Identification of a crucial amino acid in the helix position 6.51 of human tachykinin neurokinin 1 and 3 receptors contributing to the insurmountable mode of antagonism by dual NK1/NK3 antagonists

Article abstract of DOI:10.1021/jm2017072

The neurokinins are neuropeptides that elicit their effect through three GPCRs called NK₁, NK₂, and NK₃. Compounds 5 and 6 are dual hNK₁ (K_i of 0.7 and 0.3 nM) and hNK ₃ (K_i of 2

Full text of DOI:10.1021/jm2017072

Article
pubs.acs.org/jmc
Identification of a Crucial Amino Acid in the Helix Position 6.51 of
Human Tachykinin Neurokinin 1 and 3 Receptors Contributing to the
Insurmountable Mode of Antagonism by Dual NK1/NK3 Antagonists
Caterina Bissantz,† Claudia Bohnert,‡ Torsten Hoffmann,† Anne Marcuz,‡ Patrick Schnider,†
and Pari Malherbe*,‡
‡
†Medicinal Chemistry and DTA CNS, pRED, Pharma Research and Early Development, F. Hoffmann-La Roche AG,
Grenzacherstrasse 124, CH4070, Basel, Switzerland
ABSTRACT: The neurokinins are neuropeptides that elicit their effect through three GPCRs called NK1, NK2, and NK3.
Compounds 5 and 6 are dual hNK1 (Ki of 0.7 and 0.3 nM) and hNK3 (Ki of 2.9 and 1.7 nM) antagonists. Both compounds
exhibit an insurmountable mode of antagonism at hNK1, whereas at hNK3, they differ in that 5 is an insurmountable but 6 a
surmountable antagonist. Using homology modeling and site-directed mutagenesis, hNK1-Phe264 and hNK3-Tyr315 were found
to be the molecular determinants of hNK1 and hNK3 antagonism by 5 and 6. In [3H]IP studies, the mutation hNK1-F264Y
converted the mode of action of 5 from insurmountable to partial insurmountable antagonism while it had no effect on that of 6.
Conversely, the mutation hNK3-Y315F enhanced the insurmountable behavior of 5 and converted 6’s surmountable to an
insurmountable antagonism. This finding was further confirmed by characterizing additional derivatives of 5 and 6, most notably
with a hybrid structure.
The family of neurokinin (NK, also called tachykinin)
peptides is composed of substance P (SP), neurokinin A
(NKA), and neurokinin B (NKB), which act as neuro-
transmitters/neuromodulators and elicit their effects through
three types of neurokinin receptors, NK1, NK2, and NK3
(nomenclature follows Alexander et al.11). The NK receptors
belong to the class A family (rhodopsin-like) of G-protein-
coupled receptors (GPCRs) that are coupled via Gq/11 to the
activation of the phospholipase C-IP3/DAG signaling pathway
leading to an elevation of intracellular Ca2+ levels.12,13 Among
NK receptors, NK3 receptors are of particular interest because
of their possible role in the pathophysiology of schizophrenia.7,9
Preclinical studies have demonstrated the involvement of
NKB/NK3-mediated activation in the release of dopamine,
especially in the ventral and dorsal striatal regions.14
Interestingly, acute administration of compound 4 (talnetant)
produced significant increases in extracellular dopamine and
noradrenalin in the medial prefrontal cortex of guinea pigs.15
SP/NK1 signaling plays a major role in the modulation of stress
responses and in the regulation of affective behavior. It has
been shown that various emotional stressors increase SP efflux
INTRODUCTION
■
Schizophrenia is a debilitating mental disorder that affects
0.5−1% of the general population. It is characterized by positive
(delusions, hallucinations, and paranoia), negative (apathy and
lack of social interaction), and mood (anxiety, dysphoria and
suicidality) symptoms, as well as cognitive impairment
(memory and attention deficits).1 Current therapies, mainly
based on antagonizing D2/5-HT2A receptors, are largely
effective against positive symptoms but have a limited efficacy
in the treatment of negative and cognitive symptoms. These
treatments also suffer from several side effects including weight
gain, diabetes, and extrapyramidal symptoms.2 The results of
randomized double-blind placebo controlled phase II clinical
trials of (S)-(+)-N-((3-[1-benzoyl-3-(3,4-dichlorophenyl)-
piperidin-3-yl]prop-1-yl)-4-phenylpiperidin-4-yl)-N-methyla-
cetamine (osanetant, SR 142801)3,4 and (S)-(−)-N-(α-ethyl-
benzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (talnetant,
SB 223412),5,6 two potent non-peptide antagonists of the
neurokinin 3 receptor in schizophrenia patients, have suggested
a significant improvement in psychopathology including positive
symptoms, with improved efficacy, but a lower propensity to
induce side effects.7−9 Thus, neurokinin 3 receptor antagonism
represents an alternative therapeutic approach for the treatment
of schizophrenia.10
Received: December 19, 2011
Published: May 10, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of the selective NK1, NK3, and dual NK1/NK3 antagonists.
in discrete forebrain areas such as amygdala and septum.16 The
pharmacological blockade or genetic disruption of NK1
expression has been shown to result in a marked reduction of
anxiety and stress-related responses and also increased 5-HT-
mediated function.17,18 Although the NK1 receptor antagonists
2-(R)-(1-(R)-3,5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-
fluoro)phenyl-4-(3-oxo-1,2,4-triazol-5-yl)methylmorphine)
(aprepitant, MK-869),19 L-759274,20 and (+)-(2S,3S)-3-(2-
methoxy-5-trifluoromethoxybenzyl)amino-2-phenylpiperidine
(CP-122,721)21 were shown to reduce symptoms of depression
in phase II studies, this could not be confirmed for aprepitant in
a phase III trial.22 Several NK1 receptor antagonists are
currently in clinical development for depression and other
psychiatric disorders, such as anxiety.22 Moreover, recent results
from two randomized, double-blind, placebo-controlled phase
II studies with the novel NK1 antagonist (2R,4S)-4-(4-acetyl-1-
piperazinyl)-N-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl]-
2-(4-fluoro-2-methylphenyl)-N-methyl-1-piperidinecarboxa-
mide (casopitant, GW 679769)23 in patients with major
depressive disorder suggest that NK1 antagonists with nearly
complete receptor occupancy could be effective in the
treatment of depression.24 On the basis of this evidence, it
can be hypothesized that the combination of NK3 and NK1
receptor antagonism may provide a therapeutic benefit for
positive, negative, and mood symptoms associated with
schizophrenia.
(2R,3S)-2-(3,5-Bis-trifluoromethylphenyl)-N-[4-(4-fluoro-2-
methylphenyl)-6-(3-hydroxy-2-hydroxymethylpyrrolidin-1-yl)-
pyridin-3-yl]-N-methylisobutyramide (RO4583298)25 is a
novel NK1/NK3 antagonist that binds with high affinity to
hNK1 (Ki = 0.7 nM) and hNK3 (Ki = 2.9 nM). Its in vitro and
in vivo characterization has demonstrated that it had an
insurmountable (also referred as apparent noncompetitive or
pseudo-irreversible26) and long-lasting mode of antagonism at
both hNK1 and hNK3.27 Compound 5 (RO4583298) robustly
blocked the gerbil foot tapping response in a dose-dependent
manner with an ED50 of 0.4 mg/kg (po) and with a long-lasting
in vivo effect. Remarkably, this long-lasting in vivo receptor
blockade by compound 5 coincided well with its insurmount-
able mode of antagonism observed in vitro.27 Recently,
researchers at GSK have reported novel dual NK1/NK3
antagonists based on a similar pyridine scaffold as compound
5.28,29 Interestingly, among these compounds, 2-[3,5-bis-
(trifluoromethyl)phenyl]-N-{4-(4-fluoro-2-methylphenyl)-6-
[(4-(hydroxymethyl)-1-methyl-5-oxo-3-pyrrolidinyl]-3-pyridin-
yl}-N,2-dimethylpropanamide (enantiomer 2) (GSK, “com-
pound 17a”)28,29 has a high structural similarity to compound 5
and differs only in the pyrrolidine substituent (Figure 1).
Compound 17a was reported to have a high potency at NK1
(Ki = 0.06 nM) and NK3 (Ki = 1 nM), as measured in a
functional cell-based assay, and also displayed an insurmount-
able profile of antagonism at both NK1 and NK3.29
The objective of the current study was to elucidate the
molecular interaction between human NK1/NK3 and com-
pound 5, which is involved in the slow kinetics and
insurmountable behavior at both receptors. Using site-directed
mutagenesis, rhodopsin-based hNK1 and hNK3 modeling, and
functional [3H]IP accumulation assay, we have identified a
single amino acid in the helix position 6.51 of both NK1 and
NK3 as a crucial residue that determines the insurmountable
behavior of compound 5. We then hypothesized that the
hydroxymethyl (−CH2OH) group of compound 5 was needed
for this insurmountable behavior. The antagonism modes of six
close analogues with or without −CH2OH group were further
analyzed, and the results have strengthened our hypothesis.
