Elevation of ligand binding to muscarinic M2 acetylcholine receptors by bis(ammonio)alkane-type allosteric modulators

Article abstract of DOI:10.1021/jm020871e

Bis(ammonio)alkane-type compounds are archetypal muscarinic allosteric modulators. Phthalimido-substituted hexane-bis-ammonium agents were methylated in the phthalimide moieties and the lateral propyl side chains. All compounds retarded allosterically the dissociation of the orthosteric ligand [³H]N-methylscopolamine ([³H]NMS) from porcine heart M₂ receptors. [³H]NMS equilibrium binding was reduced, left unaltered, or elevated, depending on the degree and position of methylation. This is the first time that an allosteric elevation of ligand binding is demonstrated for bis(ammonio)alkane-type compounds.

Full text of DOI:10.1021/jm020871e

J . Med. Chem. 2002, 45, 3809-3812
3809
Eleva tion of Liga n d Bin d in g to Mu sca r in ic M2 Acetylch olin e Recep tor s by
Bis(a m m on io)a lk a n e-Typ e Alloster ic Mod u la tor s
Alexandra Raasch,† Olaf Scharfenstein,‡ Christian Tra¨nkle,† Ulrike Holzgrabe,‡ and Klaus Mohr*,†
Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany, and
Pharmaceutical Chemistry, Institute of Pharmacy, University of Wu¨rzburg, Am Hubland, 97074 Wu¨rzburg, Germany
Received March 7, 2002
Bis(ammonio)alkane-type compounds are archetypal muscarinic allosteric modulators. Phthal-
imido-substituted hexane-bis-ammonium agents were methylated in the phthalimide moieties
and the lateral propyl side chains. All compounds retarded allosterically the dissociation of
the orthosteric ligand [3H]N-methylscopolamine ([3H]NMS) from porcine heart M2 receptors.
[3H]NMS equilibrium binding was reduced, left unaltered, or elevated, depending on the degree
and position of methylation. This is the first time that an allosteric elevation of ligand binding
is demonstrated for bis(ammonio)alkane-type compounds.
In tr od u ction
nonsymmetrical compounds, the phthalimides 4, 6, and
7 first have to be alkylated by using an excess of
dibromohexane without any reaction solvent3 to achieve
8, 9, and 10, respectively. Alkylation of 6 with 8, 5 with
9, 4 with 10, and 5 with 10 by refluxing in acetonitrile
gave 1b, 2c, 2b, and 3b, respectively (see Scheme 1).
All bis(ammonio)alkane-type compounds 1-3 were ob-
tained in satisfactory yields. The identities were estab-
With W84 (1a ) as a lead compound, we have previ-
ously developed a number of bis(ammonio)alkane-type
allosteric agents with increased affinity for the allosteric
site in N-methylscopolamine (NMS) occupied and in free
muscarinic M2 acetylcholine receptors.1-4 The meth-
ylphthalimido derivative dimethyl-W84 (3a ) has, under
conditions of low ionic strength and in the absence of
divalent cations, a top position among allosteric agents
with respect to the affinity for NMS-occupied receptors
and was introduced as the first (and still only) radio-
ligand for the common allosteric site.1 An allosteric
elevation of NMS binding is reported for some com-
pounds such as the common site allosteric agent alcu-
ronium.5 Yet, with regard to the bis(ammonio)alkane-
type agents, even 3a maintained a weak inhibitive effect
on NMS binding despite its high affinity for the allo-
steric site of NMS-occupied M2 receptors.1 Here, how-
ever, we report that an introduction of methyl groups
into the propyl side chain of 1a and its derivatives may
lead to allosteric agents that elevate orthosteric ligand
binding in muscarinic M2 receptors. These findings
demonstrate that bis(ammonio)alkane-type agents may
serve as lead structures for the development of allosteric
enhancers of ligand binding to muscarinic receptors.
