Identification of a critical residue in the transmembrane domain 2 of tachykinin neurokinin 3 receptor affecting the dissociation kinetics and antagonism mode of osanetant (SR 142801) and piperidine-based structures

Article abstract of DOI:10.1021/jm900948q

In this study, we show that compound 3 (osanetant) binds with a pseudoirreversible, apparent noncompetitive mode of antagonism at the guinea pig NK₃, while it behaves competitively at the human NK₃. This difference is caused by a slo

Full text of DOI:10.1021/jm900948q

J. Med. Chem. 2009, 52, 7103–7112 7103
DOI: 10.1021/jm900948q
Identification of a Critical Residue in the Transmembrane Domain 2 of Tachykinin Neurokinin
3 Receptor Affecting the Dissociation Kinetics and Antagonism Mode of Osanetant (SR 142801)
and Piperidine-Based Structures
†
†
†
‡
‡
Pari Malherbe,*,† Claudia Kratzeisen, Anne Marcuz, Marie-Therese Zenner, Matthias H. Nettekoven, Hasane Ratni,
ꢀ ꢁ
Joseph G. Wettstein,† and Caterina Bissantz‡
†Discovery Research CNS and ‡Chemistry, F. Hoffmann-La Roche Ltd., PRDN, Building 69/333, CH-4070 Basel, Switzerland
Received June 29, 2009
In this study, we show that compound 3 (osanetant) binds with a pseudoirreversible, apparent
noncompetitive mode of antagonism at the guinea pig NK3, while it behaves competitively at the
human NK3. This difference is caused by a slower dissociation rate of compound 3 at the guinea pig NK3
compared to human NK3. The only amino acid difference between the human and guinea pig NK3 in the
binding site (Thr1392.58 in human, corresponding to Ala1142.58 in guinea pig) has been shown to be
responsible for the different behavior. Compound 1 (talnetant), however, behaves competitively at both
receptors. Using these data, 3D homology modeling, and site-directed mutagenesis, a model has been
developed to predict the mode of antagonism of NK3 antagonists based on their binding mode. This
model was successfully used to predict the mode of antagonism of compounds of another chemical series
including piperidine-based structures at human and guinea pig NK3.
Introduction
bronchial asthma, gastrointestinal disorders, inflammatory
bowel syndrome, urinary bladder, and psychiatric disor-
ders.9-11 Preclinical studies have demonstrated the involve-
ment of NK3-mediated activation in the release of dopamine,
especially in ventral and dorsal striatal region.12,13 (S)-(þ)-
N-((3-[1-Benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]prop-
1-yl)-4-phenylpiperidin-4-yl)-N-methylacetamine (osanetant,
SR 142801)14,15 and (S)-(-)-N-(R-ethylbenzyl)-3-hydroxy-2-
phenylquinoline-4-carboxamide (talnetant, SB 223412),16,17
which are from two distinct chemical classes, have been shown
to be potent nonpeptide antagonists of the NK3. Both
antagonists have been studied in schizophrenia patients in a
double blind placebo controlled (short-term) clinical trial that
suggested significant improvement in psychopathology, in-
cluding positive symptoms.18 The information about phase II
clinical trial of 3 (osanetant) showed the compound to be
active in schizophrenia patients with improved efficacy, side
effect profiles, and good tolerability.19 Recent clinical inves-
tigation of 1 (talnetant) in healthy volunteers has shown that it
had psychomotor and cognitive effects and was able to
improve visuomotor coordination and vigilance.20
Although compound 1 clearly displayed a reversible
and competitive mode of antagonism in the NKB-induced
Ca2þ mobilization at cloned human NK3 and in the
senktide-induced contractions in rabbit isolated iris sphincter
muscles,16,17 there have been controversial data regarding the
inhibition mode of compound 3. Investigations using
[MePhe7]NKB- and senktide-stimulated inositol phosphate
(IP) formation at the cloned human NK3 or [MePhe7]NKB-
mediated contractions of guinea pig ileum have shown a
competitive mode of antagonism by compound 3.14,21
However, many other studies that used senktide- and
[MePhe7]NKB-mediated contractions of the guinea pig iso-
lated ileum longitudinal muscle preparation or senktide-induced
The neurokinins (NKs, also called tachykininsa) belong toa
familyofneuropeptides thatmainlycomprises the substanceP
(SP), neurokinin A (NKA), and neurokinin B (NKB). They
elicit their effect through three G-protein-coupled receptors
(GPCRs) called NK1, NK2, and NK3 (nomenclature follows
Alexander et al., 20081) that are coupled via Gq/11 to the
activation of phospholipase C, leading to an elevation of
intracellular Ca2þ levels. The tachykinins rank order of
potency at the NK receptors: SP > NKA > NKB for the
NK1, NKA > NKB > SP for the NK2, and NKB > NKA >
SP for the NK3.2,3 The comparative studies using [3H]senktide
audoradiography revealed striking differences in the CNS
localization of NK3 among rat, gerbil, and guinea pig4 and
between some of these species and primates.5 In general, the
expression of NK3 (by in situ hybridization histochemisry and
NKB/senktide binding) was detected in brain regions that
include cortex (frontal, parietal, and cingulate cortex), various
nuclei of the amygdala, the hippocampus, and midbrain
structures (the substantia nigra, ventral tegmental area, and
raphe nuclei).4,6,7 The distribution of NK3 has been reported
in the prefrontal and visual cortex of the human brain by
immunohistochemistry.8
The tachykinins are involved in numerous physiological
functions including nociception, neuroimmunomodulation,
and reproduction. NK receptor dysfunction has been impli-
cated in the pathology of various diseases such as emesis,
*To whom correspondence should be addressed. Phone: þ41-61-688-
6286. Fax: þ 41-61-688-1720. E-mail: parichehr.malherbe@roche.com.
a Abbreviations: NK, neurokinin; NK3, neurokinin 3 receptor;
GPCRs, G-protein coupled receptors; NKB, neurokinin B; IP, inositol
phosphates; 7TMD, seven-transmembrane domain; WT, wild-type;
OPSD, bovine rhodopsin.
r
2009 American Chemical Society
Published on Web 10/09/2009
pubs.acs.org/jmc

7104 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Malherbe et al.
