describing the equilibration of X between the
two sites. Also shown is a competitive inhibitor (I) for the
orthosteric site having an affinity constant denoted by KI. It
can be shown that X binds to this circuit of two sites in a
manner consistent with a simple one-site model having an
observed dissociation constant (Kobs) equal to 1/(KX + KXKB)
[see Appendix, Equation (A4)]. According to this model, the
rate of alkylation of the orthosteric site would be proportional
to receptor occupancy of the two equilibrating sites, and the
half-maximal rate would occur when the concentration of
BR384 was equivalent to Kobs. In addition, at high concentra-
tions, an orthosteric ligand could completely inhibit the
alkylation.
There are four reasons, however, why this model cannot
explain our data. First, the maximal rate constant for alkyla-
tion of the orthosteric site would be less than if it alkylated
the site directly. For example, if BR384 alkylated the orthos-
teric site with a rate constant of k1, its observed rate constant
(k1-obs) would be equivalent to [1/(1 + 1/KB)]k1 [see Appendix,
Equation (A9)]. Thus, if BR384 spent 90% of its time on the
allosteric site and only 10% of its time on the orthosteric site
British Journal of Pharmacology (2010) 160 1534–1549

M1 muscarinic receptors and McN-A-343
1546
KW Figueroa et al
et al. (1994) have also given a similar explanation for the
slower rate of aklylation of the M1 receptor by AChM com-
pared to benzilylcholine mustard. A readily alkylated ground
state may account, at least in part, for the larger rate constant
of BR384 compared to that of AchM, because McN-A-343 is
less efficacious than ACh, and the same relation probably
applies to the aziridinium ions of BR384 and AChM.
Structural features of the aziridinium ion unrelated to effi-
cacy are also capable of influencing the alkylation rate. For
example, the rate constant for alkylation of cortical muscar-
inic receptors by 4-DAMP mustard (Thomas et al., 1992) is
approximately one-fourth that of benzilylcholine mustard.
Presumably, the piperidine ring of 4-DAMP mustard hinders
the alkylation reaction as compared to the more flexible ali-
phatic ethylamine moiety of benzilylcholine mustard.
measure radioligand binding at a high-enough concentration
to discriminate a competitive inhibitor from negative allos-
teric modulator. Our approach does not suffer from this con-
straint because the primary interaction is between the
electrophile and the test drug, both of which can be used over
a wide range of concentrations. Evidence of their interactive
behaviour is preserved after washing the receptor preparation
because the electrophile binds covalently. The amount of
unalkylated receptors can be determined easily using a low,
feasible concentration of radioligand. As described herein,
allosteric and competitive inhibitors differ in how they
protect radioligand-binding sites from alkylation by a site-
directed electrophile.
Kinetics are often viewed as a solution to the problem of
discriminating between competitive inhibition and high
negative cooperativity because, in the simplest case, a com-
petitive inhibitor should have no effect on kinetics, whereas a
negative allosteric modulator should increase dissociation
kinetics. But at muscarinic receptors, mere occupancy of the
allosteric site can prevent ligand traffic to and from the
orthosteric site. This means that a major component of
the effect of an allosteric modulator on the kinetics of radio-
ligand binding may be unrelated to the microscopic rate con-
stants for states of the orthosteric binding pocket, but rather,
to a simple blockade of ligand traffic (Proska and Tucek,
1994). There is also a true allosteric component related to the
change in the conformation of the receptor. Nonetheless, the
capping effect of allosteric modulators on kinetics can be the
predominant effect, and ligands that occupy the allosteric site
without causing a change in the conformation of the orthos-
teric site can have large effects on kinetics without allosteri-
cally modulating the orthosteric site. While knowledge of
whether a drug can occupy the allosteric site is useful, one can
never determine from this type of kinetic experiment to
which site the putative modulator binds with higher affinity
– orthosteric or allosteric (Birdsall and Lazareno, 2005).