RESULTS
■
Inhibition Mode of NK Antagonists on the SP- or
[MePhe7]NKB-Evoked Accumulation of [3H]IP at Human
NK1 and NK3 Receptors. Compound 5 and its derivative
(S)-2-(3,5-bis-trifluoromethylphenyl)-N-[4-(4-fluoro-2-methyl-
phenyl)-6-((S)-4-methanesulfonyl-3-methylpiperazin-1-yl)-
pyridin-3-yl]-N-methylisobutyramide (RO4908594), which or-
iginated from an internal drug discovery program,25 belong
to a novel structural class binding to both NK1 and NK3.
Their chemical structures are different from those of selective
antagonists of NK1 1 (aprepitant) and (2S,3S)-cis-2-(diphe-
nylmethyl)-N-[(2-methoxyphenyl)methyl]-1-azabicyclo[2.2.2]-
octan-3-amine (CP-96,345)30 and of NK3 3 (osanetant) and 4
(Figure 1). The biochemical characterization of compounds 5
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Figure 2. Schild plot analyses for antagonism of SP-induced accumulation of [3H]IP by compounds 1, 2, 5, and 6: concentration−response curves
for [3H]IP formation stimulated by SP in the absence and presence of various concentrations of compound 1 (A, B), compound 2 (C, D),
compound 5 (E, F), and compound 6 (G, H) in HEK293 cells expressing transiently the hNK1-WT or hNK1-F264Y6.51. Each curve represents the
mean of six concentration−response measurements from three independent transfections.
and 6 (RO4908594) has been previously described.27,31 To
compare the inhibition mode of 5, 6, and selective NK1 and
NK3 antagonists for NK1 and NK3, we measured the
concentration−response curves (CRCs) for [3H]IP formation
stimulated by SP (NK1 agonist) or [MePhe7]NKB (NK3
agonist) in the absence or presence of increasing concen-
trations of antagonist in HEK293 cells transiently transfected
with various NK receptors. In the hNK1 expressing cells, SP
elicited concentration-dependent increases in the accumulation
of [3H]IP with the EC50 of 0.6 0.0 nM. Consistent with a
previous report,19 compound 1 displayed an insurmountable
mode of antagonism at hNK1, shifting SP CRCs to the right
with a concomitant decrease in maximal response (Figure 2A
and Table 1), whereas compound 2 (CP-96,345)30 behaved as
a surmountable antagonist at hNK1, shifting the SP CRCs to
the right without changing its maximal response (Figure 2C,
and pA2 and Schild slope values shown in Table 1). Both
compounds 5 and 6 also behaved as insurmountable
antagonists at hNK1, producing a rightward shift in SP CRC
as well as a full suppression of the maximal response (Figure
2E,G and Table 1).
[MePhe7]NKB was used as agonist because it has been
shown to have a high selectivity for hNK3 (Ki = 0.3 nM)
versus hNK2 (Ki = 1597 nM) and hNK1 (Ki > 10000 nM).6
[MePhe7]NKB elicited concentration-dependent increases in
the accumulation of [3H]IP in HEK293 cells expressing hNK3
with an EC50 of 0.71
0.0 nM. Compound 5 displayed an
insurmountable mode of antagonism at the hNK3 receptor
(Figure 3C and Table 2), while compound 6 behaved in a
surmountable manner at hNK3 (Figure 3E and Table 2). Both
compounds 3 and 4 behaved as surmountable antagonists at
hNK3, shifting the NKB CRC to the right without changing its
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Table 1. Schild Analyses for the Antagonism of SP-Induced Accumulation of [3H]IP by Selective NK1 and Dual NK1/NK3
a
Antagonists in HEK293 Cells Expressing Transiently the hNK1-WT or hNK1-F264Y6.51
SP-induced [3H]IP accumulation
hNK1-WT
Schild slope
hNK1-F264Y6.51
Schild slope
NK antagonist
pA2
mode of antagonism
pA2
mode of antagonism
1
ND
8.27
ND
ND
ND
ND
ND
ND
ND
ND
1.19
ND
ND
ND
ND
ND
ND
ND
insurmountable
surmountable
8.08
7.78
7.54
7.40
7.77
ND
ND
ND
ND
1.76
0.98
2.24
2.43
2.69
ND
ND
ND
ND
surmountable
2
surmountable
5
insurmountable
insurmountable
insurmountable
insurmountable
insurmountable
insurmountable
insurmountable
partial insurmountable
partial insurmountable
partial insurmountable
insurmountable
7
9
8
6
insurmountable
10
11
insurmountable
insurmountable
a
The apparent antagonist potency (pA2, −log of the molar concentration of antagonist producing a 2-fold shift of the concentration response curve)
and Schild slope values of antagonists at hNK1-WT and hNK1-F264Y6.51 were determined using SP CRCs in the absence and presence of various
concentrations of antagonist and Schild plot analyses. ND, not determined.
maximal response (Figure 3A and pA2 and slope values shown
in Table 2).
different kinetics of compound 6 at the two receptors, we also
included them in the mutation studies to further validate our
hypothesis.
To explore which structural elements determine the
antagonistic mode of action of compounds 5 and 6 at NK1
and NK3, the five derivatives 7, 8, 9, 10, and 11 (Figure 4) were
further investigated in the SP- or NKB-induced formation of
[3H]IP assay in HEK293 cells transiently expressing hNK1 and
hNK3. Compounds 7−11 are dual NK1/NK3 antagonists,25 and
their selection was guided by our docking model that predicted
the hydroxymethyl group on compounds 5, 7, and 9 to be
involved in the insurmountable mode of antagonism at NK3. All
five antagonists 7−11 had an insurmountable profile at hNK1
(Table 1). As predicted, compounds 5, 7, and 9 acted in an
insurmountable manner at hNK3 (Figure 3C,G and Table 2),
while compounds 8, 10, and 11 displayed a surmountable
mode of antagonism at hNK3 (pA2 and slope values shown in
Table 2).
Mutant Selection. Comparing the structure of the two
NK1/NK3 antagonists 5 and 6, we concluded that the difference
in their kinetic behavior should be caused by interactions of the
amine substituents with the receptor. These substituents are
predicted by the model to be located in the pocket formed by
residues of the TMDs 3, 5, and 6. Important in terms of binding
energy, it struck us that compound 5 with its −CH2OH group
may form a hydrogen bond with the OH group of hNK3-
Tyr3156.51 while compound 6 cannot form a hydrogen bond
with the phenolic OH. Normally, hydrogen bond donors without
any interaction partner should cause a loss of binding energy and,
if this hypothesis is correct, the mutation hNK3-Y315F6.51 should
lead to an insurmountable behavior of compound 6. This would
also fit the fact that both compounds are insurmountable at
hNK1, which has Phe2646.51. In analogy, we also investigated the
mutation hNK1-F264Y6.51 with the expectation that compound 6
should then become surmountable.
Effect of the Mutation at Helix Position 6.51 on
Antagonism Mode of NK Antagonists. An alignment of the
amino acids in the seven-transmembrane domain (7TMD)
forming the NK receptor binding pockets toward the 7TMD
of bovine rhodopsin (PDB code 1F88)32 and human
β2-adrenergic receptor (PDB code 2RH1)33,34 is shown in
Figure 5. The only amino acid difference in TM6 between hNK1
and hNK3 is the residue Phe2646.51 in hNK1, which corresponds
to Tyr3156.51 in hNK3 (Figure 5). Our docking models of NK1
and NK3 predicted that the residue at helix position 6.51 might
contribute to the mode of antagonism of NK antagonists.
Hence, hNK1-Phe2646.51 was converted to Tyr2646.51 and and
hNK3-Tyr3156.51 to Phe3156.51. Since the mutation hNK1-
F264Y6.51 had no effect on SP potency and the mutation hNK3-
Y315F6.51 had a minor effect on [MePhe7]NKB potency, the
antagonism modes of compounds 1−11 were compared at the
mutated hNK1-F264Y6.51 and hNK3-Y315F6.51 receptors using
Schild analyses. As seen, the mutation hNK1-F264Y6.51
converted compound 1 from insurmountable to surmountable
(Figure 2B and Table 1) and compounds 5, 7, and 9 from
insurmountable to partially insurmountable (Figure 2F and
Table 1) and had no effect on antagonism modes of compounds
2, 6, 8, 10, 11 (Figure 2D,H and Table 1). Conversely, hNK3-
Y315F6.51 converted compound 6 from a surmountable to an
insurmountable antagonist (Figure 3F and Table 2), while it had
no effect on the modes of antagonism of compounds 3, 4, 8,
10, and 11 except for increasing their potency (Figure 3B and
Table 2). For compounds 5, 7, and 9 the mutation hNK3-
Y315F6.51 caused a further depression in maximal NKB response at
a compound concentration of 10 nM (Figure 3D,H and Table 2).