Table 1. Schild Constants for Antagonism of [MePhe7]NKB-Induced Accumulation of [3H]IP by NK3 Antagonists in the HEK293 Cells Transiently
Expressing hNK3-WT, gpNK3-WT, or gpNK3-A1142.58Ta
hNK3-WT
gpNK3-WT
gpNK3-A1142.58
T
NK3 antagonist pA2 Schild slope mode of antagonism pA2 Schild slope mode of antagonism pA2 Schild slope mode of antagonism
1
3
5
6
7
8.24
7.73
8.05
8.11
7.74
0.88
1.59
0.99
0.85
1.24
competitive
competitive
competitive
competitive
competitive
7.81
7.62
1.07
1.25
competitive
7.91
8.5
1.12
1.03
1.32
0.76
0.97
competitive
competitive
competitive
competitive
competitive
noncompetitive
noncompetitive
noncompetitive
competitive
7.96
7.83
7.53
a The apparent antagonist potency (pA2) and Schild slope values were determined from Schild plot analyses.
formation of [3H]IP in slices from the guinea pig ileum all
pointed to a noncompetitive, insurmountable, and long-
[MePhe7]NKB Schild analyses are given in Table 1. Com-
pound 1 behaved as a competitive antagonist at both hNK3
and gpNK3, shifting the NKB CRCs to the right without
changing its maximal response (Figure 1A,B and Table 1).
Compound 3 behaved in a competitive manner at hNK3
(Figure 2A and Table 1), but it displayed a noncompetitive
like mode of antagonism at gpNK3 (Figure 2B).
lasting irreversible antagonism by compound 3.15,22,23
A
recent study, which compared antagonism modes of com-
pounds 1 and 3 in cellular Ca2þ mobilization and binding
kinetics, has demonstrated that both antagonists displayed a
competitive mode of antagonism at recombinant human
NK3.24 Furthermore, site-directed mutagenesis and mole-
cular modeling studies have indicated that (S)-(-)-N-
(R-ethylbenzyl)-3-methoxy-2-phenylquinoline-4-carboxamide
(Me-talnetant) and compound 3 interact within overlapping
but not identical binding pockets in the human NK3 7TMD.25
Taken together, these studies are consistent with the notion
that the guinea pig NK3 receptor has to be responsible for the
compound 3’s apparent noncompetitive (pseudoirreversible)
mode of antagonism.
In the current study, we have compared the compound 3’s
mode of antagonism at human and guinea pig NK3 wild-type
and mutated receptors by binding kinetics and functional
Schild analysis using [MePhe7]NKB-induced formation of
[3H]IP assay. This data, together with rhodopsin-based 3D
modeling and site-directed mutagenesis, allowed development
of a model that predicts the mode of antagonism of NK3
antagonists based on their predicted docking poses. We have
validated this model by testing its capability to predict
theantagonismmode ofthree piperidine derivatives, 2-(3,4-di-
chlorophenyl)-N-(4-fluoro-3-trifluoromethylbenzyl)-2,N-di-
methyl-4-[4-(2-oxo-pyrrolidin-1-yl)-piperidin-1-yl]-butyramide
(5), 4-[4-(acetylmethyl-amino)-piperidin-1-yl]-2-(3,4-dichloro-
phenyl)-N-(4-fluoro-3-trifluoromethylbenzyl)-2,N-dimethyl-
butyramide (6), and 4-[4-(acetylmethyl-amino)-piperidin-1-yl]-
N-(2-chloro-benzyl)-2-(3,4-dichlorophenyl)-2,N-dimethylbu-
tyramide (7)26 at the human NK3 and guinea pig NK3-WT and
mutated receptors.
The alignment of the amino acids forming the NK3
binding pocket among various species is shown in Figure 3.
The only amino acid difference between human and guinea
pig is the residue Thr1392.58 in human NK3, which corre-
sponds to Ala1142.58 in guinea pig NK3 (Figure 3). Hence,
guinea pig NK3-Ala1142.58 was converted to Thr1142.58
.
Because the mutation gpNK3-A1142.58T had no effect on
[MePhe7]NKB potency (EC50 and nH values of 0.9 ( 0.1 nM
and 1.1 ( 0.1, respectively), the antagonism mode of com-
pounds 1 and 3 was compared at the mutated gpNK3-
A1142.58T receptor using Schild analyses. While the muta-
tion of gpNK3-A1142.58T had no effect on competitive mode
of antagonism by compound 1 (Figure 1C,D and Table 1), it
yet converted compound 3’s noncompetitive mode of anta-
gonism to a competitive one (Figure 2C, D and Table 1).
Binding Affinity of NK3 Antagonists at Various NK Re-
ceptors as Measured by Displacement Studies. The affinity
constants of NK3 antagonist in the membrane preparations
from HEK293 cells transiently expressing hNK1, hNK2,
hNK3, gpNK3, or gpNK3-A1142.58T are given in Table 2.
Both compounds 1 and 3 bind with similar affinity to hNK3,
gpNK3, and gpNK3-A1142.58T (Table 2). Compounds 5, 6,
and 7 (piperidine derivatives) are potent and selective hNK3
antagonists (Table 2), and their structures are shown in
Figure 4.26 As seen in Table 2, all three compounds also
displayed similar affinity for hNK3, gpNK3, and gpNK3-
A1142.58T. Therefore, the mutation gpNK3-A1142.58T had
no effect on binding affinities of NK3 antagonists.