1
lished by one- and two-dimensional H NMR (Support-
ing Information) and 13C NMR spectroscopical experi-
ments. The phthalimido moiety of the compounds tends
to hydrolyze to give amido derivatives. However, the
stability of all compounds was found to be high (t1/2
>
36 h). The log P value characterizes the compounds to
be slightly hydrophilic. Lipophilicity does not correlate
with the allosteric potency of the compounds.
P h a r m a cology
The allosteric interaction of the test compounds with
muscarinic M2 receptors was studied as described
previously6 in homogenates of porcine heart ventricles
with [3H]N-methylscopolamine as the ligand for the
orthosteric receptor site. The concentration of the test
compounds that inhibits the dissociation of [3H]NMS
half-maximally, EC50,diss, can be taken as a measure of
the equilibrium dissociation constant of allosteric modu-
lator binding to [3H]NMS-occupied receptors.1 Experi-
ments to measure the effect of the allosteric test
compounds on the equilibrium binding of [3H]NMS
yielded the cooperativity factor R and pKA, i.e., the
negative log value of the equilibrium dissociation con-
stant KA of allosteric agent binding to ligand-free
receptors. If the allosteric agent enhances [3H]NMS
binding, there is a positive cooperativity between the
allosteric and the orthosteric compound, and according
to Ehlert,7 the cooperativity factor amounts to R < 1.
In the case of negative cooperativity, [3H]NMS binding
is lowered by the allosteric agent and R > 1. Neutral
cooperativity means that [3H]NMS binding is un-
changed despite allosteric agent binding to the recep-
tors, R ) 1.
Syn th esis
Because of the availability of 3-(N,N-dimethylamino)-
propylphthalimide and the corresponding methylphthal-
imides 4 and 5, 1a and 3a could be obtained by
conversion of two molecules of the phthalimide deriva-
tives with dibromohexane.1 To achieve the phthalimides
6 and 7, phthalic acid anhydride and methylphthalic
acid anhydride, respectively, were refluxed in the pres-
ence of 1,3-diamino-N,N-2,2-tetramethylpropane in tolu-
ene using a water separator. Conversion of two mol-
ecules of 6 and 7, respectively, with dibromohexane gave
the symmetrical compounds 1c and 3c. To obtain the
* To whom correspondence should be addressed. Phone: + 228-73-
9103. Fax: + 228-73-9215. E-mail: k.mohr@uni-bonn.de.
† University of Bonn.
‡ University of Wu¨rzburg.
10.1021/jm020871e CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/11/2002

3810 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Sch em e 1. Synthesis Pathway of the Compounds Studied
Brief Articles
Ta ble 1. Parameters Characterizing the Allosteric Interaction of the Indicated Test Compounds with [3H]N-methylscopolamine at
Porcine Heart M2 Receptors (Mean Values ( SEM from Three Independent Experiments; For Details See Text)
compd
R1
R2
X1
X2
pKA
log R
R
p(RKA)
pEC50,diss
p(RKA)/pEC50,diss
1a
1b
1c
2a
2b
2c
3a
3b
3c
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
CH3
CH3
CH3
H
H
H
CH3
H
H
CH3
H
H
6.53 ( 0.02
6.90 ( 0.03a,b
7.08 ( 0.09a
6.89 ( 0.18
7.26 ( 0.03a
7.20 ( 0.22
7.08 ( 0.10a,d
7.41 ( 0.02a,b
7.21 ( 0.11a
0.48 ( 0.02
0.04 ( 0.03a
0.21 ( 0.02a
0.43 ( 0.01
-0.19 ( 0.02a,c
0.13 ( 0.02a
0.33a,d
3.02
1.10
1.62
2.69
0.65
1.35
2.14
0.93
0.68
6.05
6.86
6.87
6.46
7.45
7.07
6.75
7.44
7.38
6.00 ( 0.07
6.87 ( 0.04a
6.75 ( 0.04a
6.49 ( 0.04a
7.26 ( 0.02a
7.15 ( 0.11a
6.77 ( 0.03a,d
7.44 ( 0.06a
7.33 ( 0.10a
1.01
1b
CH3
CH3
H
CH3
H
H
CH3
CH3
1.02
1.00
1.03
0.99
1.00
1b
-0.03 ( 0.02a
-0.17 ( 0.02a,c
CH3
1.01
a
b
Significantly different from the parameter value of the parent compound 1a ; P < 0.05. Curve-fitting under the assumption that
p(RKA)/pEC50,diss ) 1. c Significantly different from log R of compound 3b; P < 0.05. Data taken from ref 13.
d
Resu lts
All test compounds reduced the apparent rate con-
nearly neutral cooperativity between this allosteric
agent and [3H]NMS. 2b enhanced [3H]NMS binding.
