The MCH1 receptor, an anti-obesity target, is allosterically inhibited by 8-methylquinoline derivatives possessing subnanomolar binding and long residence times

Article abstract of DOI:10.1111/bph.12529

Background and Purpose: Melanin-concentrating hormone receptor 1 (MCH ₁ receptor) antagonists are being considered as anti-obesity agents. The present study reports a new class of MCH₁ receptor antagonists with an 8-methylquinoline scaffold. The molecular mechanism of MCH₁ receptor blockade by these antagonists was examined. Experimental Approach: The pharmacological properties of the 8-methylquinolines as exemplified by MQ1 were evaluated by use of multiple biophysical and cell-based functional assays. Key Results: Multiple signalling pathways for Gα_i and Gα_q, and β-arrestin were inhibited by MQ1. Furthermore, MQ1 produced an insurmountable antagonism, causing a rightward shift of the curve for concentration-dependent binding of MCH along with a progressive reduction of the maximal response. The dissociation kinetics for MQ1 were determined from washout experiments as well as by affinity selection-MS. In short, MQ1 was shown to be a slowly dissociating reversible MCH₁ receptor blocker with a low K_off value. Conclusion and Implications: This is the first time that a slowly dissociating negative allosteric modulator of the MCH₁ receptor has been demonstrated to inhibit the numerous signalling pathways of this receptor. The characteristics of MQ1 are superior and distinct from previously reported MCH₁ receptor antagonists, making members of this chemotype attractive as drug candidates.

Full text of DOI:10.1111/bph.12529

DOI:10.1111/bph.12529
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Correspondence
RESEARCH PAPER
Taku Sakurai, Pharmaceutical
Research Division, Takeda
Pharmaceutical Company
Limited, 26-1 Muraoka-higashi
2-chome, Fujisawa, Kanagawa
251-8555, Japan.
The MCH1 receptor, an
anti-obesity target, is
allosterically inhibited
by 8-methylquinoline
derivatives possessing
subnanomolar binding and
long residence times
E-mail: taku.sakurai@takeda.com
----------------------------------------------------------------
Keywords
melanin-concentrating hormone
receptor 1 (MCH1 receptor);
antagonist; time-dependent
inhibition; slow dissociation;
negative allosteric modulator;
obesity
----------------------------------------------------------------
Received
24 April 2013
Revised
16 October 2013
Accepted
14 November 2013
T Sakurai, K Ogawa, Y Ishihara, S Kasai and M Nakayama
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Kanagawa, Japan
BACKGROUND AND PURPOSE
Melanin-concentrating hormone receptor 1 (MCH1 receptor) antagonists are being considered as anti-obesity agents. The
present study reports a new class of MCH1 receptor antagonists with an 8-methylquinoline scaffold. The molecular mechanism
of MCH1 receptor blockade by these antagonists was examined.
EXPERIMENTAL APPROACH
The pharmacological properties of the 8-methylquinolines as exemplified by MQ1 were evaluated by use of multiple
biophysical and cell-based functional assays.
KEY RESULTS
Multiple signalling pathways for Gαi and Gαq, and β-arrestin were inhibited by MQ1. Furthermore, MQ1 produced an
insurmountable antagonism, causing a rightward shift of the curve for concentration-dependent binding of MCH along with a
progressive reduction of the maximal response. The dissociation kinetics for MQ1 were determined from washout experiments
as well as by affinity selection-MS. In short, MQ1 was shown to be a slowly dissociating reversible MCH1 receptor blocker with
a low Koff value.
CONCLUSION AND IMPLICATIONS
This is the first time that a slowly dissociating negative allosteric modulator of the MCH1 receptor has been demonstrated to
inhibit the numerous signalling pathways of this receptor. The characteristics of MQ1 are superior and distinct from previously
reported MCH1 receptor antagonists, making members of this chemotype attractive as drug candidates.
Abbreviations
MCH1 receptor, melanin-concentrating hormone receptor 1; MQ1, 4-(cyclopropylmethoxy)-N-(8-methyl-3-((1R)-1-
(pyrrolidin-1-yl)ethyl)quinolin-7-yl)-benzamide; NAM, negative allosteric modulator
© 2013 The British Pharmacological Society
British Journal of Pharmacology (2014) 171 1287–1298 1287

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The antagonism induced by MQ1 (Kasai et al., 2012) is
allosteric, time-dependent and reversible affording MQ1 dis-
tinctive advantages over previously reported antagonists
(McBriar, 2006; Luthin, 2007; Cheon, 2012). MQ1 is a highly
selective slowly dissociating negative allosteric modulator
(NAM) of MCH1 receptors with a residence time (half-life) of
78 min (95% CI: 72–86 min).