Comparison of Binding Kinetics of Radioligand [3H]3
([3H]SR 142801) to hNK3-WT, gpNK3-WT, and gpNK3-
A1142.58T. Binding kinetics of radioligand [3H]3 to mem-
brane preparations from HEK293 cells transiently expres-
sing hNK3-WT, gpNK3-WT, and gpNK3-A1142.58T are
shown in Figure 5 and the kinetic parameters in Table 3.
Binding of radioligand [3H]3 to the hNK3-WT was rapid
with half-maximal binding occurring at 4 min and reaching
equilibrium within 30 min (Figure 5A and Table 3). The
association bindings of radioligand [3H]3 to the gpNK3-WT
and gpNK3-A1142.58T were similar with half-maximal bind-
ing, t1/2 values of 10.2 and 10.0 min, respectively (Figure 5A
and Table 3). The data for three receptors were fit to a one-
phase exponential model (Figure 5A).
Results
Comparison of the Mode of Antagonism of Compounds 1
and 3 at Human and Guinea Pig NK3 Receptors. To compare
the inhibition mode of NK3 antagonists at hNK3 and
gpNK3, the concentration-response curves (CRCs) for
[3H]IP formation stimulated by [MePhe7]NKB were mea-
sured in the absence or presence of increasing concentrations
of antagonist in the HEK cells transiently transfected with
hNK3 or gpNK3. [MePhe7]NKB was used as agonist because
it has a high selectivity for hNK3 (Ki value of 0.3 nM) versus
hNK2 (Ki value of 1597 nM).17 [MePhe7]NKB elicited
concentration-dependent increases in the accumulation of
[3H]IP in the hNK3 expressing cells with the EC50 and nH
values of 0.6 ( 0.0 nM and 1.0 ( 0.0, respectively, and in the
gpNK3 expressing cells with the EC50 and nH values of 0.6 (
0.0 nM and 1.0 ( 0.1, respectively. The apparent antagonist
potency (pA2) and the Schild slope values calculated from
The dissociation rates for radioligand [3H]3 bindings to the
hNK3-WT, gpNK3-WT, and gpNK3-A1142.58T were deter-
mined by the addition of an excess amount of (S)-(-)-N-(R-
ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide (SB

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7105
Figure 1. Schild plot analyses for antagonism of [MePhe7]NKB-induced accumulation of [3H]IP by compound 1 (talnetant). Concentration-
response curves (CRCs) for [3H]IP formation stimulated by [MePhe7]NKB in the absence and presence of various concentrations of com-
pound 1 in HEK293 cells expressing transiently the gpNK3-WT (A) and gpNK3-A1142.58T (C). Schild plots for antagonism by compound 1 at
gpNK3-WT (B) and gpNK3-A1142.58T (D). The EC50 and EC50 values, which derived from NKB CRCs in the absence and presence of
0
0
increasing fixed concentrations of compound 1 (A,C) were used to calculate the dose ratios (DR = EC50 /EC50) and plotted according to Schild
regression in (B) and (D). Each curve represents the mean of six concentration-response measurements from three independent transfections.
222200)16 after equilibrium was reached. The reversals of
bindings from hNK3-WT and gpNK3-A1142.58T were com-
plete, with t1/2 values of 10 and 17 min, respectively (Figure 5B
and Table 3). The rate of radioligand [3H]3 dissociation from
the gpNK3 was decreased as compared to the hNK3-WT
(Figure 5B and Table 3). The calculations of the apparent Kd
values derived from the kinetic experiments are given in
Table 3. The radioligand [3H]3 had an apparent Kd value
of 0.22 ( 0.06 nM at WT receptor, which is in good agreement
with the equilibrium Kd value (0.20 ( 0.01). However,
the apparent Kd values of radioligand [3H]3 at the gpNK3
(0.08 ( 0.01 nM) was lower than that of equilibrium Kd values
(0.15 ( 0.03). Conversely, it had a higher apparent Kd value
(0.12 ( 0.03 nM) than that of equilibrium Kd value (0.08 (
0.01 nM) at gpNK3-A1142.58T.
Inhibition Mode of Compounds 5, 6, and 7 on the
[MePhe7]NKB-Evoked Accumulation of [3H]IP at hNK3,
gpNK3, and gpNK3-A1142.58T. To explore the chemical
moiety that is involved in NK3 antagonists’ mode of action,
three piperidine derivatives, compounds 5, 6, and 7, were
further investigated in the NKB-induced formation of [3H]IP
assay in HEK293 cells expressing transiently the hNK3,
gpNK3 and gpNK3-A1142.58T. The choice of these anta-
gonists was guided by our docking model that predicted
compounds 5 and 6 to have a noncompetitive mode of
antagonism and compound 7 a competitive one. All three
NK3 antagonists behaved competitively at hNK3 (Table 1).
As predicted, both compounds 5 and 6 acted in a noncom-
petitive manner at gpNK3 and a competitive one when the
mutated guinea pig receptor was used (Figure 6A-D and
Table 1), while compound 7 displayed a competitive mode of
antagonism at both gpNK3 and gpNK3-A1142.58
(Figure 6E,F and Table 1).
T
Discussion and Conclusion
Phase II clinical results of compounds 1 and 3 have
indicated that blocking the NK3 receptor could be beneficial
for the treatment of schizophrenia and possibly other psy-
choses.18,19 Therefore, NK3 antagonists represent an alter-
native therapeutical approach for the treatment of
schizophrenia.27 Species-related differences in the pharmaco-
logy of NK3 antagonists have been reported, especially be-
tween human and mouse/rat NK3. Thus, two residues in
TM2, Met1342.53 and Ala1462.65 of hNK3, which correspond
to Val1212.53 and Gly1332.65 of mouse, are shown to be
involved in the species-selectivity of N-[(S)-4-(4-acetylamino-
4-phenyl-piperidin-1-yl)-2-(3,4-dichloro-phenyl)-butyl]-N-
methyl-benzamide (SR 48968, 4) (a close derivative of com-
pound 3) for hNK3.28,29 However, there is no such report
concerning guinea pig and selective NK3 antagonists. As
compounds 1 and 3 have displayed similar potency at human
and guinea pig NK3 receptors, human-like pharmacology of
guinea pig NK3 has made an appropriate species for char-
acterization of the invivo efficacy ofNK3 antagonists.30 Inthe
current study, we have attempted to resolve the controversy
about compound 3’s antagonism mode of action between
human and guinea pig receptors.