Effect of the Mutation at Helix Position 6.51 on
Binding Affinity of NK Antagonists As Measured by
Displacement Studies. The affinity constants of NK
antagonists in the membrane preparations from HEK293 cells
transiently expressing hNK1-WT, hNK1-F264Y, hNK3-WT, or
hNK3-Y315F are given in Table 3. The affinity constants of
compounds 1 and 2 were not affected by mutation hNK1-
F264Y (Figure 6A,B and Table 3). The mutation hNK3-Y315F
led to an increase in the Ki of compound 3 by 18-fold
(P = 0.0006), yet caused an increase in affinity of compound 4
In order to get additional validation of this hypothesis from
the ligand side, we tested the five structurally closely related
compounds 7−11 on these mutations, as well as two NK1 selec-
tive antagonists and two NK3 selective antagonists.
Depending on the exact conformation of His6.52, the −CH2OH
group could alternatively bind to a nitrogen of this side chain.
Also, the −OH function could bind to the phenolic OH of
Tyr5.38. Although these residues are conserved between hNK1
and hNK3, and thus these interactions would not explain the
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Figure 3. Schild plot analyses for antagonism of [MePhe7]NKB-induced accumulation of [3H]IP by compounds 4, 5, 6, and 9: concentration−
response curves for [3H]IP formation stimulated by [MePhe7]NKB in the absence and presence of various concentrations of compound 4 (A, B),
compound 5 (C, D), compound 6 (E, F), and compound 9 (G, H) in HEK293 cells expressing transiently the hNK3-WT or hNK3-Y315F6.51. Each
curve represents the mean of six concentration−response measurements from three independent transfections.
by a factor of 2.1 (P = 0.012) (Figure 6C,D and Table 3).
As seen in Figure 6 and Table 3, both compounds 5 and 6 bind
with similar affinity to hNK1-WT and hNK1-F264Y, but their
affinities were increased for mutation hNK3-Y315F by 4.1- and
8.5-fold (statistical significance of P = 0.0019 and P = 0.0063),
respectively, in comparison to the WT. Similarly, the mutation
hNK1-F264Y did not affected the affinity constants of
compounds 7−11, while the mutation hNK3-Y315F caused
increases in affinities of compounds 7−11 that were statistically
significant (Table 3).
Effect of the Mutations at Helix Positions 5.38 and
6.52 of NK3 Receptor on Antagonism Mode and Binding
Affinity of NK Antagonists. To investigate the role of hNK3
residues at helix positions 5.38 and 6.52 on the mode of
antagonism of NK antagonists, the NKB-induced formation
of [3H]IP was tested in HEK293 cells expressing transiently the
WT and mutated hNK3 receptors. The EC50, nH, and relative
Emax values, calculated from concentration−response curves of
[MePhe7]NKB, are given in Table 4. NKB was completely
unable to evoke accumulation of [3H]IP in HEK293 cells
transiently expressing the NK3 mutants: Y247A5.38, Y247W5.38
,
and H316F6.52. Except for a decrease in efficacy of NKB at
mutant Y247F5.38 (relative Emax was decreased by 46%), the
mutations Y247F5.38 and H316W6.52 had no effect on the
potency of NKB-induced accumulation of [3H]IP (Table 4).
Therefore, the mutants Y247F5.38 and H316W6.52 were further
chosen to study the antagonism modes of compounds 3, 4, 5,
and 6 by Schild analyses. The Schild constants (pA2 values and
Schild slope) for antagonism of NKB-induced accumulation of
[3H]IP by compounds 3, 4, 5, and 6 in HEK293 cells expressing
hNK3-WT, Y247F5.38, or H316W6.52 are given in Table 5.
The mutation Y247F5.38 had no effect on the surmountable
antagonism mode of compounds 3, 4, and 6 or on the
insurmountable mode of compound 5. Except for a decrease in
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Table 2. Schild Analyses for the Antagonism of [MePhe7]NKB-Induced Accumulation of [3H]IP by Selective NK3 and Dual
a
NK1/NK3 Antagonists in HEK293 Cells Expressing Transiently the hNK3-WT or hNK3-Y315F6.51
[MePhe7]NKB-induced [3H]IP accumulation
hNK3-WT
Schild slope
hNK3-Y315F6.51
NK antagonist
pA2
mode of antagonism
pA2
Schild slope
mode of antagonism
3
7.73
8.24
ND
ND
ND
6.92
7.79
6.91
7.01
1.59
0.88
ND
ND
ND
0.7
surmountable
surmountable
insurmountable
insurmountable
insurmountable
surmountable
surmountable
surmountable
surmountable
7.53
8.63
ND
ND
ND
7.68
ND
8.61
7.43
0.96
0.76
ND
ND
ND
0.71
ND
2.00
1.2
surmountable
4
surmountable
5
more insurmountable
more insurmountable
more insurmountable
surmountable
7
9
8
6
1.12
0.63
0.61
insurmountable
surmountable
10
11
surmountable
a
The apparent antagonist potency (pA2) and Schild slope values of antagonists at hNK3-WT and hNK3-Y315F6.51 were determined using NKB
CRCs in the absence and presence of various concentrations of antagonist and Schild plot analyses. ND, not determined.
Figure 4. Chemical structures of the dual NK1/NK3 antagonists, 2-phenyl-N-(pyridin-3-yl)-N-methylisobutyramide derivatives, compounds 5−11.
The group that controls the mode of antagonism is indicated by a yellow highlighted circle.
Figure 5. Alignment of the amino acids forming the binding site of NK1, NK2, and NK3 toward the 7TMD of bovine rhodopsin (PDB code 1F88)32
and human β2-adrenergic (PDB code 2RH1)33,34 receptors. The Ballesteros−Weinstein numbering scheme of the amino acids47 are given to
facilitate the comparison with other GPCRs (see Experimental Section). The conserved residue in each TM of rhodopsin that is assigned as 50 is
shown in the bottom row. The numbers above the NK3R_HUMAN and NK1R_HUMAN receptors give the sequence number of the position of
the mutation carried out in this study. The residue at helix position 6.51, which is different between NK1 and NK3 binding pockets, is boxed and
yellow highlighted. Four residues at helix positions 5.47, 6.48, 6.51, and 6.52 that have been shown in the high resolution X-ray structures to be
involved in the toggle switch mechanism of hβ2AR activation33,34 and bovine rhodopsin activation32 are indicated in bold underlined with their
amino acid sequence number shown above.
potency of compound 4, the mutation of H316W6.52 had no
effect on the mode of antagonism of compounds 3 and 4, but it
completely abolished the ability of compounds 5 and 6 to shift
NKB CRC to the right (Table 5).
To characterize the binding properties of the hNK3 mutants
at helix positions 5.38 and 6.52, the saturation isotherm and
competition binding analyses were performed at binding
equilibrium using radioligand [3H]3 ([3H]SR 142801) on
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Table 3. Radioligand Binding Affinity of NK Antagonists in Membrane Preparations from HEK293 Cells Transiently Expressing
a
hNK1-WT, hNK1-F264Y6.51, hNK3-WT, or hNK3-Y315F6.51
[3H]SP competition binding
hNK1-WT
hNK1-F264Y6.51
[3H]3 competition binding
hNK3-Y315F6.51
hNK3-WT
Ki, nM
NK
antagonist
fold affinity
Ki, nM
nH
Ki, nM
nH
nH
Ki, nM
ND
increase
nH
1
03 0.0
0.4 0.2
209.6 24.6
>10000
1.1 0.0
0.5 0.0
0.9 0.2
0.8 0.1
0.3 0.4
ND
0.8 0.1
0.9 0.3
ND
454.1 66.1
>10,000
1.0 0.1
ND
ND
ND
2
ND
ND
3
0.6 0.1
3.6 0.7
2.9 0.3
1.5 0.2
1.2 0.2
11.9 1.1
1.7 0.3
2.4 0.37
3.5 0.3
1.1 0.0
1.0 0.0
1.1 0.1
1.1 0.07
1.1 0.02
1.1 0.04
1.1 0.0
1.0 0.04
1.3 0.15
10.8 0.8
1.7 0.4
0.7 0.1
0.6 0.1
0.6 0.1
2.5 0.4
0.2 0.0
0.7 0.01
1.0 0.2
0.06***
2.1*
4.1**
2.5*
0.9 0.1
1.0 0.1
1.1 0.2
1.2 0.09
1.5 0.1
1.0 0.04
1.2 0.1
1.1 0.02
1.2 0.08
4
ND
ND
5
0.7 0.1
1.4 0.3
0.7 0.2
1.3 0.4
0.3 0.1
0.4 0.1
1.4 0.3
1.0 0.1
0.9 0.06
1.1 0.05
1.0 0.05
0.7 0.1
0.8 0.04
1.0 0.1
1.1 0.2
2.8 0.9
1.3 0.1
2.1 0.3
0.5 0.4
1.4 0.1
1.7 0.2
0.7 0.1
1.8 0.4
2.4 0.5
1.9 0.1
1.6 0.1
1.8 0.1
1.5 0.1
7
9
2*
8
4.8**
8.5**
3.4**
3.5**
6
10
11
a
The affinity constant (Ki) and Hill slope (nH) values for various NK antagonists determined using either [3H]SP or [3H]3 were calculated as
described in the Experimental Section. Radioligands concentrations were the following: 0.3 and 0.8 nM [3H]SP for hNK1-WT and hNK1-F264Y6.51
membranes, respectively; 0.2 and 1.8 nM [3H]3 for hNK3-WT and hNK3-Y315F6.51 membranes, respectively. Values are the mean SE of the Ki and
were calculated from three independent experiments, each performed in duplicate. Statistical significance was determined using a two-tailed unpaired
Student’s t test: (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.001. ND, not determined.