Although our results illustrate clear differences between
allosteric and competitive inhibitors, the site-directed electro-
philes that we used (AChM and BR384) are less than ideal
because of their moderately low affinity (log K1 = 4.5–5.0).
This places an upper limit of about 100-fold on the highest
concentration (1 mM) of these agents that can be used rela-
tive to their dissociation constants. This working concentra-
tion range establishes a limit for the maximum amount of
negative cooperativity that could be distinguished from com-
petitive inhibition using our approach. Highly potent alkylat-
ing agents, like benzilylcholine mustard, are undesirable
because of their bulky size and slow equilibration. For
example, gallamine prevents the access and egress of [3H]NMS
from the orthosteric-binding pocket of the M2 receptor, and
greatly slows down its binding kinetics (Stockton et al., 1983).
It is expected to do the same for benzilylcholine mustard. An
ideal agent would be one of small size and moderate affinity
(e.g. log K1 = 7.0). These properties would ensure rapid equili-
bration and reduce the possibility that bulky allosteric modu-
lators would interfere with trafficking of the electrophile to
and from the receptor. Various oxotremorine analogs display
a broad range of potency and efficacy at muscarinic receptors,
including antagonists with moderate affinity (log K1 = 7.0)
that differ from oxotremorine only by the addition of a single
In addition, the enantiomers of
analog of N-methyloxotremorine
a
nitrogen mustard
(BM130, N-[4-(2-
chloromethylpyrrolidine)-2-butynyl]-2-pyrrolidone) having
an asymmetric carbon atom in the quaternary aziridinum
ring exhibit similar agonist activity on the guinea pig ileum
and similar affinity for rat cortical muscarinic receptors, but
the (+) enantiomer alkylates muscarinic receptors at a fourfold
faster rate (Ringdahl et al., 1989).
We found that the rate constant for alkylation of the M1
receptor by AChM was faster in cellular homogenates than in
intact cells. We observed similar behaviour at the M2 receptor
and have suggested that the mechanism might involve recy-
cling of internalized receptors after treatment with AChM
(Suga et al., 2008). While this complication causes an under-
estimation in the rate constant for alkylation, it does not
appear to alter the pattern of receptor protection causes by
competitive or allosteric ligands.
The structural similarity of McN-A-343 with the aziridinium
ion of BR384 (Figure 1) strongly suggests that the two ligands
interact at the same site on the M1 muscarinic receptor. Our
estimates of the log affinity constants of the two ligands were
approximately the same (about 5.0), but varied somewhat
across different experimental paradigms.
Our experiments with BR384 on rat cerebral cortex (Fig-
ure 10) are consistent with the behaviour that we observed at
the human M1 receptor. The rat cortex is known to express M1
and M4 receptors in substantial abundance (35 and 40%,
respectively) in addition to a moderate amount of the M2
receptor (15%) and less of the M3 and M5 subtypes (Yasuda
et al., 1993). Our results suggest an orthosteric interaction
with at least a majority of the sites in cortex. Studying recom-
binant muscarinic receptors in isolation should yield a more
unambiguous conclusion. It has previously been shown that
prior treatment of the rat cerebral cortex with the aziridinium
ion of BR384 causes an irreversible decrease in the binding
capacity of [3H]NMS without affecting its binding affinity
(Ringdahl et al., 1990).
Measurement of the ability of a test drug to affect the
irreversible binding of a site-directed electrophile is a power-
ful means of determining whether the drug interacts at allos-
teric or orthosteric sites on the receptor (Ehlert and Jenden,
1985; Suga et al., 2008). In the conventional binding experi-
ment, the interaction of a drug with a radioligand having a
defined binding site is usually studied. But, in cases where
there is high negative cooperativity, it may not be feasible to
British Journal of Pharmacology (2010) 160 1534–1549

M1 muscarinic receptors and McN-A-343
KW Figueroa et al
1547
Lazareno S, Popham A, Birdsall NJ (2002). Analogs of WIN 62 577
define a second allosteric site on muscarinic receptors. Mol Pharma-
col 62: 1492–1505.