This indicates positive cooperativity; R ) 0.65 means
that the receptor-bound allosteric agent increases the
binding affinity of [3H]NMS almost 2-fold. The cooper-
ativity factors for all test compounds are compiled in
Table 1. Because R values are known to be log-normally
distributed,9 log R values were used for computing mean
values ( SEM and for statistical testing.
If an allosteric agent affects the equilibrium binding
of the orthosteric radioligand, allosteric curve fitting also
yields the equilibrium dissociation constant KA for the
interaction of the allosteric agent with ligand-free
receptors. The product RKA represents the equilibrium
dissociation constant of alloster binding to [3H]NMS-
occupied receptors.7 As mentioned above, the dissocia-
tion experiments also provide a measure of this binding
constant, i.e., EC50,diss. To check whether the indepen-
dent experimental approaches yielded consistent find-
ings, the ratio p(RKA)/pEC50,diss was computed, which
should amount to unity. As can be seen in Table 1, this
was the case. If an allosteric agent does not affect
stant k-1 of [3H]NMS dissociation and displayed con-
centration/effect curves with the slope factors not dif-
ferent from unity and bottom plateaus not significantly
different from k-1 ) 0. The curves (available as Sup-
porting Information) reflect binding of the allosteric
agents to [3H]NMS-occupied receptors. EC50,diss values
are listed in Table 1. As described in the Experimental
Section, the EC50,diss values were used to find out for
which concentrations of the allosteric agents [3H]NMS
equilibrium binding could be attained within an incuba-
tion time of up to 180 min. 1a displayed the expected8
submaximal inhibition of [3H]NMS equilibrium binding,
which is indicative of a negative cooperativity between
the allosteric and the orthosteric ligand, R ) 3.02 (Table
1). 1b had almost no effect on [3H]NMS equilibrium
binding. Since the dissociation experiments proved that
there is an interaction with the receptor, it can be
concluded that receptor occupation of this agent does
not affect the binding affinity of [3H]NMS. The cooper-
ativity factor R ) 1.10 is close to unity, which indicates

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3811
equilibrium binding, as found with 1b and 3b, curve-
fitting with R and KA as variable parameters does not
work. In that instance, we replaced KA by EC50,diss/R
(since RKA/EC50,diss ) 1, as mentioned earlier). Because
EC50,diss was known, the remaining variable R could be
obtained from the curve fit and KA was then derived
from EC50,diss/R.
pyl-substituted bis(ammonio)alkane-type compounds
endows these agents with the property to elevate
allosterically the binding of an orthosteric ligand to
muscarinic receptors.
Exp er im en ta l Section
Melting points (Supporting Information) were determined
with a Dr. Tottoli and a Gallenkamp melting point apparatus
(Bu¨chi, Switzerland) and were not corrected. 1H NMR spectra
were recorded on a Varian XL 300 instrument (1H 299.956
MHz; Supporting Information). IR spectra (Supporting Infor-
mation), recorded as KBr disks, were obtained using a Perkin-
Elmer 298 spectrometer, and UV spectra were recorded on a
Hewlett-Packard 8452A diode array spectrometer. Dry sol-
vents were used throughout. 1a was synthesized according to
ref 2, 2a according to ref 3, and 3a according to ref 1, 4 was
purchased from Aldrich (Steinheim, Germany), and 5 was
synthesized according to ref 1. Analyses indicated by the
symbols of the elements were within (0.4% of the theoretical
value (Supporting Information).