Figure 6. Effects of the mutation at helix position 6.51 on the competition binding of compounds 1, 2, 3, 4, 5, and 6 in membrane preparations from
HEK293 cells transiently expressing hNK1-WT (A), hNK1-F264Y6.51 (B), hNK3-WT (C), or hNK3-Y315F6.51 (D). The [3H]SP and [3H]3 at
concentrations equal to their KD values were used in these competition binding experiments. Each data point is the mean SE (bars) of three
individual experiments performed in duplicate.
membranes isolated from HEK293 cells transiently transfected
with hNK3-WT and mutated receptors. The dissociation
constants (KD) and the maximum number of binding sites
(Bmax) derived from the saturation isotherms of [3H]3 at hNK3-
WT and mutated receptors are given in Table 6. The binding
affinity of [3H]3 was lost at mutant Y247A5.38. The mutation
H316W6.52 caused decreases in the binding affinity of [3H]3 by
and H316F6.52 had no effect on the KD values of [3H]3
(Table 6). Therefore, four mutations Y247F5.38, Y247W5.38
,
H316F6.52, and H316W6.52, which did not affect or only partially
affected the binding affinity of [3H]3, were chosen further for
the competition binding studies with compounds 3, 4, 5, and 6.
Table 6 summarizes the affinity constant (Ki) and Hill slope
(nH) values for the [3H]3 displacement by 3, 4, 5, or 6 on
HEK293 cell membranes expressing four point-mutated hNK3.
11.0-fold (P = 0.01), while the mutations Y247F5.38, Y247W5.38
,
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Table 4. Effect of Mutation on the [MePhe7]NKB-Evoked
mode of antagonism at hNK3 (parallel rightward shifts of agonist
dose−response curves with no alteration of Emax). To better
understand the different behavior of compounds 5 and 6, we
looked at the docking of these compounds onto the NK1- and
NK3-7TMD binding cavities (Figure 8). The docking modes
suggested several mutations that might be relevant for the
different behavior of the compounds investigated in this study.
In agreement with our hypothesis, we identified hNK3-Tyr6.51
as a contributing cause for the different modes of antagonism of
compounds 5 and 6. The mutation hNK3-Y315F6.51 converted
the surmountable mode of antagonism of compound 6 to an
insurmountable one, indicating that the presence of the OH
function of hNK3-Tyr6.51 is plausibly the reason for the
difference between hNK1 and hNK3. This goes in parallel with
the 8.5-fold increase of binding affinity through this mutation.
The same mutation led to an even stronger insurmountable
mode of antagonism of compound 5 compared to the hNK3-
WT and to a 4.1-fold increase in binding affinity. The observed
effect of this mutation on compound 6 agrees with our
hypothesis. However, for compound 5, on the basis of our
hypothesis, we would have expected at first that this mutation
would lead to a decrease of the insurmountable mode of
antagonism. A possible explanation why it is not the case might
be that the formation of the hydrogen bond between a phenolic
OH and an aliphatic OH is not as favorable as the presence of a
coordinatively unsaturated aliphatic OH group in the ligand,
possibly as consequence of a high desolvation penalty of the
phenolic OH when it binds to the ligand’s alcoholic OH group
or that the alcoholic OH group can then interact with another
hydrogen bonding partner such as, for example, a water
molecule. In any case, hNK3-Y315F6.51 seems to represent a
preferred energetic situation. Nevertheless, the hypothesis
described above is only one among other possible explanations
for this observation. The experimental results agree with our
hypothesis that the Y6.51F difference between the hNK1 and
hNK3 receptors is responsible for the different antagonism
modes of compounds 5 and 6. In this context, it is interesting to
note that compound 17a (GSK), which exhibited an
insurmountable mode of antagonism at NK1/NK3, also contains
a −CH2OH group, just like compound 5.29
a
Accumulation of [3H]IP
[MePhe7]NKB
position in
hNK3
WT
7TMD
EC50, nM
nH
relative Emax
100
0.71 0.04
2.22 1.08
inactive
0.8 0.0
0.7 0.0
inactive
Y247F
Y247A
Y247W
H316F
H316W
5.38
5.38
5.38
6.52
6.52
46.37 5.17
inactive
inactive
inactive
inactive
inactive
inactive
inactive
3.48 1.61
0.9 0.0
146.32 4.55
a
EC50, Hill slope (nH), and relative efficacy (Emax) values for the NKB-
induced formation of [3H]IP in HEK293 cells expressing transiently
the WT or mutated NK3 receptors. The data are the mean SE of
eight concentration−response measurements (each performed in
duplicate) from four independent transfections.
In the competition binding experiments by compounds 3−6
(Figure 7 and Table 6), the mutation Y247F5.38 had no effect
on the competition bindings by compounds 3−6. The mutation
Y247W5.38 decreased only the binding affinity of compound 4
by 10.3-fold (statistical significance of P = 0.03). H316F6.52
caused only a decrease in binding affinity of compound 6 by
11.6-fold (statistical significance of P = 0.0035), while the
mutation H316W6.52 caused decreases in binding affinities of
compounds 3, 4, 5, and 6 by 8.0-, 33.9-, 693.1-, and >10000-
fold (statistical significance of P of 0.01, 0.0002, 0.0001, and
<0.0001), respectively. In general, there was a good agreement
between the functional [3H]IP accumulation (Table 5) and
competition binding results (Table 6) for compounds 3−6 at
mutants Y247F and H316W.
DISCUSSION AND CONCLUSION
■
In the current study, the −CH2OH substituted compounds 5, 7,
and 9 exhibited an insurmountable mode of antagonism at both
hNK1 and hNK3. This mode of action was characterized by a
parallel rightward shift of the SP or [MePhe7]NKB concen-
tration−response curves (increase in EC50 values) in the presence
of increasing antagonist concentration, with a concomitant large
decrease in the maximal agonist-evoked response (Emax). A
possible explanation for this insurmountable antagonism of
compounds 5, 7, and 9 at NK1 and NK3 determined by Schild
plot analyses might be their slow dissociation rate from NK1 and
NK3. Consequently, a large portion of the NK1 and NK3
receptors are not available for activation by SP or [MePhe7]NKB
and, thereby, the maximally achievable response of SP or
[MePhe7]NKB drops dramatically in comparison to the fast
dissociating surmountable antagonist.35 In contrast, compounds
6, 8, 10, and 11 lacking the −CH2OH group had an
insurmountable profile at hNK1, but all had a surmountable
To further test the importance of the −CH2OH group,
we also included compounds 7, 8, 9, 10, and 11 in our study.
As expected, the −CH2OH substituted compound 7 was also
insurmountable at hNK3 while compounds 8, 10, and 11
lacking this functional group displayed a surmountable mode of
antagonism. Most remarkable was the result that the hybrid
structure compound 9 behaved indeed as insurmountable
antagonist at hNK3. Thus, we have identified the presence
of an alcoholic function in the ligand as trigger that renders
the pyridine series insurmountable at hNK3, possibly by the
Table 5. Schild Constants for Antagonism of [MePhe7]NKB-Induced Accumulation of [3H]IP by Compounds 3, 4, 5 and 6 in
a
HEK293 Cells Expressing Transiently the hNK3-WT and Mutants
3
4
6
position in
7TMD
Schild
Slope
mode of
Schild
Slope
mode of
5, mode of
Schild
mode of
hNK3
WT
pA2
antagonism
pA2
antagonism
antagonism
pA2
7.79
Slope
antagonism
7.73
8.23
7.77
1.59
0.94
1.13
surmountable
surmountable
surmountable
8.24
8.54
6.64
0.88
0.84
1.14
surmountable
surmountable
surmountable
insurmountable
insurmountable
inactive
1.12
surmountable
surmountable
inactive
Y247F
5.38
6.52
7.32
2.73
H316W
inactive
inactive
a
The apparent antagonist potency (pA2) and Schild slope values of compounds 3, 4, and 6 at hNK3-WT and mutants were determined from NKB
CRCs in the absence and presence of various concentrations of antagonist and Schild plot analyses.