Introduction
Obesity is defined as abnormal or excessive accumulation of
fat that may impair health and nearly 400 million adults
worldwide are obese, a number that has more than doubled
since 1980 (Low et al., 2009). Obesity is a serious problem
because it causes or aggravates various diseases, such as type
2 diabetes, hypertension, stroke, depression, sleep disorders
and certain types of cancer, resulting in a huge socio-
economic burden (Bray and Bellanger, 2006). Despite the
intensive research efforts made by many pharmaceutical and
biotechnology companies to develop anti-obesity drugs, only
a few drugs are currently available to treat obesity and this
remains a massive unmet medical need (Rodgers et al., 2012).
Melanin-concentrating hormone (MCH) is a disulfide-
linked cyclic nonadecapeptide that is mainly expressed in the
lateral hypothalamus and zona incerta, which are regions
with extensive projections throughout the brain (Vaughan
et al., 1989; Presse et al., 1990; Bittencourt et al., 1992; Saper
et al., 2002). After the discovery of MCH, a number of studies
demonstrated that it is involved in regulating food intake,
energy homeostasis and weight gain (Qu et al., 1996;
Della-Zuana et al., 2002; Ito et al., 2003; Pereira-da-Silva et al.,
2003).
Methods
Materials
MQ1
[4-(cyclopropylmethoxy)-N-(8-methyl-3-((1R)-1-
(pyrrolidin-1-yl)ethyl)quinolin-7-yl)-benzamide] (Kasai et al.,
2012), MQ2 [4-(4-hydroxybutoxy)-N-(8-methyl-3-((1R)-1-
(pyrrolidin-1-yl)ethyl)quinolin-7-yl)benz-amide] and MQ3
[4-(cyclopropylmethoxy)-N-(8-methyl-3-(((1-oxidotetrahydro
-2H-thiopyran-4-yl)amino)methyl)quinolin-7-yl)benzamide]
(Figure 1) were synthesized by Takeda Pharmaceutical
Company (Osaka, Japan). The synthesis of MQ2 and MQ3, as
well as the sources of other materials used in this study, are
summarized in the Supporting Information.
Establishment of stable cell lines
The development of two stable cell lines expressing human
MCH1 receptors, CHO (dhfr-)-hMCH1 receptor and CHO-K1-
BAEA-hMCH1 receptor is described in the Supporting
Information.
Two GPCRs for MCH have been reported so far. Melanin-
concentrating hormone receptor 1 (MCH1 receptor), which
was originally cloned and named SLC-1, has been character-
ized as
a receptor with a high affinity for MCH by
several groups (Chambers et al., 1999; Lembo et al., 1999;
Shimomura et al., 1999; Saito, 2001). The MCH1 receptor is
primarily expressed in the CNS, including the hippocampus,
nucleus accumbens and hypothalamus (Pissios et al., 2006). It
has been shown that the MCH1 receptor couples with multi-
ple G-proteins, including Gαi and Gαq (Chambers et al., 1999;
Lembo et al., 1999; Hawes et al., 2000; Pissios et al., 2006).
Also there have been a number of reports suggesting a role
for MCH1 receptors in feeding and energy expenditure. For
example, MCH1 receptor-deficient mice are reported to be
lean and resistant to dietary obesity due to their hyperactivity
and increased energy expenditure (Chen et al., 2002; Marsh
et al., 2002). In addition, administration of low molecular
weight antagonists that block MCH1 receptors suppresses
MCH-induced food intake (Takekawa et al., 2002) and reduces
weight gain (Borowsky et al., 2002). These findings indicate
that MCH1 receptor plays a key role in the regulation of
feeding and energy expenditure, suggesting that an MCH1
receptor antagonist could be a promising anti-obesity agent.