7106 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Malherbe et al.
Figure 2. Schild analyses showing the competitive like mode of antagonism by compound 3 (osanetant) at hNK3-WT and gpNK3-A1142.58
T
and noncompetitive one at gpNK3-WT. CRCs for [3H]IP formation stimulated by [MePhe7]NKB in the absence or presence of increasing
concentrations of compound 3 in HEK293 cells expressing transiently the hNK3-WT (A), gpNK3-WT (B), and gpNK3-A1142.58T (C). Schild
plot for antagonism by compound 3 at gpNK3-A1142.58T is shown in (D). Each curve represents the mean of six concentration-response
measurements from three independent transfections.
Figure 3. Alignment of the amino acids forming the binding site. The first row gives the Ballesteros-Weinstein numbering scheme. The
numbers above the NK3R_HUMAN and NK3R_CAVPO receptors give the sequence number of the positions of the mutations carried out in
this study. The amino acid sequences of the rat NK3 (accession number: P16177), mouse NK3 (accession number: P47937), gerbil NK3
(accession number: AM157740), human NK3 (accession number: P29371), guinea pig NK3 (accession number: P30098), dog NK3 (accession
number: AM423140), cynomolgus monkey NK3 (in-house data), human NK1 (accession number: P25103), and human NK2 (accession
number: P21452) were retrieved from the Swiss-Prot database.
Investigation of the antagonistic mechanism of compounds
1 and 3 revealed that both compounds act as competitive
antagonists at hNK3. Compound 1 still had a competitive
mode of antagonism at gpNK3, yet compound 3 behaved in a
noncompetitive manner at gpNK3. This mode of action was
characterized by a rightward shift of the [MePhe7]NKB
concentration-response curves (increase in EC50 values) in
the presence of increasing compound 3 concentrations with
a concomitant large decrease in the maximal effect (Emax).
To better understand the mechanism of compound 3’s appa-
rent noncompetitive behavior at gpNK3, binding kinetics of
radioligand [3H]3 were compared between guinea pig and
human receptors. As expected, radioligand [3H]3 had a
slower dissociation rate at guinea pig receptor (the reversal
of binding was incomplete and did not reached the baseline
even after 2 h) in comparison to human one. This could be
a possible explanation for the apparent noncompetitive
and pseudoirreversible mode of antagonism by compound 3
at gpNK3 determined by Schild plot analyses. Because of
compound 3’s slow dissociation, a large portion of the gpNK3
is not available for activation by NKB, consequently, the
maximally achievable response of NKB drops dramatically

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7107
Table 2. Binding Affinity of NK3 Antagonists in the Membrane Preparations from HEK293 Cells Transiently Expressing hNK1, hNK2, hNK3, gpNK3,
or gpNK3-A1142.58Ta
[3H]3
[3H]SP
hNK1
[3H]4
hNK2
hNK3
Ki nM
gpNK3
Ki nM nH
gpNK3-A1142.58
Ki nM nH
3.8 ( 0.2 1.0 ( 0.0
T
NK3 antagonist
Ki nM
>10000
nH
Ki nM
>10000
nH
nH
1
3
5
6
7
3.0 ( 0.4 0.9 ( 0.1 5.0 ( 1.5 1.0 ( 0.1
0.5 ( 0.0 1.0 ( 0.0 0.6 ( 0.2 1.0 ( 0.1
0.8 ( 0.2 0.9 ( 0.1 1.6 ( 0.3 1.7 ( 0.5
3.1 ( 0.4 1.1 ( 0.1 3.5 ( 0.3 1.2 ( 0.1
209.6 ( 24.6 0.9 ( 0.2
181.6 ( 20.8 0.9 ( 0.0
151.8 ( 15.1 0.9 ( 0.1
70.9 ( 21.6 0.8 ( 0.1
35.5 ( 1.1
108.6 ( 10.1
515.3 ( 24.9
1.0 ( 0.0
0.9 ( 0.0
0.9 ( 0.1
0.8 ( 0.0
1.8 ( 0.2
2.7 ( 0.2
1.5 ( 0.0
1.5 ( 0.1
1.4 ( 0.0
952.6 ( 156.8 0.7 ( 0.1 10.4 ( 5.0 1.1 ( 0.0 8.0 ( 3.5 1.0 ( 0.0 11.9 ( 2.8 1.2 ( 0.1
a The affinity constant (Ki) and Hill slope (nH) values for radioligands [3H]SP, [3H]4 ([3H]SR 48968), and [3H]3 ([3H]SR 142801) binding inhibitions by
various antagonists were calculated as described in the Experimental Section. Values are mean ( SE of the Ki calculated from three independent
experiments, each performed in duplicate.
Figure 4. Chemical structures of NK antagonists. The moiety that controls the mode of antagonism is indicated by the filled circle.
Figure 5. Time course for the association (A) and dissociation (B) of radioligand [3H]3 binding to the hNK3-WT, gpNK3-WT, and gpNK3-
A1142.58T membranes. Each data point is mean ( SE (bars) of three individual experiments performed in quadruplet.