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formation of a hydrogen bond between hNK3-Tyr6.51 and the
hydroxy group of the ligand. While compounds 7 and 9 also
showed the expected strengthening of their insurmountable
behavior at mutation hNK3-Y315F6.51, compounds 8, 10, and
11 stayed surmountable at mutation hNK3-Y315F6.51, yet with
large increases in their pA2 values and binding affinities. This
shows that for these compounds removal of the −CH2OH
group is also energetically favorable.
The other mutations did not affect the mode of antagonism.
Mutation of hNK3-Y247F5.38 did not affect the binding affinity
of the two dual NK1/NK3 antagonists 5 and 6, but mutation of
hNK3-H316W6.52 affected the binding affinities of both
compounds, especially compound 6. This confirms the binding
pose, where compound 6 can get closer to hNK3-His3166.52
than compound 5 because of its sulfonamide substituent.
Compounds 3 and 4 were unaffected in their surmountable
mode of antagonism by any mutation, which is in agreement
with our assumption that both do not form a hydrogen bond
with hNK3-Tyr3156.51. Binding of compound 3 is affected by
hNK3-Y315F6.51, although it is more likely due to a change of
electronic properties of the aromatic ring and thus its interaction
with the basic N in compound 3. Otherwise, it could be due to a
hydrogen bond which would show that the observed effect is
specific to this chemical class. As reported previously,36
compound 3 displayed an insurmountable behavior at the
guinea pig NK3 receptor, and the TM2 residue Ala-1142.59 in
guinea pig NK3 (threonine in all other species) was shown to be
involved in this compound’s insurmountable behavior by
slowing its dissociation kinetics. Interestingly, compound 5
also showed an insurmountable behavior at the guinea pig NK3
receptor,27 and both mutations hNK3-T139A2.59 and gpNK3-
A114T2.59 had no effect on the insurmountable behavior of
compound 5 at human and guinea pig NK3 receptors. Why
hNK3-H316W6.52 significantly affects the binding of compound
4 cannot be explained by a direct interaction. It is interesting to
note that the helix position 6.52 is also a key position for
receptor stability, as shown for bovine rhodopsin (Ala2696.52),32
human β2-adrenergic receptor (Phe2906.52),33,34 human A2A
adenosine receptor (His2506.52),37 and human dopamine D3
receptor (Phe3466.52).38
In agreement with the above hypothesis, the mutation hNK1-
F264Y6.51 weakened the insurmountable mode of antagonism
of compound 5 to a partial insurmountable-like mode of
antagonism, resembling the situation in the hNK3 receptor. In
this mode of antagonism, an increase of compound 5
concentrations first depressed the maximal response to a limit
and then only produced parallel rightward shifts of the SP
concentration−response curves. Similar behavior was observed
with compounds 7 and 9. The NK1 selective antagonist
compound 1 had previously been shown to have a very slow rate
of dissociation from the hNK1 receptor (t1/2 of 154 min) and to
behave in an insurmountable manner.19,39 The insurmountable
behavior of compound 1 was explained by its slow dissociation
rate from the receptor. In the current study, the insurmountable
behavior of compound 1 was converted to a surmountable one
at mutation hNK1-F264Y6.51. This is not surprising because
compound 1 also has a hydrogen bond acceptor that can
interact with the phenolic OH group. As expected, the mutation
hNK1-F264Y6.51 had no effect on the surmountable mode of
antagonism of compound 2, another selective NK1 antagonist.30
Nevertheless, surprisingly, the modes of antagonism of
compounds 6, 8, 10, and 11 were not affected by the hNK1-
F264Y6.51 mutation. A comparison of the ligand binding pockets
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Figure 7. Effects of the mutations at helix positions 5.38 and 6.52 on the competition binding of compounds: 3 (A), 4 (B), 5 (C), and 6 (D) in
membrane preparations from HEK293 cells transiently expressing hNK3-WT, hNK3-Y247F5.38, hNK3-Y247W5.38, hNK3-H316F6.52, or hNK3-
H316W6.52. The [3H]3 at a concentration equal to its KD value was used in these competition binding experiments. Each data point is the mean SE
(bars) of three individual experiments performed in duplicate.
of hNK1 and hNK3 indicates that there are eight amino acids
differences in TM region and ECL2 loop between two
receptors, four of them being nonconservative. Therefore,
additional factors that are not explained by our model must
control the insurmountable mode of antagonism at the hNK1
receptor of these compounds.
compound 5 (half-life t1/2 = 14 h).27 As it has already been well
documented for the angiotensin AT1 receptor and many other
GPCRs,42−46 the ability of antagonists to form slowly
dissociating complexes with their cognate receptors is being
proposed to be responsible for their long-lasting clinical actions.
It remains to be determined whether the residue at helix
position 6.51 has a similar key contribution to the
insurmountable behavior of other antagonists in interactions
with their cognate peptide GPCRs. This would be of high
importance because it might further assist the development of
drugs with an insurmountable mode of antagonism and a long
duration of in vivo action.
Finally, a high resolution X-ray structure of hβ2AR has
revealed that four residues Trp2866.48 (key residue of the
rotamer toggle switch), Phe2896.51, Phe2906.52 in TM6 and
Phe2085.47 in TM5 are involved in the toggle switch mechanism
of β2AR activation.33,34 A rotameric change of Trp2866.48 is
thought to occur in the activation step, and the residue at helix
position 6.51 (Phe289), as part of a ring face aromatic
interaction, can stabilize the active conformation. It is also
interesting to note that recent findings have demonstrated that
four out of nine multiplex families affected by hypogonado-
tropic hypogonadism carry loss-of-function mutations in NKB
or the hNK3 receptor, indicating that this system also plays a
key role in the central neuroendocrine control of reproduction
in humans.40 Remarkably, the homozygous loss-of-function
mutation hNK3-Y315C found in these patients with repro-
ductive dysfunction41 is located at the helix position 6.51, which
corresponds to the residue hNK3-Tyr3156.51 (or hNK1-
Phe2646.51) that we have found to contribute to an
insurmountable mode of antagonism of NK1/NK3 antagonists.
Taken together, hNK3-Tyr3156.51 (or hNK1-Phe2646.51) occu-
pies a critical position in the TM6, which is involved in receptor
stability and activation.
EXPERIMENTAL SECTION
■
Chemistry. General. Solvents and reagents of commercial quality
were used as purchased. Column chromatography was carried out on
silica gel 60 (32−60 mesh, 60 Å) or on prepacked columns (Isolute
Flash Si). 1H NMR spectra were recorded on a Bruker AvanceIII 600
cryo-TCI spectrometer with the residual solvent peak as the internal
1
reference (i.e., CDCl3 = 7.26 ppm). H resonances are reported to
the nearest 0.01 ppm. The multiplicities of signals are given as s =
singlet, d = doublet, t = triplet, q = quartet, br = broad, and com-
binations thereof. Coupling constants (J) are reported to the nea-
rest 0.01 Hz. High resolution mass spectrometry (HRMS) was per-
formed on a Agilent 6520 spectrometer using time-of-flight (TOF)
with positive ESI or EI at 70 eV within a tolerance of 5 ppm of the
theoretical value. The purity of compounds 5−11 was determined
as >95% by HPLC analysis. Parameters were as follows: system,
Agilent 1290 LC with CTC PAL coupled to a Agilent 6520 QTOF;
column, Zorbax Eclipse Plus C18 2.1 mm × 50 mm, 1.8 μm (Agilent
purchase no. 959757-902); eluent A, 1 L of H2O + 0.2 mL
of HCOOH; eluent B, 80% ACN, 20% 3i-PrOH, 0.01% HCOOH;
initial, 5% B; column temperature, 55 °C; detection wavelength,
220 5 nm.
In conclusion, we have shown in molecular details the
likely mechanism of the insurmountable behavior of the dual
NK1/NK3 antagonist 5 and a few structural analogues. Inter-
estingly, this insurmountable mode of antagonism observed
in vitro translates to a long-lasting in vivo receptor blockade by
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Figure 8. Compounds 5 (magenta) and 6 (cyan) docked into the homology model of the human NK3 receptor (top) and human
NK1 receptor (bottom). Carbon atoms of the protein are shown in green.
Reference Compounds 1−4. 2-(R)-(1-(R)-3,5-Bis(trifluoro-
methyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-oxo-1,2,4-triazol-
5yl)methylmorphine) (1, aprepitant, MK-869),19 (2S,3S)-cis-2-(diphe-
nylmethyl)-N-[(2-methoxyphenyl)methyl]-1-azabicyclo[2.2.2]octan-3-
amine (2, CP-96,345),30 (S)-(+)-N-((3-[1-benzoyl-3-(3,4-dichloro-
phenyl)piperidin-3-yl]prop-1-yl)-4-phenylpiperidin-4-yl)-N-methylacet-
amine (3, osanetant, SR 142801),3,4 (S)-(−)-N-(α-ethylbenzyl)-3-hy-
droxy-2-phenylquinoline-4-carboxamide (4, talnetant, SB 223412),5,6 and
(S)-(−)-N-(α-ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide
(SB 222200)5 were synthesized according to procedures described
in the literature, and their purity was confirmed (>95% by HPLC
analysis).