Discu ssion
In the present study we show for the first time that
allosteric enhancers of muscarinic ligand binding may
be derived from bis(ammonio)alkane-type agents by
rather small structural modifications. The parent com-
pound 1a has an inhibitive effect on the binding of NMS,
which indicates negative cooperativity between these
ligands. A seemingly minor structural modification, i.e.,
the introduction of methyl groups in two positions (2b),
was sufficient to elicit positive cooperativity with the
orthosteric ligand (Table 1). Compared with 1a , meth-
ylation generally tended to increase binding affinity
both at the ligand free receptor (see pKA in Table 1) and
at the NMS occupied receptor (see EC50,diss in Table 1).
With respect to positive cooperativity (R < 1), however,
it is pivotal that the affinity of the allosteric agent for
the orthoster-occupied receptor is higher than for the
ligand-free receptor. This is because binding of the
allosteric agent to the orthoster-occupied receptor in-
hibits orthoster dissociation and, thereby, promotes
orthoster equilibrium binding. Binding of the allosteric
agent to unoccupied receptors inhibits the orthosteric
ligand association and, thus, tends to diminish orthoster
equilibrium binding. With regard to allosteric agent
binding to NMS-occupied receptors, there is a strict
dependence on the position of methylation (Table 1).
Introduction of a methyl group into the phthalimide
moiety, either on one (2a ) or on both sides of the
molecule (3a ), hardly reduces the extent of negative
cooperativity compared with the parent compound 1a .
Methylation of the propyl chains can induce a more
pronounced diminution of negative cooperativity. Re-
markably, methylation in one of the propyl chains
allows for neutral cooperativity (1b), whereas methyl-
ation of both propyl chains still goes with negative
cooperativity (1c). A combination of both phthalimide
ring methylation and propyl chain methylation may
evoke positive cooperativity (2b, 3c) but not in all cases
(2c, 3b). Comparison of 2b and 2c indicates that propyl
chain methylation and phthalimide methylation on the
same side of the molecule induce positive cooperativity,
whereas these methylations carried out on opposite
sides do not. In fact, phthalimide plus propyl methyla-
tions on one side of the molecule (2b) yield a level of
positive cooperativity that is not overcome by additional
methylations on the other side of the molecule (3b, 3c).
The effect of an allosteric modulator on orthosteric
ligand binding is known to depend on the type of
orthosteric ligand.10 Future investigations will reveal
how much bis(ammonio)alkane-type compounds also
elevate the binding of muscarinic agonists such as
acetylcholine.
Syn th esis of 2-[3-(N,N-Dim eth yla m in o)-2,2-d im eth yl-
p r op yl]isoin d ole-1,3-d ion e (6). Phthalic acid anhydride
(7.41 g, 50 mmol) was dissolved in toluene (100 mL) at 80 °C,
then 1,3-diamino-N,N-2,2-tetramethylpropane (6.51 g, 50 mmol)
was added dropwise, and the resulting solution was refluxed
using a water separator. After about 3 h, the solvent was
evaporated, affording an oil that was crystallized from ethanol
and a few drops of petroleum ether to give 6: 63% yield, mp
75-78 °C.
Syn th esis of 2-[3-(N,N-Dim eth yla m in o)-2,2-d im eth yl-
p r op yl]-5-m eth ylisoin d ole-1,3-d ion e (7). 4-Methylphthalic
acid anhydride (6.49 g, 40 mmol) was dissolved in toluene (100
mL), then 1,3-diamino-N,N-2,2-tetramethylpropane (5.21 g, 40
mmol) was added dropwise, and the resulting solution was
refluxed using a water separator. After 4 h, the solvent was
evaporated, affording the product that was purified by means
of column chromatography (silica gel; eluent CH2Cl2/MeOH )
5:1, Rf ) 0.65) to give 7: 68% yield, mp 81-83 °C.