Table 3. Kinetic Parameters for Association and Dissociation of Radioligand [3H]3 in the hNK3-WT, gpNK3-WT, and gpNK3-A1142.58T Membranesa
association kinetic
dissociation kinetic
NK3 receptor
human
guinea pig
Kob min-1
Kon nM-1min-1
t1/2 min
Koff min-1
t1/2 min
apparent Kd nM
equilibrium Kd nM
0.17 ( 0.02
0.068 ( 0.01
0.07 ( 0.01
0.33 ( 0.04
0.32 ( 0.06
0.32 ( 0.05
4.01 ( 0.73
10.23 ( 0.20
9.9 ( 1.2
0.07 ( 0.01
0.022 ( 0.01
0.04 ( 0.01
10.10 ( 1.07
31.5 ( 4.10
17.43 ( 0.5
0.22 ( 0.06
0.08 ( 0.01
0.12 ( 0.03
0.20 ( 0.01
0.15 ( 0.03
0.08 ( 0.01
gpNK3-A1142.58
T
a The Kob (observed on rate), Koff (observed off rate), Kon, t1/2 (half-maximal binding), and Kd (apparent dissociation constant) values are mean ( SE,
calculated from three independent experiments (each performed in quadruplet) as described in the Experimental Section.
in comparison to fast dissociating antagonist such as com-
pound 1.
When the sequences of the NK3 7TMD were compared
among seven species, it showed that, with the exception of
gpNK3, which carries an alanine at position 114 (helix posi-
tion 2.58), all other species have threonine at this position.
This is also the only amino acid difference found in the 7TMD
region between guinea pig and human. Indeed, the mutation

7108 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Malherbe et al.
Figure 6. Schild plot analyses for antagonism of [MePhe7]NKB-induced accumulation of [3H]IP by compounds 5, 6, and 7. Concentra-
tion-response curves for [3H]IP formation stimulated by [MePhe7]NKB in the absence and presence of various concentrations of compound 5
(A,B), compound 6 (C,D), and compound 7 (E,F) in HEK293 cells expressing transiently the gpNK3-WT or gpNK3-A1142.58T. Each curve
represents the mean of six concentration-response measurements from three independent transfections.
gpNK3 A1142.58T converted compound 3’s apparent non-
competitive behavior to a competitive one and its slow
dissociation to a fast one, very similar to human receptor.
Interestingly, as we have shown previously, mutation of hu-
man NK3 Thr1392.58 to alanine has conversely caused com-
affected by this mutation, does not locate any of its atoms
close enough to residue2.58to generate a repulsive interaction
with hNK3 Thr1392.58 or a favorable one with gpNK3
Ala1142.58. Moreover, searches in the Cambridge Structural
Database (www.ccdc.cam.ac.uk/products/csd/) validated our
˚
pound 3’s competitive mode to be converted to
a
opinion that a distance below 3 A is indeed repulsive for an
oxygen-aryl interaction, the shortest observed distance for
such interactions being 3.16 A. Additionally, we analyzed the
noncompetitive one while it had no effect on compound 1’s
competitive mode.25 Therefore, Ala1142.58 was identified as
the critical residue for the apparent noncompetitive mode of
antagonism by compound 3 at the guinea pig versus its
competitive one at human NK3 Thr1392.58 receptor.
˚
distance between alkyl-aryl groups. Here, the most favorable
distance determined by a peak in the distance histogram is
˚
between 3.6 and 4.2 A. It was thus concluded that the two
Tovisualize thesedata, wecarefullyanalyzed the previously
reported docking poses ofcompounds1 and3 ontothe 7TMD
binding cavity of the human NK3 model (Figure 7A).25
Strikingly, compound 3 locates two of its carbon atoms at a
carbon atoms of compound 3 located in the region close to
2.58are responsiblefor the apparentnoncompetitivebehavior
and slow dissociation rate of compound 3 at the guinea pig
receptor. We then hypothesized that in general NK3 anta-
gonists with a heavy atom located in this region of the binding
pocket should slow the dissociation rate at gpNK3 and there-
fore show an apparent noncompetitive binding kinetics.
Possible heavy atoms should be any that can form hydro-
phobic interactions with alanine and are repulsive to oxygen
distance below 3 A to the oxygen of hNK3 Thr1392.58
˚
˚
(Figure 7B). At such a short distance (<3 A), the oxygen-
aryl interaction is repulsive. On the other side, the distance
to Cβ of gpNK3 Ala1142.58 is within the favorable range
of an alkyl-aryl interaction. Thus, compound 3 forms in
gpNK3, an additional attractive interaction that is not present
in hNK3. This change between an unfavorable situation in
hNK3 to a favorable one in gpNK3 can explain the obser-
ved switch from fast to slow dissociation rate from hNK3
to gpNK3. On the other hand, compound 1, which is not
˚
at a distance below 3 A, therefore besides carbon, fluorine
should also fulfill this criterion. Compounds of the piperidine
series should hence be suitable to validate this hypothesis. In
their predicted docking pose, derivatives with a para-substi-
tuted phenyl ring locate the para substituent onto compound

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7109
Figure 7. (A) Proposed docking poses of 3 (osanetant) (magenta) and 1 (talnetant) (blue) as described in our previous work.25 (B) Zoom on the
region of difference in the human and guinea pig receptor. The distances of the two carbon atoms of compound 3 that are closest to hNK3
Thr1392.58 are displayed and given in A: in red the distances to the oxygen atom of hNK3 Thr1392.58, in blue the distances to the Cβ of gpNK3
˚
Ala1142.58. (C) Compound 6 (piperidine series, orange) docked additionally to compounds 1 (blue) and 3 (magenta) into the human NK3.
(D) Zoom on the region of difference between the two species with the same ligands as shown in (C). The distances of the fluorine atom of
compound 6 to the residue in position 2.58 are displayed and given in A: in red the distance to the oxygen atom of hNK3 Thr1392.58, in blue the
˚
distance to the Cβ of gpNK3 Ala1142.58
.