In contrast, little is known about the physiological role of
MCH2 receptors (An et al., 2001; Hill et al., 2001; Mori et al.,
2001; Rodriguez et al., 2001; Sailer et al., 2001).
cAMP assay
The cAMP assay was carried out by using an AlphaScreen
cAMP Assay Kit from PerkinElmer (Covina, CA, USA) accord-
So far, numerous pharmaceutical and biotechnology com-
panies have discovered low molecular weight MCH1 receptor
antagonists and extensive clinical trials have been conducted
(McBriar, 2006; Luthin, 2007; Cheon, 2012). Several groups
have reported on the in vitro characterization of some of these
MCH1 receptor antagonists (Borowsky et al., 2002; Chaki
et al., 2005; David et al., 2007), but in-depth studies on the
molecular mechanism of MCH1 receptor blockade have not
yet been conducted.
Figure 1
Here we describe a potent low molecular weight MCH1
receptor blocker that inhibits multiple signalling pathways.
Chemical structures of MCH1 receptor antagonists.
1288 British Journal of Pharmacology (2014) 171 1287–1298

Slowly dissociating NAM of the MCH1 receptor
BJP
ing to the manufacturer’s instructions with minor modifica-
tions (Supporting Information).
Data analysis and statisitics
Data analysis was performed with GraphPad Prism5 software
(GraphPad, San Diego, CA). The values of EC50, IC50, and
Emax were calculated by fitting the data to a sigmoidal dose-
response equation. For analysis of dissociation kinetics, the
dissociation rate constant (Koff) was calculated by fitting the
data to the formula Y = Ae-kx, which is an exponential decay
model. Unless otherwise stated, EC50, IC50, and Kd values are
expressed as the mean with 95% confidence interval (CI) for
four experiments (n = 4), each of which was performed more
than twice. Unless otherwise stated, statistical significance
was determined by using an Student’s unpaired t-test. ANOVA
with a Dunnett’s test was used for the results presented in
Tables 2 and 3. A value of P < 0.05 was taken as statistically
significant.
Calcium flux assay
The calcium flux assay was performed by using a FLIPR Tetra
(Molecular Devices, Sunnyvale, CA, USA) according to the
manufacturer’s instructions with minor modifications (Sup-
porting Information).
PathHunter β-arrestin recruitment assay
The PathHunter β-arrestin recruitment assay was performed
according to the manufacturer’s instructions with minor
modifications (Supporting Information).
[125I]-MCH-(4-19) binding assay
Preparation of human MCH1 receptor membranes, prepara-
tion of radiolabelled [125I]-MCH-(4-19), and the receptor-
binding assay were all performed as described previously
(Takekawa et al., 2002) with minor modifications (Supporting
Information).
Results
Antagonistic effect of MQ1 on multiple
signalling pathways
It was previously reported that screening for MCH1 receptor
antagonists and subsequent chemical modification of lead
compounds resulted in the discovery of MQ1 (Figure 1) (Kasai
et al., 2012). In the membrane binding assay using radiola-
belled MCH-(4-19), which is a structural analogue of MCH
with agonistic activity, EC50 1.6 nM (95% CI: 0.44–5.7 nM) in
the cAMP assay, MQ1 inhibited the binding of [125I]-MCH-(4-
19) to human MCH1 receptor membrane fractions with an
IC50 value of 2.2 nM (95% CI: 1.8–2.7 nM) (Table 1).
We subsequently investigated the antagonistic effect of
MQ1 in functional assays using cells that stably expressed
human MCH1 receptor. It has already been indicated that the
MCH1 receptor couples with multiple G-proteins, including
Gαi, Gαo and Gαq (Hawes et al., 2000). We established a cAMP
assay and a calcium flux assay in which MCH stimulated
these G-protein signalling pathways with an EC50 value of
[125I]-MCH-(4-19) dissociation assay
The [125I]-MCH-(4-19) dissociation assay was performed as
described in the Supporting Information.
Equilibrium binding and dissociation assay
with affinity selection-MS
The equilibrium binding assay and dissociation assay with
affinity selection-MS were performed by using a LC-MS
system (API5000 LC/MS/MS system; AB SCIEX, Tokyo, Japan)
(Supporting Information).
Mutation analysis
Site-directed mutagenesis and transient transfection were per-
formed as described in the Supporting Information.