Gen er a l P r oced u r e for th e Syn th esis of (6-Br om o-
h exyl)[3-(1,3-d ioxo-1,3-d ih yd r o-isoin d ol-2-yl)p r op yl]d i-
m eth yla m m on iu m Br om id e (8), (6-Br om oh exyl)[3-(1,3-
d ioxo-1,3-d ih yd r oisoin d ol-2-yl)-2,2-d im et h ylp r op yl]d i-
m eth yla m m on iu m Br om id e (9), a n d (6-Br om oh exyl)[3-
(1,3-d i o x o -1,3-d i h y d r o -5-m e t h y li s o i n d o l-2-y l)-2,2-
d im eth ylp r op yl]d im eth yla m m on iu m Br om id e (10). A
mixture of the corresponding amines (0.01 mol), 4, 6, and 7,
respectively, and a 3-fold excess of dibromohexane was allowed
to stand at least 5 days at ambient temperature until a
precipitate appeared. The crystals were filtrated, washed with
ethyl acetate, recrystallized from 2-propanol/ethanol (5:1) to
give 8 (yield 63%; mp 189-90 °C), recrystallized from acetone
and petroleum ether to give 9 (yield 35%; mp 157-69 °C (dec)),
and recrystallized from 2-propanol/ethanol (5:1) and a few
drops of diethyl ether to give 10 (yield 10%; mp 131-133 °C).
Syn th esis of 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(1,3-d ioxo-
1,3-d ih yd r oisoin d ol-2-yl)-2,2-d im eth ylp r op yl]a m m on io}-
h exyl)-1,1-d im et h yla m m on io]-2,2-d im et h ylp r op yl}-1,3-
isoin d olin d ion e Dib r om id e (1c) a n d 2-{3-[1-(6-{1,1-Di-
m et h yl-1-[3-(1,3-d ioxo-1,3-d ih yd r o-5-m et h ylisoin d ol-2-
yl)-2,2-d im eth ylp r op yl]a m m on io}h exyl)-1,1-d im eth yla m -
m on io]-2,2-d im et h ylp r op yl}-5-m et h yl-1,3-isoin d olin d i-
on e Dib r om id e (3c). Compounds
6 and 7 (10 mmol),
respectively, and dibromohexane (5 mmol) were dissolved in
acetonitrile (100 mL), and a catalytic amount of a mixture of
KI and K2CO3 (1:1) was added. The reaction solution was
refluxed for 3 days (TLC control, silica gel, mobile phase )
CH3OH/0.2 M NH4NO3 solution (aqueous) ) 3:2). The obtained
precipitate was filtered, and the filtrate was recrystallized
several times from a solvent mixture composed of ethanol and
a few drops of petroleum ether to give 1c (yield 60%) and 3c
(yield 61%), respectively.
Con clu sion
The present study shows for the first time that
methylation in appropriate positions of phthalimidopro-

3812 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Brief Articles
Gen er a l P r oced u r e for th e Syn th esis of 2-{3-[1-(6-{1,1-
Dim et h yl-1-[3-(1,3-d ioxo-1,3-d ih yd r oisoin d ol-2-yl)-2,2-
dim eth ylpr opyl]am m on io}h exyl)-1,1-dim eth ylam m on io]-
p r op yl}-1,3-isoin d olin d ion e Dib r om id e (1b ), 2-{3-[1-(6-
{1,1-Dim eth yl-1-[3-(1,3-dioxo-1,3-dih ydr o-5-m eth ylisoin dol-
2 -y l ) -2 , 2 -d i m e t h y l p r o p y l ] a m m o n i o }h e x y l ) -1 , 1 -
dim eth ylam m on io]pr opyl}-1,3-isoin dolin dion e Dibr om ide
(2b), 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(1,3-d ioxo-1,3-d ih yd r o-
isoin d ol-2-yl)-2,2-d im et h ylp r op yl]a m m on io}h exyl)-1,1-
d im et h yla m m on io]p r op yl}-5-m et h yl-1,3-isoin d olin d i-
on e Dibr om id e (2c), a n d 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-
(1,3-d i o x o -1,3-d i h y d r o -5-m e t h y li s o i n d o l-2-y l)-2,2-
dim eth ylpr opyl]am m on io}h exyl)-1,1-dim eth ylam m on io]-
p r op yl}-5-m et h yl-1,3-isoin d olin d ion e Dib r om id e (3b).