McKinney M, Miller JH, Gibson VA, Nickelson L, Aksoy S (1991).
Interactions of agonists with M2 and M4 muscarinic receptor sub-
types mediating cyclic AMP inhibition. Mol Pharmacol 40: 1014–
1022.
Martin JR (1996). Pressor effect of the putative M1 muscarinic
receptor agonist MCN-A-343 in the conscious rat. Life Sci 59: 1839–
1852.
Matsui H, Lazareno S, Birdsall NJ (1995). Probing of the location of the
allosteric site on M1 muscarinic receptors by site-directed mutagen-
esis. Mol Pharmacol 47: 88–98.
methyl group (Ringdahl and Jenden, 1983; Ringdahl, 1988).
Nitrogen mustard derivatives of these compounds may have
utility in studying allosterism using the approach described
here. The working concentration range of an alkylating agent
having a dissociation constant of 0.1 mM could easily be
10 000-fold. This large range would greatly extend the
maximum detectable amount of negative cooperativity
between it and an interacting ligand. When used in combi-
nation with site-directed mutagenesis, our method has con-
siderable power in discriminating competitive inhibition
from negative allosteric modulation.
May LT, Avlani VA, Langmead CJ, Herdon HJ, Wood MD, Sexton PM
et al. (2007). Structure–function studies of allosteric agonism
at M2 muscarinic acetylcholine receptors. Mol Pharmacol 72: 463–
476.
Mellin C, Vargas HM, Ringdahl B (1989). Dimethylsulfonium ana-
logues of the muscarinic agent McN-A-343: [4-[[N-(3- or
4-halophenyl)carbamoyl]oxy]-2-butynyl] dimethylsulfonium per-
chlorates. J Med Chem 32: 1590–1593.
Acknowledgements
Initial feasibility studies were carried out with Don Jenden
and the late Bjorn Ringdahl at UCLA under the support of the
National Institutes of Health grant MH 17691. The work on
rat cerebral cortex described here was supported by the
National Institutes of Health grant NS 26511, and the remain-
der of the work was supported by the National Institutes of
Health Grant GM 69829 and a Predoctoral Fellowship from
the PhRMA Foundation (K.W.F.).
Proska J, Tucek S (1994). Mechanisms of steric and cooperative actions
of alcuronium on cardiac muscarinic acetylcholine receptors. Mol
Pharmacol 45: 709–717.
Ringdahl B (1988). 5-Methyl-2-pyrrolidone analogues of oxotremo-
rine as selective muscarinic agonists. J Med Chem 31: 683–688.
Ringdahl B, Jenden DJ (1983). Pharmacological properties of
oxotremorine and its analogs. Life Sci 32: 2401–2413.
Ringdahl B, Katz ED, Roch M, Jenden DJ (1989). Muscarinic
actions and receptor binding of the enantiomers of BM 130, an
alkylating analog of oxotremorine. J Pharmacol Exp Ther 249: 210–
215.
Ringdahl B, Mellin C, Ehlert FJ, Roch M, Rice KM, Jenden DJ (1990).
Tertiary 2-haloethylamine derivatives of the muscarinic agent McN-
A-343, [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethyl
ammonium chloride. J Med Chem 33: 281–286.
Conflicts of interest
F.J.E. and H.S. have no conflicts of interest to declare. After
completing this work, one of us (K.W.F.) took up a position
with Johnson & Johnson.