Table 1
Affinity and potency for MCH and three antagonists in multiple assays
Kd value
(95% CI)
(nM)
MCH EC50
(95% CI)
(nM)
MQ1 IC50
(95% CI)
(nM)
MQ2 IC50
(95% CI)
(nM)
MQ3 IC50
(95% CI)
(nM)
Assay type
[
125I]-MCH-(4-19) binding assay
(1 h of incubation)
–
–
–
–
2.2 (1.8–2.7)
28 (15–52)
21 (11–41)
16 (10–25)
11 (7.8–17)
[
125I]-MCH-(4-19) binding assay
(at equilibria)
0.32 (0.26–0.41)
Ki
0.058 (0.031–0.085)
–
0.16
11
5.6
cAMP assay
–
–
–
1.8 (0.81–4.3)
0.70 (0.53–0.92)
2.5 (1.9–3.3)
5.7 (2.7–12)
31 (18–51)
1.7 (1.4–2.0)
27 (12–58)
230 (200–280)
53 (32–87)
45 (15–136)
64 (54–75)
6.8 (4.6–10)
Calcium flux assay
PathHunter B-arrestin assay
The inhibitory effect (IC50) of MQ1, MQ2 and MQ3 was assessed with the [125I]-MCH-(4-19) binding assay with 1 and 8 h of incubation. The
calcium flux assay, PathHunter β-arrestin recruitment assay and cAMP assay were performed in the presence of 2, 10 and 5 nM of MCH
respectively. Values are means of three independent experiments and 95% confidence intervals (CI) conducted in duplicate (n = 2) for the
[
125I]-MCH-(4-19) binding assay and quadruplicate (n = 4) for the other assays.
British Journal of Pharmacology (2014) 171 1287–1298 1289

T Sakurai et al.
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1.8 nM (95% CI: 0.81–4.3 nM) and 0.70 nM (95% CI: 0.53–
0.92 nM) respectively. The IC50 value obtained for MQ1 in the
cAMP assay and the calcium flux assay was 5.7 nM (95% CI:
2.7–12 nM) and 31 nM (95% CI: 18–51 nM) respectively
(Table 1).
While activated GPCRs transduce G-protein signals, they
generally also activate β-arrestin-mediated signalling, which
is thought to be involved in various physiological functions
(Xiao et al., 2010). Therefore, we examined the antagonistic
effect of MQ1 on β-arrestin-mediated signalling by establish-
ing a PathHunter β-arrestin recruitment assay, in which the
interaction of GPCR and β-arrestin was detected by using
enzyme fragment complementation technology (Eglen,
2002). In this assay, MCH induced the recruitment of
β-arrestin to the MCH1 receptor with an EC50 value of 2.5 nM
(95% CI: 1.9–3.3 nM) and MQ1 showed antagonism with an
IC50 value of 1.7 nM (95% CI: 1.4–2.0 nM) (Table 1). These
results demonstrated that MQ1 had the ability to inhibit
multiple signalling pathways mediated by Gαi, Gαq and
β-arrestin.
Time dependence of inhibition by MQ1
The inhibitory effect of MQ1 was slightly weaker in the
calcium flux assay than in the other assays. This discrepancy
motivated us to investigate the possibility that inhibition by
the compound was time dependent, because it was consid-
ered likely that the calcium flux assay was conducted at
hemi-equilibrium, while the other three assays (the binding
assay, cAMP assay and PathHunter β-arrestin recruitment
assay) were performed with a long enough incubation time
for equilibrium to be reached. Accordingly, the PathHunter
β-arrestin recruitment assay was performed with various incu-
bation times to examine whether MQ1 exhibited time-
dependent inhibition. We found that the IC50 value decreased
as the incubation time became longer. Without pre-
incubation, the IC50 value was 14 nM (95% CI: 9.6–20 nM),
whereas after pre-incubation for 30, 60 or 120 min, the value
was significantly decreased to 4.1 nM (95% CI: 2.8–6.0 nM)
(P < 0.01), 2.6 nM (95% CI: 1.9–3.5 nM) (P < 0.01) and 1.9 nM
(95% CI: 1.3–2.6 nM) (P < 0.01) respectively (Figure 2A). In a
similar manner, we also evaluated the time dependence of
inhibition by MQ1 in the [125I]-MCH-(4-19) binding assay and
observed that its inhibitory effect increased in a time-
dependent manner. The IC50 value was 2.5 nM (95% CI:
1.6–4.0 nM) without pre-incubation, whereas after pre-
incubation for 30, 60 or 120 min, the IC50 was 1.0 nM (95%
CI: 0.45–2.3 nM), 0.57 nM (95% CI: 0.36–0.89 nM) (P < 0.01)
and 0.39 nM (95% CI: 0.26–0.59 nM) (P < 0.01) respectively
(Figure 2B). Taken together, these results suggested that MQ1
is an MCH1 receptor antagonist that shows time-dependent
inhibition.