Equimolar amounts of 8, 9, and 10 (1 mmol), respectively, and
the corresponding dimethylaminopropylimides 6, 5, and 4 were
dissolved in acetonitrile (100 mL), and a catalytic amount of
a mixture of KI and K2CO3 (1:1) was added. The mixture was
refluxed for 2-4 days (TLC control, silica gel, mobile phase )
CH3OH/0.2 M NH4NO3 solution (aqueous) ) 3:2). The obtained
precipitate was filtered and washed several times with hot
acetonitrile to give about 60% of each compound.
Sta bility. Tris-Mg buffer consisted of 3.6 mM MgHPO4, 50
mM Tris (pH 7.3 attained by using HCl), and 500 mL of
distilled water. A 10-4 M solution of each compound was
prepared in Tris buffer; the absorption spectra of these
solutions were recorded between 220 and 800 nm every 15 min
for a period of 24 h. The decrease in the absorption at the
maximum wavelength at 300 nm was registered. Nonlinear
regression analysis (GraphPad software) yielded the half-life
t1/2 of the hydrolysis. The half-lives were found to be higher
than 36 h.
Pad. Two-tailed t tests with Welch correction when necessary
were carried out with the Instat software (version 1.11a,
GraphPad, San Diego, CA).
Ack n ow led gm en t. Thanks are due to the DFG
(Grants MO 821/1-2 and HO 1368/7-1), the Fonds der
Chemischen Industrie for financial support, and to an
anonymous reviewer for his valuable advice.
Su p p or tin g In for m a tion Ava ila ble: Analytical data, 1H
NMR data, concentration/effect-curves for the inhibition of
[3H]NMS dissociation by representative test compounds, and
concentration/effect curves for the effect of representative
test compounds on [3H]NMS equilibrium binding. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
(1) Tra¨nkle, C.; Mies-Klomfass, E.; Botero Cid, H. M.; Holzgrabe
U.; Mohr, K. Identification of a [3H]ligand for the common
allosteric site of muscarinic acetylcholine receptors. Mol. Phar-
macol. 1998, 54, 139-145.
(2) Nassif-Makki, T.; Tra¨nkle, C.; Zlotos, D.; Bejeuhr, G.; Cambareri,
A.; Kostenis, E.; Mohr, K.; Holzgrabe, U. Bisquaternary ligands
of the common allosteric site of M2 acetylcholine receptors:
Search for the minimum essential distances between the phar-
macophoric elements. J . Med. Chem. 1999, 42, 849-858.
(3) Bender, W.; Staudt, M.; Tra¨nkle, C.; Mohr, K.; Holzgrabe, U.
Probing the size of a hydrophobic binding pocket within the
allosteric site of muscarinic acetylcholine M2-receptors. Life Sci.
2000, 66, 1675-1682.
(4) Botero Cid, H. M.; Tra¨nkle, C.; Baumann, K.; Pick, R.; Mies-
Klomfass, E.; Kostenis, E.; Mohr, K.; Holzgrabe, U. Structure-
activity relationships in a series of bisquaternary bisphthal-
imidine derivatives modulating the muscarinic M2-receptor
allosterically. J . Med. Chem. 2000, 43, 2155-2164.
Octa n ol/Bu ffer P a r tition Coefficien t . A solution of each
compound (10-5 M) was prepared in phosphate buffer (pH 7.4,
0.078 M), saturated with 1-octanol, and the absorbance was
measured at the maximum wavelength (222-228 nm). This
solution (1 mL) was shaken for 4 h with 1-octanol (1 mL)
saturated with buffer. After centrifugation, the aqueous layer
was separated and again the absorption measured. From the
differences of the absorptions, the octanol/buffer partition
coefficient was calculated, and as can be seen from the log P
values, the compounds were found to be slightly hydrophilic:
1b ) -0.72; 1c ) -0.82; 2b ) -0.33; 2c ) -0.63; 3b ) -0.79;
3c ) -0.39.