Synthesis. (2R,3S)-2-(3,5-Bis-trifluoromethylphenyl)-N-[4-(4-
fluoro-2-methylphenyl)-6-(3-hydroxy-2-hydroxymethylpyrro-
lidin-1-yl)pyridin-3-yl]-N-methylisobutyramide (5, RO4583298).25
A mixture of 11.0 g (20.6 mmol) of 2-(3,5-bis-trifluoromethyl-phenyl)-
N-[6-chloro-4-(4-fluoro-2-methylphenyl)pyridin-3-yl]-N-methylisobu-
tyramide,25 14.3 g (103 mmol) of potassium carbonate, and 12.2 g
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(75.7 mmol) of (2R,3S)-2-(hydroxymethyl)-3-hydroxypyrrolidine in
110 mL dimethyl sulfoxide was stirred at 130 °C for 68 h. The reaction
mixture was diluted with 800 mL of ethyl acetate and washed with
800 mL of saturated sodium carbonate solution, 500 mL of water, and
750 mL of brine. The combined aqueous layers were extracted with
two 800 mL portions of ethyl acetate. The combined organic layers
were dried over sodium sulfate and concentrated in vacuo. Purification
by flash chromatography gave 8.48 g (67%) of 5 as off-white foamy
solid. 1H NMR (600 MHz, CDCl3) δ 7.86 (d, J = 11.69 Hz, 1H), 7.76
(s, 1H), 7.63 (br s, 2H), 7.19−7.34 (m, 1H), 6.95 (br s, 2H), 6.31 (br
s, 1H), 5.29−6.10 (m, 1H), 4.13−4.47 (m, 2H), 3.79−3.96 (m, 1H),
3.37−3.73 (m, 3H), 1.90−2.69 (m, 8H), 1.07−1.73 (m, 6H); HRMS
calcd for C30H30F7N3O3, 613.2175; found, 613.218.
7.21−7.31 (m, 1H), 6.94 (br s, 2H), 6.19 (s, 1H), 4.62 (br s, 1H),
3.45−3.71 (m, 4H), 2.04−2.68 (m, 8H), 1.17−1.86 (m, 6H); HRMS
calcd for C29H28F7N3O2, 583.207; found, 583.2075.
(S)-2-(3,5-Bis-trifluoromethylphenyl)-N-[4-(4-fluoro-2-meth-
ylphenyl)-6-(2-hydroxymethyl-4-methanesulfonylpiperazin-1-
yl)wpyridin-3-yl]-N-methylisobutyramide (Compound 9,
RO4861497).25 Off-white solid after purification by silica gel column
chromatography. 1H NMR (600 MHz, CDCl3) δ 7.96 (br s, 1H), 7.77
(s, 1H), 7.64 (br s, 2H), 7.18−7.32 (m, 1H), 6.96 (br s, 2H), 6.52 (br
s, 1H), 4.42−4.77 (m, 1H), 4.11−4.16 (m, 1H), 4.25 (br s, 1H), 3.91−
4.01 (m, 2H), 3.81 (d, J = 11.48 Hz, 2H), 3.18−3.43 (m, 1H), 2.98
(dd, J = 3.63, 12.09 Hz, 1H), 2.88−2.95 (m, 1H), 2.77−2.87 (m, 3H),
1.98−2.72 (m, 6H), 1.18−1.71 (m, 6H); HRMS calcd for
C31H33F7N4O4S, 690.2111; found, 690.211.
(S)-2-(3,5-Bis-trifluoromethylphenyl)-N-[4-(4-fluoro-2-meth-
ylphenyl)-6-((S)-4-methanesulfonyl-3-methylpiperazin-1-yl)-
pyridin-3-yl]-N-methylisobutyramide (6, RO4908594).25 (a) A
mixture of 0.20 g (0.38 mmol) of 2-(3,5-bis-trifluoromethylphenyl)-N-
[6-chloro-4-(4-fluoro-2-methylphenyl)pyridin-3-yl]-N-methylisobutyr-
amide,25 0.11 g (1.1 mmol) of (S)-2-methylpiperazine, and 0.10 g
(0.72 mmol) of potassium carbonate in 0.3 mL of dimethyl sulfoxide
was heated at 180 °C under microwave irradiation in a sealed tube for
30 min. After cooling to room temperature, the reaction mixture was
diluted with a 0.3 M aqueous solution of sodium hydroxide and
extracted with three portions of tert-butyl methyl ether. The combined
organic extracts were dried over sodium sulfate and concentrated.
Purification by silica gel column chromatography gave 0.12 g (51%) of
(S)-2-(3,5-bis-trifluoromethylphenyl)-N-[4-(4-fluoro-2-methylphen-
yl)-6-(3-methylpiperazin-1-yl)pyridin-3-y1]-N-methylisobutyramide as
an off-white solid. MS m/e (%): 597 (M + H+, 100). (b) To a solution
of 0.19 g (0.31 mmol) of (S)-2-(3,5-bistrifluoromethylphenyl)-N-[4-
(4-fluoro-2-methylphenyl)-6-(3-methylpiperazin-l-yl)pyridin-3-yl]-N-
methylisobutyramide and 38 mg (0.38 mmol) of triethylamine in 4 mL
of dichloromethane was added 38 mg (0.33 mmol) of methanesulfonyl
chloride at 0 °C. After the addition was completed, the reaction
mixture was allowed to warm to room temperature. Conversion was
monitored by thin layer chromatography. After complete consumption
of the starting material the reaction mixture was diluted with water and
extracted with three portions of tert-butyl methyl ether. The combined
organic layers were dried over sodium sulfate and concentrated in
vacuo. Purification by silica gel column chromatography gave 0.15 g
2-(3,5-Bis-trifluoromethylphenyl)-N-[4-(4-fluoro-2-methyl-
phenyl)-6-(4-methanesulfonylpiperazin-1-yl)pyridin-3-yl]-N-
methylisobutyramide (Compound 10, RO4858154).25 White
1
solid after purification by silica gel column chromatography. H NMR
(600 MHz, CDCl3) δ 8.01 (s, 1H), 7.77 (s, 1H), 7.65 (br s, 2H),
7.18−7.32 (m, 1H), 6.96 (br s, 2H), 6.50 (s, 1H), 3.54−3.85 (m, 4H),
3.33 (t, J = 5.04 Hz, 4H), 2.81 (s, 3H), 2.01−2.65 (m, 6H), 1.17−1.67
(m, 6H); HRMS calcd for C30H31F7N4O3S, 660.2005; found, 660.201.
2-(3,5-Bis-trifluoromethylphenyl)-N-[6-(4,4-dioxo-1-oxa-4λ6-
thia-8-aza-spiro[4.5]dec-8-yl)-4-(4-fluoro-2-methylphenyl)-
pyridin-3-yl]-N-methylisobutyramide (Compound 11,
RO4896062).25 White solid after purification by silica gel column
1
chromatography. H NMR (600 MHz, CDCl3) δ 7.99 (s, 1H), 7.77 (s,
1H), 7.65 (br s, 2H), 7.19−7.33 (m, 1H), 6.78−7.06 (m, 2H), 6.51 (s, 1H),
4.34 (t, J = 6.90 Hz, 2H), 4.16 (br s, 1H), 4.01 (br s, 1H), 3.48 (br s, 2H),
3.25 (t, J = 6.90 Hz, 2H), 2.59 (br s, 1H), 2.35 (br s, 2H), 2.27 (br s, 1H),
2.12 (br s, 4H), 1.94 (d, J = 13.30 Hz, 2H), 1.46−1.61 (m, 3H), 1.31−1.44
(m, 3H); HRMS calcd for C32H32F7N3O4S, 687.2002; found, 687.2002.
Materials. Radioligands [3H]3 ([3H]SR 142801) (catalog no.
TRK1035, specific activity of 74.0 Ci/mmol, radiochemical purity of
99.7% by HPLC, Novapak C18 column) and [3H]SP ([3H]substance P,
catalog no. TRK786, specific activity of 40.0 Ci/mmol) were purchased
from GE Healthcare UK Ltd., Chalfont St. Giles, U.K. [MePhe7]-
Neurokinin B (Asp-Met-His-Asp-Phe-Phe-NMe-Phe-Gly-Leu-Met-NH2,
catalog no. SC981) was purchased from NeoMPS SA (Strasbourg,
France). Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-
NH2, catalog no. 1156) was obtained from Tocris Biosciences (Bristol,
U.K.). [myo-1,2-3H]Inositol with PT6-271 (TRK911, specific activity of
16.0 Ci/mmol) and yttrium silicate (Ysi) RNA binding SPA beads
(RPNQ0013) were purchased from GE Healthcare.
Plasmids, Cell Culture, and Membrane Preparation. The
cDNAs encoding human NK1 receptor (accession number P25103) and
human NK3 receptor (accession number P29371) were subcloned into
pCI-Neo expression vectors (Promega Corporation, Madison, WI). All
point mutants were constructed using the QuickChange site-directed
mutagenesis kit (catalog 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.31 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 16 000
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.