3’s carbon that is responsible for the apparent noncompetitive
kinetics (Figure 7C,D). Thus, it was predicted that the para
substituted derivatives, compounds 5 and 6, should have an
apparent noncompetitive behavior at gpNK3 while para-H
derivatives such as compound 7 should be competitive.
Compounds 5, 6, and 7 are potent and selective hNK3
antagonists, their structures belonging to a chemical class differ-
ent from compounds 1 and 3. As predicted by modeling, com-
pounds 5 and 6 showed an apparent noncompetitive mode of
action, very much like compound 3, at the gpNK3, while
compound 7 behaved competitively. The mutation A1142.58T
in gpNK3 also converted the apparent noncompetitive behavior
of compounds 5 and 6 to a competitive one, very similar to that
of hNK3-WT. This confirmed the hypothesis that the placement
of a heavy atom close to Ala2.58 can trigger the apparent non-
competitive behavior at gpNK3. Compounds 6 and 5, differing
in the piperidine substituent, showed that this part does not
contribute to the special binding kinetics. Consequently, we have
discovered a region in the binding site of the NK3 receptor that is
responsible for different modes of antagonism at hNK3 and
gpNK3 when occupied by an antagonist carrying a hydrophobic
atom like carbon or fluorine. In the hNK3 receptor, antagonists
with a heavy atom in this space show a fast dissociation rate;
hence a competitive behavior because the interaction with hNK3
Thr1392.58 is repulsive. In the gpNK3, the interaction of a heavy
atom in this region with gpNK3 Ala1142.58 is attractive. By
providing an anchor point, this additional favorable hydro-
phobic interaction is leading to a slower dissociation rate and
thus an apparent noncompetitive behavior.
In conclusion, a single point mutation of guinea pig NK3
receptor changes the mechanism of action of compound 3 by
affecting its dissociation kinetics. To the best of our know-
ledge, this is the first time that binding kinetics is predictable
across various chemical series on the rational basis of a
common specific protein-ligand interaction. It remains to
be shown whether similar key protein-ligand interactions can
be found for the human NK3 receptor. This would be of high
importance because, as it has already been well documented
in the case of angiotensin AT1 receptor and many other
GPCRs,31-35 a noncompetitive (slow dissociating) antagonist
should possess in vivo a much longer duration of action and
greater efficacy than a competitive (rapidly dissociating) one.
Experimental Section
Chemistry. All reactions were carried out under argon atmo-
sphere. Dry solvents and reagents of commercial quality were
used as purchased. Column chromatography was carried out on
silica gel 60 (32-60 mesh) or on prepacked columns (Isolute
Flash Si). Proton NMR spectra were obtained on Bruker 300 or
400 MHz instrument with chemical shifts relative to tetra-
methylsilane as internal standard. Mass spectra were recorded
on SSQ 7000 (Finnigan-MAT) for electron impact ionization.
The purity of all final compounds was assessed by LC-MS and
found above 99%.
(S)-(þ)-N-((3-[1-Benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]-
prop-1-yl)-4-phenylpiperidin-4-yl)-N-methylacetamine (3, osane-
tant, SR 142801),14 (S)-(-)-N-(R-ethylbenzyl)-3-methyl-2-phenyl-
quinoline-4-carboxamide (2, SB 222200),16 (S)-(-)-N-(R-ethylben-
zyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (1, talnetant, SB

7110 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Malherbe et al.
223412),16 (2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-
methyl]-1-azabicyclo[2.2.2]octan-3-amine (CP-96,34),36 and 1-{2-[-
(R)-3-(3,4-dichloro-phenyl)-1-(3,4,5-trimethoxy-benzoyl)-pyrro-
lidin-3-yl]-ethyl}-4-phenyl-piperidine-4-carboxylic acid amide
(MDL 105,212)37 were synthesized according to procedures
described in published literature and their purity was confirmed
(>99% by LC-MS analysis).
The preparation, NMR, and MS spectroscopic description of
compounds 2-(3,4-dichlorophenyl)-N-(4-fluoro-3-trifluoromethyl-
benzyl)-2,N-dimethyl-4-[4-(2-oxo-pyrrolidin-1-yl)-piperidin-
1-yl]-butyramide (5),26 4-[4-(acetyl-methyl-amino)-piperidin-1-
yl]-2-(3,4-dichloro-phenyl)-N-(4-fluoro-3-trifluoromethyl-ben-
zyl)-2,N-dimethyl-butyramide (6),26 and 4-[4-(acetyl-methyl-
amino)-piperidin-1-yl]-N-(2-chloro-benzyl)-2-(3,4-dichloro-
phenyl)-2,N-dimethyl-butyramide (7)26 are described in the Sup-
porting Information.
Nonspecific binding was measured in the presence of 10 μM
compound 2. The radioactivity on the filter was counted (5 min)
on a Packard Top-count microplate scintillation counter with
quenching correction after addition of 45 μL of microscint 40
€
(Canberra Packard SA, Zurich, Switzerland) and shaking for
1 h. Saturation experiments were analyzed by Prism 4.0
(GraphPad software, San Diego, CA) using the rectangular
hyperbolic equation derived from the equation of a bimolecular
reaction and the law of mass action, B = (Bmax[F])/(KD þ [F]),
where B is the amount of ligand bound at equilibrium, Bmax is
the maximum number of binding sites, [F] is the concentration
of free ligand, and Kd is the ligand dissociation constant. For
inhibition experiments, membranes were incubated with [3H]3
at a concentration equal to Kd value of radioligand and 10
concentrations of the inhibitory compound (0.0003-10 μM).