(5) Prosˇka, J .; Tucˇek, S. Mechanisms of steric and cooperative
actions of alcuronium on cardiac muscarinic acetylcholine recep-
tors. Mol. Pharmacol. 1994, 45, 709-717.
(6) Daiss, J . O.; Duda-J ohner, S.; Burschka, C.; Holzgrabe, U.; Mohr,
K.; Tacke, R. N+/Si replacement as
a tool for probing the
pharmacophore of allosteric modulators of muscarinic M2 recep-
tors: synthesis, allosteric potency, and positive cooperativity of
silicon-based W84 derivatives. Organometallics 2002, 21, 803-
811.
(7) Ehlert, F. J . Estimation of the affinities of allosteric ligands using
radioligand binding and pharmacological null methods. Mol.
Pharmacol. 1988, 33, 187-194.
(8) Tra¨nkle, C.; Andresen, I.; Lambrecht, G.; Mohr, K. M2 receptor
binding of the selective antagonist AF-DX 384: possible involve-
ment of the common allosteric site. Mol. Pharmacol. 1998, 53,
304-312.
Ra d ioliga n d Bin d in g Stu d ies. Porcine cardiac homoge-
nates were prepared as described previously.1 Binding of [3H]-
N-methylscopolamine ([3H]NMS) (0.2 nM; specific activity
70.0-83.5 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA)
(9) Christopoulos, A. Assessing the distribution of parameters in
models of ligand-receptor interaction: to log or not to log.
Trends Pharmacol. Sci. 1998, 19, 351-357.
was measured in 45 mM Tris-HCl and 2.6 mM MgHPO4
:
pH 7.3, 37 °C. Nonspecific [3H]NMS binding was marked by
1 µM atropine and was less than 5% of the total binding.
Membranes were separated by vacuum filtration after 2-3 h
of incubation. Radioactivity was determined by liquid scintil-
lation counting. The pKD of NMS binding amounted to 9.50 (
0.08 (mean ( SEM, n ) 4). [3H]NMS dissociation was
monophasic (t1/2,control ) 3.0 ( 0.1 min; mean ( SEM, n ) 31).
After 30 min of preincubating the membranes with [3H]NMS,
its dissociation was made visible by addition of 1 µM atropine
alone or in combination with a test compound. Two-point
kinetic experiments11 were applied with measurements of [3H]-
NMS binding at t ) 0 and t ) 10 min. To investigate [3H]-
NMS equilibrium binding, the appropriate time of incubation
was determined according to Lazareno and Birdsall12 (eq 31
therein). For the time needed for equilibrium binding, we took
5 half-lives. Equilibrium binding data were analyzed according
to Ehlert7 as described previously (eq 3 in ref 8). For nonlinear
regression analysis, we used the software Prism 3.0, Graph
(10) Lee, N. H.; El-Fakahany, E. E. Influence of ligand choice on the
apparent binding profile of gallamine to cardiac muscarinic
receptors. Identification of three main types of gallamine-
muscarinic receptor interaction. J . Pharmacol. Exp. Ther. 1988,
246, 829-838.
(11) Kostenis, E.; Mohr, K. Two-point kinetic experiments to quantify
allosteric effects on radioligand dissociation. Trends Pharmacol.
Sci. 1996, 17, 280-283.
(12) Lazareno, S.; Birdsall, N. J . M. Detection, quantitation, and
verification of allosteric interactions of agents with labeled and
unlabeled ligands at G protein-coupled receptors: interactions
of strychnine and acetylcholine at muscarinic receptors. Mol.
Pharmacol. 1995, 48, 362-378.
(13) Mies-Klomfass, E. Suche nach einem potentiellen Radioliganden
zur Markierung der allosterischen Bindungsstelle am muskari-
nischen Acetylcholin-Rezeptor vom Subtyp M2 (Search for a
potential radioligand to label the allosteric site of M2 muscarinic
acetylcholine receptors). Ph.D. Thesis, University of Bonn,
Germany, 1997.
J M020871E