1
(75%) of 6 as a white solid. H NMR (600 MHz, CDCl3) δ 7.98 (s,
1H), 7.77 (s, 1H), 7.56−7.70 (m, 2H), 7.22−7.25 (m, 1H), 6.95 (br s,
2H), 6.44 (s, 1H), 4.16−4.35 (m, 2H), 3.96−4.15 (m, 1H), 3.67 (d, J =
12.79 Hz, 1H), 3.34 (t, J = 11.23 Hz, 1H), 3.26 (d, J = 10.48 Hz, 1H),
2.99−3.13 (m, 1H), 2.90 (s, 3H), 2.58 (br s, 1H), 2.35 (br s, 2H), 2.26
(br s, 1H), 2.11 (br s, 2H), 1.45−1.60 (m, 3H), 1.28−1.45 (m, 6H);
HRMS calcd for C31H33F7N4O3S, 674.2162; found, 674.2179.
(2S)-2-(3,5-Bis-trifluoromethylphenyl)-N-[4-(4-fluoro-2-
methylphenyl)-6-(2-hydroxymethylpyrrolidin-1-yl)pyridin-3-
yl]-N-methylisobutyramide (Compound 7, RO4603325).25
A
mixture of 0.20 g (0.38 mmol) of 2-(3,5-bis-trifluoromethylphenyl)-N-
[6-chloro-4-(4-fluoro-2-methylphenyl)pyridin-3-yl]-N-methylisobutyr-
amide,25 0.23 g (2.3 mmol) of L-prolinol, and 0.15 g (1.1 mmol) of
potassium carbonate in 0.5 mL of dimethyl sulfoxide was heated at
180 °C under microwave irradiation for 30 min in a sealed tube. After
cooling to room temperature, the reaction mixture was diluted with
water and extracted with three portions of tert-butyl methyl ether. The
combined organic extracts were washed with water and brine, dried
over sodium sulfate, and concentrated in vacuo. Purification by silica
gel column chromatography gave 0.18 g (78%) of 7 as off-white solid.
1H NMR (600 MHz, CDCl3) δ 7.81−7.96 (m, 1H), 7.76 (s, 1H), 7.63
(br s, 2H), 7.19−7.35 (m, 1H), 6.77−7.05 (m, 2H), 6.25 (s, 2H),
4.24−4.47 (m, 1H), 3.55−3.86 (m, 2H), 3.14−3.51 (m, 2H), 1.88−
2.74 (m, 9H), 1.74 (br s, 1H), 1.15−1.66 (m, 6H); HRMS calcd for
C30H30F7N3O2, 597.2226; found, 597.222.
Radioligand Binding. After thawing, the membrane homogenates
were centrifuged at 48000g for 10 min at 4 °C. The pellets were
resuspended in the binding buffer. The assay buffers consisted of the
following: for NK1, 50 mM Hepes, 3 mM MnCl2, 2 μM
phosphoramidon, 16.8 μM Leupeptin, 0.04% BSA binding buffer at
pH 7.4; for NK3, 50 mM Tris-HCl, 4 mM MnCl2, 1 μM phos-
phoramidon, 0.1% bovine serum albumin at pH 7.4. Final assay
(S)-2-(3,5-Bis-trifluoromethylphenyl)-N-[4-(4-fluoro-2-meth-
ylphenyl)-6-(3-hydroxypyrrolidin-1-yl)pyridin-3-yl]-N-methyli-
sobutyramide (Compound 8, RO4746092).25 Off-white foamy
1
solid after purification by silica gel column chromatography. H NMR
(600 MHz, CDCl3) δ 7.96 (s, 1H), 7.76 (s, 1H), 7.65 (br s, 2H),
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concentration for hNK1-WT and hNK1-F264Y expressing membranes
was 2.5 μg protein/well, and for hNK3-WT and hNK3-Y315F
expressing membranes it was 5 μg protein/well. Saturation isotherms
were determined by addition of various concentrations of radioligand
[3H]SP (NK1, 0.04−18 nM) or [3H]3 (NK3, 0.009−3 nM) to
membranes (in a total reaction volume of 500 μL) for 90 min,
respectively, at room temperature (rt). Nonspecific binding was
determined with 10 μM compound 2 (NK1) and 10 μM SB 222200
(NK3). At the end of the incubation, membranes were filtered onto
96-well white microplates (preincubated for 1 h in 0.3% poly-
ethylenimine + 0.1% bovine serum albumin) with a bonded GF/C filter
for [3H]SP and [3H]3 binding (PerkinElmer Life and Analytical
Sciences, Waltham, MA), with a FilterMate-96 well harvester
(PerkinElmer Life and Analytical Sciences), and washed 4 times with
ice-cold 50 mM Tris-HCl, pH 7.4 buffer. 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
and then 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
Analytical Sciences). For PI hydrolysis, activation and inhibition curves
were fitted according to the equation y = A + (B − A)/[1 + (C/x)D],
where A is ymin, B is ymax, C is EC50, and D is the Hill slope factor,
using ExcelFit 3.0 (IDBS software). The relative efficacy (Emax) values
of SP or [MePhe7]NKB was calculated as the fitted maximum of the
concentration−response curve of each mutated receptor expressed as a
percentage of fitted maximum of the wild type (WT) concentration−
response curve from cells transfected and assayed on the same day.
Residue Numbering Scheme. The position of each amino acid
residue in the 7TMD is identified both by its sequence number and by
its generic numbering system proposed by Ballesteros and Weinstein47
which is shown as superscript. In this numbering system, amino acid
residues in the 7TMD are given two numbers. The first refers to the
TM number, and the second indicates its position relative to a highly
conserved residue of class A GPCRs in that TM, which is arbitrarily
assigned 50. The amino acids in the extracellular loop ECL2 are
labeled 45 to indicate their location between the helixes 4 and 5. The
highly conserved cysteine thought to be disulfide bonded was given
the index number 45.50 (SWISS-PROT: opsd_bovin C187), and the
residues within the ECL2 loop are then indexed relative to the “50”
position.
Model Building. The amino acid sequences of the human NK3
(accession number P29371), rat NK3 (accession number P16177),
mouse NK3 (accession number P47937), gerbil NK3 (accession
number AM157740), human NK1 (accession number P25103), and
human NK2 (accession number P21452) were retrieved form the
Swiss-Prot database. These amino acid sequences were aligned to the
sequence of bovine rhodopsin (accession number P02699) using the
ClustalW multiple alignment program. A slow pairwise alignment
using the BLOSUM matrix series48 and a gap opening penalty of 15.0
were chosen for aligning the amino acid sequences. Other parameters
were those given as default. The sequences were aligned in two steps:
(i) from the N-terminus to the first five residues of the third
intracellular loop (ICL3) and (ii) from the last five residues of the
ICL3 loop to the C-terminus. The ICL3 loop was excluded from
the alignment, since it shows too high variability in amino acid
composition and length. The alignments were then verified to ensure
that conserved residues of the transmembrane regions were aligned
and manually adjusted in the second extracellular loop (ECL2) in
order to align the conserved cysteine which takes part in the disulfide
bridges occurring between the third transmembrane segment (TM3)
and the second extracellular loop (ECL2).
By use of this alignment and the X-ray structure of bovine
rhodopsin32 as template, the software package MOE (MOE, version
2005.05, Chemical Computing Group, Montreal, Quebec, Canada)
was used to generate a three-dimensional model of the hNK3. Ten
intermediate models were generated, and the best one was selected as
the final MOE model. No minimization was used in order to keep the
backbone coordinates as in the X-ray structure. After the heavy atoms
were modeled, all hydrogen atoms were added in appropriate locations
with the preparatory program PROTONATE of AMBER, version 6
(University of California, San Francisco). Compound 3 was then
manually docked into the transmembrane cavity of the hNK3 model.
The docking mode was based on the following hypotheses: (1) The
ligand should make a direct interaction with Met1342.53, since this
residue has been shown to be responsible for species selectivity of N-
[(S)-4-(4-acetylamino-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)-
butyl]-N-methylbenzamide (saredutant, SR 48968).49 (2) Phenyl-
piperidine substructures are privileged fragments for the subpocket
formed by the transmembrane domains 3, 5, and 6. The resulting
protein−ligand complex was then minimized using AMBER, version 6.
The minimization was carried out using 5000 steps of steepest descent
followed by conjugate gradient minimization until the rms (root-
mean-square) gradient of the potential energy was less than 0.05 kcal
mol−1 Å−1. A twin cutoff (10.0, 15.0 Å) was used to calculate non-
bonded electrostatic interactions at every minimization step and the
nonbonded pair list was update every 25 steps. A distance-dependent
40 (Canberra Packard S.A., Zurich, Switzerland) and 1 h of agitation.