Figure 2
Time-dependent inhibition by MQ1. (A) Concentration-dependent
inhibition by MQ1 was assessed in the PathHunter β-arrestin recruit-
ment assay. CHO-K1-BAEA-hMCH1 receptor cells were pre-incubated
with MQ1 for 0, 30, 60 or 120 min before incubation for 30 min with
MCH (10 nM). Data points are the mean SD of four values from
a representative experiment of three separate experiments. (B)
Concentration-dependent inhibition by MQ1 was assessed in the
[
125I]-MCH-(4-19) binding assay. Human MCH1 receptor membrane
fractions were pre-incubated with MQ1 for 0, 30, 60 or 120 min
before incubation for 30 min with [125I]-MCH-(4-19) (50 pM). Each
data point (n = 2) is plotted on the graph. Results are from repre-
sentative experiments that were performed twice.
value of 28 nM (95% CI: 15–52 nM) in the [125I-MCH-(4-19)
binding assay (Table 1, Supporting Information Figure S1b).
MQ1 showed saturable binding to membrane fractions
expressing MCH1 receptors in the absence of MQ2, whereas it
was completely displaced by an excess of MQ2 (Figure 3).
These findings indicate that the binding of MQ1 to MCH1
receptors is reversible.
Inhibitory effect of MQ1 after washout
Time-dependent reversible inhibition is generally considered
to be caused by slow dissociation of a compound from its
receptor. To confirm that this applied to MQ1, we performed
washout experiments using the PathHunter β-arrestin recruit-
ment assay. Pretreatment with various concentrations of test
compounds for 2 h inhibited MCH-induced recruitment of
β-arrestin in a concentration-dependent manner (Figure 4).
The inhibitory effect of MQ1 was still observed even after the
cells were washed twice before addition of MCH (Figure 4A).
In contrast, MQ2 did not show time-dependent inhibition in
the [125I]-MCH-(4-19) binding assay (Supporting Information
Reversible inhibition by MQ1
The time-dependence of inhibition by MQ1 (demonstrated
above) raised the possibility that it might be an irreversible
antagonist. Therefore, we investigated whether the inhibitory
effect of MQ1 was based on covalent binding to MCH1 recep-
tors. We developed an equilibrium binding assay using affin-
ity selection-MS to test whether MQ1 that had bound to
MCH1 receptors could be displaced by MQ2, a structurally
related MCH1 receptor antagonist (Figure 1) with an IC50
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Figure 3
Saturation of the binding of MQ1 to MCH1 receptors assessed by
affinity selection-MS. Human MCH1 receptor membrane fractions
were incubated with various concentrations of MQ1 in the absence
(total binding) or presence (non-specific) of MQ2 (30 μM) for
210 min at room temperature. Specific binding was determined as
the difference between binding in the absence or presence of MQ2.
The analyte peak area is displayed versus the concentration of MQ1.
Each data point (n = 2) is plotted on the graph. Results are from
representative experiments that were performed twice.
Figure 5
Dissociation kinetics of MCH1 receptor antagonists. Specific binding
of MQ1 and MQ2 to human MCH1 receptor membrane fractions was
measured over time as described in the Methods section. Initial
binding of each compound and the plateau of specific binding were
set as 100 and 0% respectively. Data points are the mean SD of
six values from
experiments.
a representative experiment of two separate
Figure S1b), and its binding was significantly reduced after
the same washout procedure (Figure 4B). These results
suggest that slow dissociation from the receptor contributed
to the time-dependence of inhibition by MQ1.