IC50 values were derived from the inhibition curve and the
affinity constant (Ki) values were calculated using the Cheng-
Prussoff equation Ki = IC50/(1 þ [L]/Kd) where [L] is the
concentration of radioligand and Kd is its dissociation constant
at the receptor, derived from the saturation isotherm. The
association and dissociation kinetics for radioligand [3H]3 in
the membrane preparations from HEK293 cells transiently
expressing hNK3-WT, gpNK3-WT, or gpNK3-A1142.58T were
measured as previously described.25 Binding kinetics para-
meters, Kob and Koff values (observed on and off rates), were
derived from association-dissociation curves using the one
phase exponential association and decay equations (Prism 4.0,
GraphPad software), respectively. Kon, half-life, and Kd were
calculated using the Kon = (Kob - Koff)/[ligand], t1/2 = ln2/K
and Kd = Koff/Kon equations, respectively.
Materials. Radioligands [3H]3 ([3H]SR 142801, catalogue
no. TRK1035, specific activity: 74.0 Ci/mmol, radiochemical
purity of 99.7% by HPLC, Novapak C18 column), [3H]SP
([3H]Substance P, catalogue no. TRK786, specific activity:
40.0 Ci/mmol), and [3H]4 ([3H]SR 48968, catalogue no.
TRK398, specific activity: 27.0 Ci/mmol, radiochemical purity
of 97.8% by HPLC Aquapore RP-300 octyl 7 μm) were pur-
chased from GE Healthcare UK limited, Chalfont St. Giles,
UK. [MePhe7]Neurokinin
B
(Asp-Met-His-Asp-Phe-Phe-
NMe-Phe-Gly-Leu-Met-NH2, catalogue no. SC981) was pur-
chased from NeoMPS SA (Strasbourg, France). [myo-
1,2-3H]Inositol with PT6-271 (TRK911, specific activity: 16.0
Ci/mmol) and yttrium silicate (Ysi) RNA binding beads
(RPNQ0013) were purchased from GE Healthcare.
Plasmids, Cell Culture, and Membrane Preparation. The
cDNAs for guinea pig NK3 receptor, gpNK3 (accession number:
P30098), was isolated by RT-PCR from a midbrain cDNA
library. cDNA encoding the human NK3 receptor, hNK3
(accession No. P29371), hNK2 (accession number: P21452),
hNK1 (accession number: P25103), and gpNK3 were sub-
cloned into pCI-Neo expression vectors (Promega Corporation,
Madison, WI). All point mutants were constructed using the Quick-
Change site-directed mutagenesis kit (catalogue no. 200518,
Stratagene, La Jolla, CA).The entire coding regions of all point
mutants were sequenced from both strands using an automated
cycle sequencer (Applied Biosystems, Foster City, CA).
Human embryonic kidney (HEK)293 cells were transfected as
previously described.25 Forty-eight hours posttransfection, the
cells were harvested and washed three times with ice-cold PBS
and frozen at -80 °C. The pellet was suspended in ice-cold
50 mM Tris-HCl pH 7.4 buffer containing 10 mM EDTA (10 ꢀ
volume) and homogenized with a polytron (Kinematica AG,
Basel, Switzerland) for 30 s at 16000 rpm After centrifugation at
48000g for 30 min at 4 °C, the pellet was suspended again in ice-
cold 10 mM Tris pH 7.4 buffer containing 0.1 mM EDTA (10 ꢀ
volume), homogenized, and spun again as above. The pellet was
resuspended in ice-cold 10 mM Tris pH 7.4 buffer containing
0.1 mM EDTA and 10% sucrose (5 ꢀ volume). The membrane
homogenate was frozen at -80 °C before use.
Radioligands [3H]SP and [3H]4 ([3H]SR 48968) Bindings.
hNK1 and hNK2 receptor binding experiments were performed
as described above for radioligand [3H]3 binding, using radio-
ligands [3H]SP and [3H]4 and membrane isolated from
HEK293 transiently expressing recombinant hNK1 and hNK2
receptors, respectively. Briefly, after thawing, the membrane
homogenates were resuspended and homogenized using a poly-
tron in the 50 mM Hepes, 3 mM MnCl2, 2 μM phosphoramidon,
16.8 μM Leupeptin, and 0.04% BSA binding buffer at pH 7.4 for
the hNK1 membrane and in the 50 mM Tris-HCl, 3 mM MnCl2,
4 μg/mL Chymostatin, and 0.04% BSA binding buffer at pH 7.4
for the hNK2 membrane to a final assay concentration of 2.5 μg
protein/well for both NK membranes. For inhibition experi-
ments, hNK1 and hNK2 membranes were incubated with
0.6 nM of radioligand [3H]SP (Kd =0.6 nM) and 0.32 nM of
radioligand [3H]4 (Kd = 0.32 nM), respectively and 10 concen-
trations of the inhibitory compound (0.0003-10 μM) (in a total
reaction volume of 500 μL) for 75 min at room temperature
(RT). Nonspecific bindings for radioligands [3H]SP and [3H]4
were measured in the presence of 10 μM (2S,3S)-cis-
2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-azabicyc-
lo[2.2.2]octan-3-amine (CP-96,345)36 and 1-{2-[(R)-3-(3,4-dich-
loro-phenyl)-1-(3,4,5-trimethoxy-benzoyl)-pyrrolidin-3-yl]-eth-
yl}-4-phenyl-piperidine-4-carboxylic acid amide (MDL 105,
212),37 respectively.
Radioligand [3H]3 Binding. After thawing, the membrane
homogenates were centrifuged at 48 000g for 10 min at 4 °C,
and the pellets were resuspended in the binding buffer (50 mM
Tris-HCl, 4 mM MnCl2, 1 μM phosphoramidon, 0.1% bovine
serum albumin at pH 7.4) to a final assay concentration of 5 μg
protein/well. Saturation isotherms were determined by addition
of various concentrations of radioligand [3H]3 (0.009 to 3 nM)
to these membranes (in a total reaction volume of 500 μL) for 75
min at room temperature (RT). At the end of the incubation,
membranes were filtered onto unitfilter (96-well white micro-
plate with bonded GF/C filter preincubated 1 h in 0.3% poly-
ethylenimine þ 0.3% bovine serum albumin; PerkinElmer Life
and Analytical Sciences, Waltham, MA) with a FilterMate-96
harvester (PerkinElmer Life and Analytical Sciences) and
washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer.