̈
Saturation experiments were analyzed by Prism 5.0 (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 + [F]), where B is the amount of ligand
bound at equilibrium, Bmax (the maximum number of binding sites) is
the specific binding extrapolated to very high concentrations of
radioligand, [F] is the concentration of free ligand, and KD (the ligand
dissociation constant) is the radioligand concentration needed to
achieve a half-maximum binding at equilibrium. For inhibition
experiments, membranes were incubated with radioligand at a
concentration equal to KD of the radioligand and 10 concentrations
of the competing compound (0.0003−10 μM). The IC50 values
(inhibition concentration at which 50% specific binding of the
radioligand was displaced) were derived from the inhibition curve
using a sigmoidal dose−response (variable slope), which is also known
as a four-parameter/nonlinear regression analysis (GraphPad Prism 5)
as shown in the equation Y = Ymin + (Ymax − Ymin)/(1 + 10(logIC −X)nH)),
50
where Y is the % specific binding, Ymin is the minimum Y, Ymax is the
maximum Y, X is the concentration of the competing compound, and
nH is the slope of the curve (the Hill coefficient). The affinity constant
(Ki) values were calculated using the Cheng−Prussoff equation Ki =
IC50/(1 + [L]/KD) where [L] is the concentration of radioligand and
KD is its dissociation constant at the receptor, derived from the
saturation isotherm. Statistical significance was determined using a two-
tailed unpaired Student’s t test (Prism 5.0, GraphPad software).
[3H]Inositol Phosphate (IP) Accumulation Assay. [3H]IP
accumulation was measured as described previously with the following
adaptations.36 HEK293 cells were transfected with various NK cDNAs
in pCl-Neo using Lipofectamine Plus reagent (Invitrogen) according
to the manufacturer’s instructions. After 24 h posttransfection, cells
were washed twice in labeling medium: Dulbecco’s modified Eagle’s
medium without inositol (MP Biomedicals, 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 labeling medium supplemented with 5 μCi/mL myo-[1,2-3H]-
inositol. On the day of assay (48 h posttransfection), cells were washed
three times with the buffer (1× HBSS, 20 mM HEPES, pH 7.4) and
then incubated for 10 min at rt in assay buffer (1× HBSS, 20 mM
HEPES, pH 7.4, plus 8 mM LiCl to prevent phosphotidylinositide
breakdown) prior to the addition of agonists or antagonists.
Antagonists were incubated for 20 min at 37 °C prior to stimulation
with the agonist, either SP (NK1) or [MePhe7]NKB (NK3), at con-
centrations ranging from 1 μM to 0.01 nM. For surmountable or
insurmountable antagonism assessment, Schild analyses were
performed. The agonist curves were generated in the presence of
increasing concentrations of antagonist, and the shift in agonist EC50
or depression in maximum response was determined. After 45 min of
incubation at 37 °C with the agonist, the assay was terminated by the
aspiration of the assay buffer and the addition of 100 μL of 20 mM
formic acid to lyse the cells. After shaking for 30 min at 23 °C, a 40 μL
aliquot was mixed with 80 μL of yttrium silicate RNA binding SPA
beads (12.5 mg/mL, bind the inositol phosphates but not the inositol)
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(ε = 4r) dielectric function was used. Removing the ligand from the
complex yielded the final coordinates of the hNK3 model. (S)-(−)-N-
(α-Ethylbenzyl)-3-methoxy-2-phenylquinoline-4-carboxamide (Me-tal-
netant, analogue of compound 4)31 was then manually docked into the
receptor. The proposed docking mode is based on the SAR
(structure−activity relationships) of compound 4 according to which
at position 3 similar side chains as used in the compound 3 series can
be added. The 3-methoxy group of Me-talnetant has thus to point into
the direction of the subpocket TM3, -5, and -6. We additionally docked
compound 6.31 This compound was previously aligned onto compound
3 by comparison of the observed SAR of the compound 3 series50 and
the compound 6 chemical series.25 Thus, its docking mode was guided
by the docking pose of compound 3 and this ligand alignment. The
resulting docking poses have been used for site-directed mutagenesis
studies. The results of these studies were in agreement with our proposed
docking poses and have been published previously.31 However, we state
clearly that this is not at all proof that the docking hypotheses represent
the true binding mode.
The described docking mode of the NK1/NK3 antagonist 6 was
used to align and dock the close analogue compound 5 and further
derivatives of this chemical series used in the present study. We have
also reconstructed a hNK3 model based on the human β2-adrenergic
receptor crystal structure (PDB code 2RH1),33,34 but this model did
not fit the NK3 antagonists as well as the model based on rhodopsin. It
relates to the fact that TM2 and especially TM5 are moved further
inside the transmembrane cavity in hβ2AR than in the rhodopsin
structure, thus limiting the space. The hNK3 model was used to build a
homology model of the hNK1 using MOE based on the alignment
described previously.31 The NK1 antagonists 1 and 2 were docked into
the model in analogy to the NK1/NK3 antagonists.
et al. SR 142801, the first potent non-peptide antagonist of the
tachykinin NK3 receptor. Life Sci. 1995, 56, PL27−PL32.
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controlled evaluation of four novel compounds for the treatment of
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(9) Spooren, W.; Riemer, C.; Meltzer, H. Opinion: NK3 receptor
antagonists: the next generation of antipsychotics? Nat. Rev. Drug
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AUTHOR INFORMATION
Corresponding Author
*Phone: +41-61-4612858. E-mail: parichehr.malherbe@
roche.com.
■
(11) Alexander, S. P.; Mathie, A.; Peters, J. A. Guide to receptors and
channels (GRAC), 5th edition. Br. J. Pharmacol. 2011, 164 (Suppl. 1),
S1−S324.
Notes
(12) Almeida, T. A.; Rojo, J.; Nieto, P. M.; Pinto, F. M.; Hernandez,
M.; Martin, J. D.; Candenas, M. L. Tachykinins and tachykinin
receptors: structure and activity relationships. Curr. Med. Chem. 2004,
11, 2045−2081.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Janine Holger and Marie-Ther
■
́
es
̀
e Zenner for their
(13) Severini, C.; Improta, G.; Falconieri-Erspamer, G.; Salvadori, S.;
Erspamer, V. The tachykinin peptide family. Pharmacol. Rev. 2002, 54,
285−322.
(14) Marco, N.; Thirion, A.; Mons, G.; Bougault, I.; Le Fur, G.;
Soubrie, P.; Steinberg, R. Activation of dopaminergic and cholinergic
neurotransmission by tachykinin NK3 receptor stimulation: an in vivo
microdialysis approach in guinea pig. Neuropeptides 1998, 32, 481−
488.
(15) Dawson, L. A.; Cato, K. J.; Scott, C.; Watson, J. M.; Wood, M.
D.; Foxton, R.; de la Flor, R.; Jones, G. A.; Kew, J. N.; Cluderay, J. E.;
Southam, E.; Murkitt, G. S.; Gartlon, J.; Pemberton, D. J.; Jones, D. N.;
Davies, C. H.; Hagan, J. In vitro and in vivo characterization of the
non-peptide NK3 receptor antagonist SB-223412 (talnetant):
potential therapeutic utility in the treatment of schizophrenia.
Neuropsychopharmacology 2008, 33, 1642−1652.
(16) Ebner, K.; Muigg, P.; Singewald, G.; Singewald, N. Substance P
in stress and anxiety: NK-1 receptor antagonism interacts with key
brain areas of the stress circuitry. Ann. N.Y. Acad. Sci. 2008, 1144, 61−
73.
(17) Blier, P.; Gobbi, G.; Haddjeri, N.; Santarelli, L.; Mathew, G.;
Hen, R. Impact of substance P receptor antagonism on the serotonin
and norepinephrine systems: relevance to the antidepressant/
anxiolytic response. J. Psychiatry Neurosci. 2004, 29, 208−218.
(18) Santarelli, L.; Gobbi, G.; Debs, P. C.; Sibille, E. T.; Blier, P.;
Hen, R.; Heath, M. J. Genetic and pharmacological disruption of
neurokinin 1 receptor function decreases anxiety-related behaviors and
increases serotonergic function. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
1912−1917.
excellent technical assistance; Cosimo Dolente, Andreas Koblet,
Beat Bucher, and Julien Heusser for the skilled synthesis of
compounds 5−11; and Dr. Josef Schneider for analytical
assistance. We are grateful to Dr. Andrew Thomas for his
critical reading of the manuscript.
ABBREVIATIONS USED
■
SP, substance P; NK, neurokinin; NKA, neurokinin A; NKB,
neurokinin B; NK1, neurokinin 1 receptor; NK2, neurokinin 2
receptor; NK3, neurokinin 3 receptor; GPCR, G-protein-
coupled receptor; IP, inositol phosphate; DAG, diacylglycerol;
7TMD, seven-transmembrane domain; 5-HT, 5-hydroxytrypt-
amine; 5-HT2A, 5-hydroxytryptamine type 2A receptor; D2,
dopamine type 2 receptor; WT, wild type; HEK, human
embryonic kidney; OPSD, bovine rhodopsin; β2AR, β2-
adrenergic receptor
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