Dissociation kinetics of MQ1
To better understand the dissociation kinetics of MQ1, we
performed a dissociation assay based on the binding assay
with affinity selection-MS. After MQ1 was pre-incubated with
human MCH1 receptor membrane fractions, dissociation was
assessed over time following the addition of 50 μM MQ3
(Figure 1), which is a structurally related MCH1 receptor
antagonist with an IC50 value of 16 nM (95% CI: 10–25 nM)
in the [125I]-MCH-(4-19) binding assay (Table 1, Supporting
Information Figure S1c). MQ3 was used because we had
previously observed that it did not demonstrate time-
dependent inhibition in the [125I]-MCH-(4-19) binding assay
(Supporting Information Figure S1c). MQ1 slowly dissociated
from the receptor (Figure 5), and the dissociation rate con-
stant (Koff) was calculated to be 0.53 h−1 (95% CI: 0.48-
0.58 h−1). In contrast, MQ2 was not expected to undergo slow
dissociation based on the results of the washout experiments
in Figure 4. As predicted, it showed relatively rapid dissocia-
tion from the receptor (Figure 5) and its Koff value was calcu-
lated to be 2.6 h−1 (95% CI: 2.2–3.0 h−1). These findings were
consistent with the results of the washout experiments
(Figure 4).
Figure 4
Inhibitory effect of two MCH1 receptor antagonists after washout.
CHO-K1-BAEA-hMCH1 receptor cells were pretreated with various
concentrations of (A) MQ1 or (B) MQ2 dissolved in Opti-MEM with
0.1% BSA for 2 h at 37°C under 5% CO2. Then, Opti-MEM medium
containing the compounds was removed and the cells were washed
twice with 50 μL of PBS. Next, the cells were stimulated with 25 μL
of MCH (10 nM) for 2 h at 37°C under 5% CO2. All data points are
the mean SD of four values from a representative experiment of
two separate experiments.
Insurmountable antagonism induced by MQ1
To investigate the mechanism of the inhibition induced by
MQ1, the effect of MQ1 on concentration-dependent binding
of MCH was assessed in the PathHunter β-arrestin recruit-
ment assay. To exclude the possibility that the results might
be influenced by the slow dissociation of MQ1, we performed
the experiments with an incubation time of 8 h, because we
had already confirmed that the system reached equilibrium
British Journal of Pharmacology (2014) 171 1287–1298 1291

T Sakurai et al.
BJP
within 4 h as the EC50 values of MCH obtained at each con-
centration of MQ1 did not change between 4 and 8 h of
incubation (Supporting Information Table S1). We also moni-
tored the inhibitory effects of MQ1 over time and confirmed
that the IC50 values remained the same after 4 h of incubation
(Supporting Information Figure S2), in accordance with the
results of Supporting Information Table S1. The addition of
increasing concentrations of MQ1 caused both a rightward
shift of the curve for concentration-dependent binding of
MCH and a progressive reduction of the maximal response
(Figure 6A, Table 2). In addition, MQ2 showed a rapid disso-
ciation from the MCH1 receptor (Figure 5) and also demon-
strated insurmountable antagonism (Figure 6B, Table 2). In
contrast, a peptide antagonist (Gva-Cys-Met-Leu-Gly-Arg-Val-
Tyr-Ava-Cys-NH2) that was expected to be competitive with
MCH caused a parallel rightward shift without reducing the
maximal response (Figure 6C, Table 2). These results suggest
that MQ1 and MQ2 act as NAMs, while the peptide antago-
nist was a competitive inhibitor.
To confirm that the insurmountable antagonism demon-
strated by MQ1 and MQ2 was not an artefact specific to the
PathHunter β-arrestin recruitment assay, we performed a
similar experiment using the cAMP assay. We again observed
a rightward shift of the curve for concentration-dependent
binding of MCH and suppression of the maximal response
with increasing concentrations of MQ1 and MQ2 (Figure 7,
Table 3), consistent with the results obtained in the Path-
Hunter β-arrestin recruitment assay. Thus, the results of the
cAMP assay further supported the possibility that MQ1 and
MQ2 are NAMs.