[3H]Inositol Phosphates (IP) Accumulation Assay. [3H]Ino-
sitol phosphates accumulation was measured as described
previously with the following adaptations.25 The HEK293 cell
was transfected with various NK3 cDNAs in pCl-Neo using
Lipofectamine Plus reagent (Invitrogen) according to the
manufacturer’s instruction. Twenty-four hours after trans-
fection, cells were washed twice in labeling medium: Dulbec-
co’s modified Eagle’s medium without inositol (MP Bio-
medicals, Irvine, CA), 10% fetal calf serum, 1% penicillin/
streptomycin, and 2 mM glutamate. Cells were seeded at 8 ꢀ
104 cells/well in poly-D-lysine-treated 96-well plates in the label-
ing medium supplemented with 5 μCi/mL myo-[1,2-3H]-inositol.
On the day of assay (48 h post-transfection), cells were washed
three times with the buffer (1ꢀ HBSS, 20 mM HEPES, pH 7.4)
and then incubated for 10 min at RT in assay buffer (1ꢀ HBSS,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7111
20 mM HEPES, pH 7.4 containing 8 mM LiCl, final concentra-
tion, to prevent phosphotidyl-inositide breakdown) prior to the
addition of agonists or antagonists. When present, antagonists
were incubated for 20 min at 37 °C prior to stimulation with
agonist [MePhe7]NKB, concentrations ranged from 10 μM to
0.1 nM. After 45 min incubation at 37 °C with [MePhe7]NKB,
the assay was terminated by the aspiration of the assay buffer
and the addition of 100 μL 20 mM formic acid to the cells. After
shaking for 30 min at 23 °C, a 40 μL aliquot was mixed with
80 μL of yttrium silicate beads (12.5 mg/mL) that bind to the
inositol phosphates (but not inositol) and shaken for 30 min at
23 °C. Assay plates were centrifuged for 2 min at 750g prior to
counting on a Packard Top-count microplate scintillation
counter with quenching correction (PerkinElmer Life and Ana-
lytical Sciences).
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Suzuki, H. Distribution and pharmacological characterization of
primate NK-1 and NK-3 tachykinin receptors in the central
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Model Building. The hNK3 7TMD model, based on the X-ray
structure of bovine rhodopsin,38 was previously described.25 In
short, the amino acid sequences of hNK3 and gpNK3 were
aligned to the sequence of bovine rhodopsin using the ClustalW
multiple alignment program.25 The alignments were then veri-
fied to ensure that conserved residues of the transmembrane
regions were aligned and manually adjusted in the second
extracellular loop (E2) in order to align the conserved cysteine,
which takes part in the disulfide bridges occurring between the
third transmembrane segment (TM3) and the second extracel-
lular loop (E2).25 Using this alignment and the X-ray structure
of bovine rhodopsin (PDB code: 1u19) as template, the software
package MOE (MOE v.2005.05, Chemical Computing Group,
Montreal, Quebec, Canada) was used to generate a three-
dimensional model of the human NK3.25 Compound 3 was then
manually docked into the binding site according to known
structure-activity relationship data and the protein-ligand
complex minimized.25 Finally, compound 1 has also been
manually docked into the thus obtained hNK3 model, again
according to known structure-activity relationship data.25
These docking modes have been validated in our previous work
by site-directed mutagenesis data.25 This original hNK3 model
was subsequently used in the current work to link the observed
differences in mode of antagonism with a chemical substructure
of compound 3. Besides, it was used to dock piperidine-based
NK3 antagonists (5, 6 and 7)26 into the binding site. This was
achieved by aligning the chemical structures onto the already
docked compound 3 and optimizing the conformation of the amide
side chain to properly fit into the binding cavity. The Ballesteros-
Weinstein numbering scheme of the amino acids in the binding
site is given to facilitate the comparison with other GPCRs.39
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D. N.; Davies, C. H.; Hagan, J. In vitro and in vivo characterization
of the non-peptide NK3 receptor antagonistSB-223412(talnetant):
potential therapeutic utility in the treatment of schizophrenia.
Neuropsychopharmacology. 2008, 33, 1642–1652.
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Soubrie, P.; Steinberg, R. Activation of dopaminergic and choli-
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an in vivo microdialysis approach in guinea pig. Neuropeptides
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B.; Poncelet, M.; Proietto, V.; Van Broeck, D.; Vilain, P.; Neliat,
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tachykinin NK3 receptor. Life Sci. 1995, 56, PL27–PL32.
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C.; Medhurst, A. D.; Graziani, D.; Schmidt, D. B.; Rigolio, R.;
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223412). J. Med. Chem. 1999, 42, 1053–1065.
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D. B.; Webb, E. F.; Martin, L. D.; Brawner, M. E.; Elshourbagy,
N. A.; Medhurst, A. D.; Giardina, G. A.; Hay, D. W. Nonpeptide
tachykinin receptor antagonists: I. Pharmacological and pharmaco-
kinetic characterization of SB 223412, a novel, potent and selective
neurokinin-3 receptor antagonist. J. Pharmacol. Exp. Ther. 1997,
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antagonists: the next generation of antipsychotics? Nat. Rev. Drug
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Acknowledgment. We are grateful to Dr. Josef Schneider
for performing NMR spectra. We thank Philippe Jablonski
and Walter Vivian for their expert technical assistance.
Supporting Information Available: Preparation, NMR, and
MS spectroscopic description of compounds 5, 6, and 7. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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