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Appendix
[
XR
RT
]
[ ]
X
=
1+ XKX
KXKB
1+ XαKX
1+ XKX
(A6)
[
X
]
+
1+ IKI
It is often suggested that because of its high reactivity, a
nitrogen mustard can bind reversibly with its primary target,
but then reacts covalently with a different nucleophilic
target nearby. We have adapted this idea to consider how
BR384 (X) would alkylate the M1 receptor if it bound revers-
ibly with an allosteric site, but then alkylated the nearby
orthosteric site coordinated by D105 in the M1 sequence (see
Scheme 2). In this model, the affinity constant of BR384 for
the allosteric site is denoted by KX. We assume that BR384
rapidly equilibrates with both sites in the form of reversible
receptor complexes (i.e. XR and RX, orthosteric and allos-
teric, respectively) as described by the unimolecular con-
stant, KB (KB = [RX]/[XR]). The rate constant for alkylation of
the orthosteric site is denoted by k1, and the affinity of the
competitive inhibitor (I) for the orthosteric site is denoted
by KI. The cooperativity between the binding of I and X is
denoted by a. The covalent complex of the aziridinium ion
of BR384 with the orthosteric site is denoted by X-R. The
affinity constants are defined in the conventional manner as
the product of the products divided by that of the reactants
[e.g. KX = [RX]/([X][R])]. Fractional receptor occupancy of
this circuit of two sites by the aziridinium ion of BR384 in
the form of reversible complexes (XR and RX) and in the
presence of I is given by:
(
)
The observed rate constant for alkylation of the orthosteric
site (kobs) in the presence of I is equivalent to the product of k1
and the fractional occupancy of the orthosteric site by the
aziridinium ion of BR384, which is derived by multiplying
Equation (A6) by k1:
[ ]
X
kobs
=
k1
1+ XKX
KXKB
1+ XαKX
1+ XKX
(A7)
[
X
]
+
1+ IKI
(
)
In the absence of I, Equation (A7) reduces to:
[ ]
X
1+ XKX
KXKB
kobs
=
k1
(A8)
[ ]
X +
The maximal observed rate constant (kobs-max) for receptor alky-
lation in the absence of I can be determined by taking the
limit of Equation (A8) as X approaches infinity:
1
kobs-max
=
k1
1
KB
(A9)
1+
If KB is very small, and the aziridinium ion of BR384 spends
most of its time on the allosteric site, then the maximal rate
constant is approximately equal to:
[
XR
]
+
RT
[
RX
]
[
XR
]
[
+
IR
[
RX
]
=
(A1)
[
XR
]
+
[
RX
]
+
]
+
[
] [ ]
IRX + R
kobs-max ≈ KBk1
(A10)
in which RT denotes the total amount of receptors. Each
receptor complex in Equation (A1) can be replaced with an
expression in terms of affinity constants and ligand concen-
trations. For example, RX = XKX[R]. Making analogous substi-
tutions for the other receptor complexes and factoring out the
term R yield:
In contrast, if KB is large, then the observed rate constant for
alkylation is approximately equal to:
kobs-max ≈ k1
(A11)
In this case, however, it is inappropriate to refer to BR384 as
an allosteric ligand because it primarily binds to the orthos-
teric site and uses the allosteric site as a relay site for access to
the orthosteric site.
In the presence of I, the maximal observed rate constant for
alkylation (kobs-max′) can be derived by taking the limit of Equa-
tion (A7) as X approaches infinity:
[
XR
]
+
RT
[
RX
]
[ ]
X
=
1
(A2)
X
]
+
[
1
+ IKI + X KX
)
]
(
1
α
[
KX + KXKB
In the absence of I, Equation (A2) reduces to:
XR RX
[
]
+
[
]
[ ]
X
1
IαKI
KB
=
(A3)
′
kobs-max
=
k1
RT
[ ]
X + Kobs
(A12)
1+
in which Kobs denotes the observed dissociation constant:
Equation (A12) shows that as the concentration of I increases
to very high levels, the maximal observed rate constant for
alkylation is approximately equal to 0. Thus, at high concen-
trations, an orthosteric ligand can completely prevent the
alkylation of the orthosteric site by X in a manner that is
insurmountable by an increasing concentration of X.
It is possible to use the model in Scheme 2 to predict how a
reversible ligand, like McN-A-343 (D), inhibits the binding of
a radioligand (L) to the orthosteric site. It is assumed that D
binds reversibly in the same manner as the aziridinium ion of
BR384, but does not alkylate the orthosteric site (k1 = 0).
Fractional receptor occupancy (y) of orthosteric radioligand L
in the presence of D is given by:
1
Kobs
=
(A4)
KX + KXKB
Fractional occupancy of the orthosteric site by the aziri-
dinium ion of BR384 in the form of a reversible complex (XR)
and in the presence of the orthosteric competitive inhibitor
(I) is given by:
[
XR
RT
]
[
+
XR
IR
]
]
=
(A5)
[
XR
]
+
[
RX
]
[
[ ] [ ]
+ IRX + R
Replacing each receptor complex with an expression in terms
of ligands and affinity constants yields:
British Journal of Pharmacology (2010) 160 1534–1549

M1 muscarinic receptors and McN-A-343
KW Figueroa et al
1549
effective concentration of D can be predicted by taking the
limit of Equation (A17) as D approaches infinity:
[
LR
]
+
[
LRD
]
[
y =
(A13)
[
LR
]
+
[
LRD
]
+
[
RD
]
+
DR
]
+
[
R
]
α
Replacing each receptor complex in Equation (A13) with an
expression in terms of ligands and affinity constants, and
then simplifying yields:
F =
(A18)
1+ KB
The maximal inhibition of radioligand binding by D (Imax
)
when L is very low can be estimated by subtracting Equa-
tion (A13) from 1:
L
y =
1
KL
DK + DK K +1
(A14)
⎛
⎞
D
D
B
L +
⎜
⎝
⎟
⎠
KL + DαKDKL
α
Imax = 1−
(A19)
1+ KB
In this equation, KD and KL are used to denote the affinity
constants of D and L in place of the corresponding values for
X and I (KA and KI) used in Scheme 2. The fractional binding
of the radioligand at a fixed concentration (F) in the presence
of various concentrations of D can be described as the ratio of
the right side of Equation (A14) divided by the same expres-
sion with the variable D = 0:
The concentration of D causing half of its maximal inhibition
of the binding of L (IC50) can be estimated by subtracting
one-half of Imax from 1, and setting the result equal to the
right-hand side of Equation (A17), with D replaced with the
variable IC50:
1
2
α
1+ IC50αKD
IC50KD + IC50KDKB +1
⎛
⎝
⎞
⎠
1−
1−
=
(A20)
L
1+ KB
1
KL
DKD + DKDKB +1
L +
Solving for IC50 yields:
IC50
(
)
1+ DαKD
L
F =
(A15)
1
=
(A21)
1
KL
KD + KDKB
L +
Because this equation was derived under the condition of a
very low concentration of L, the IC50 in Equation (A21) rep-
resents the observed dissociation constant of D for the recep-
tor system illustrated in Scheme 2. It is analogous to that
described above in Equation (A4) for the reversible binding of
the aziridinium ion.
Equation (A16) can also be used to determine the effect of
an increase in the radioligand concentration on the ability of
D to inhibit radioligand binding. It can be shown that as the
concentration of L increases, the maximal inhibition of radio-
ligand binding decreases. At sufficiently high concentrations
of L, D has no effect on radioligand binding. This behaviour is
inconsistent with the influence of McN-A-343 on [3H]NMS
binding (see Figure 4).
This equation reduces to:
LKL +1
DKD + DKDKB +1
F =
(A16)
LKL +
1+ DαKD
By taking the limit of Equation (A16) as L approaches 0, it is
possible to predict how various concentrations of D inhibit
the binding of radioligand L when the latter is at a very low
fixed concentration:
1+ DαKD
F =
(A17)
DKD + DKDKB +1
Under this condition (i.e. L approaches 0), the fractional
binding of the radioligand in the presence of a maximally
British Journal of Pharmacology (2010) 160 1534–1549