Effect of MQ1 on the dissociation of
[125I]-MCH-(4-19) from MCH1 receptors
One characteristic of allosteric modulators is their ability
to alter the binding kinetics of orthosteric ligands
(Christopoulos and Kenakin, 2002). Accordingly, we exam-
ined the dissociation kinetics of [125I]-MCH-(4-19) from MCH1
receptors to obtain further evidence about the allosteric inter-
action of MQ1. The dissociation of [125I]-MCH-(4-19) was
detected after the addition of a substantial excess of unla-
belled MCH-(4-19) to prevent the association of [125I-MCH-
(4-19) with the receptor. The dissociation of [125I-MCH-(4-19)
from MCH1 receptors occurred with a Koff value of 0.13 min−1
(95% CI: 0.070–0.19 min−1) (Figure 8). While MCH-(1-19)
[which was expected to compete with MCH-(4-19)] did not
have any effect on the dissociation rate of [125I]-MCH-(4-19),
MQ1 significantly decreased the Koff value to 0.011 min−1
(95% CI: 0.0044–0.018 min−1) (P < 0.01) (Figure 8). We con-
firmed that MQ2 also significantly decreased the Koff value to
0.026 min−1 (95% CI: 0.0013–0.040 min−1) (P < 0.01). This
demonstrates that MQ1 and MQ2 alter the dissociation kinet-
ics of [125I]-MCH-(4-19) and clearly show that these com-
pounds have an allosteric interaction with MCH1 receptors.
Figure 6
Effect of antagonists on concentration-dependent activity of MCH in
the PathHunter β-arrestin recruitment assay. Concentration-
dependent inhibition by (A) MQ1 and (B) MQ2 in the presence
of increasing concentrations of MCH was measured in the Path-
Hunter β-arrestin recruitment assay after incubation for 8 h. (C)
Concentration-dependent inhibition by
a peptide antagonist
(Gva-Cys-Met-Leu-Gly-Arg-Val-Tyr-Ava-Cys-NH2) in the presence of
increasing concentrations of MCH was measured in the PathHunter
β-arrestin recruitment assay after overnight incubation. The magni-
tude of the response at each concentration was compared with the
amplitude of the response to a maximally efficacious concentration
of MCH (10 μM). Thus, results are expressed as % of Emax. All data
points are the mean
SD of four values from a representative
experiment of three separate experiments.
Site-directed mutagenesis
To further confirm that MQ1 binds to a different site from
that of MCH, we performed mutation analysis using a con-
ventional alanine scan on transmembrane (TM) helices 3, 5
and 6 based on the information that the corresponding TM
helices of the crystal structure of corticotrophin-releasing
factor receptor 1 forms an allosteric pocket (Hollenstein et al.,
2013). Eighteen residues predicted to be within this region
were separately mutated to alanine or valine, and potency of
MCH and MQ1 at each of these mutants was investigated in
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Table 2
Emax values of MCH at different concentrations of three antagonists in the PathHunter β-arrestin recruitment assay
MQ1
Control (nM)
100
1
10
100
1000
77 1.2***
52 1.0***
33 1.0***
24 1.1***
MQ2
Control (nM)
100
100
1000
3000
10 000
109 4.6
95 5.3
80 5.5
44 4.9***
Peptide antagonist
Control (nM)
100
10
100
1000
3000
96 1.9
98 2.2
104 3.2
92 3.2
Emax values of MCH with MQ1, MQ2 and a peptide antagonist were assessed with the PathHunter β-arrestin recruitment assay. All values
are expressed as mean SEM of four values from a representative experiment of three separate experiments. Statistical comparison was
performed using ANOVA with a Dunnett’s test. (*** P < 0.001 vs. Emax control values).
Information Table S2) whereas the mutants A136V and
H147A interestingly showed significant increases in the pIC50
value (Supporting Information Table S2). Furthermore, muta-
tion of threonine 209 and glutamine 276, which are consid-
ered to be involved in the ligand binding (Macdonald et al.,
2000), displayed significant decreases in the pEC50 value of
MCH but showed no statistical changes in the pIC50 value of
MQ1. These data further support the possibility that the
binding mode of MQ1 is different from that of MCH.
Selectivity of MQ1 for MCH1 receptors
To assess the selectivity of MQ1 for MCH1 receptors, the effect
of this compound on other drug targets was experimentally
examined at Ricerca Biosciences (Concord, OH, USA) using
equilibrium binding assays and enzyme activity assays. MQ1
(1 μM) did not show a strong effect on over 100 targets,
including various GPCRs, enzymes and ion channels (data
not shown). In particular, it had no significant effect on
MCH2 receptors, somatostatin receptor 1 and μ-opioid recep-
tor, all of which exhibit a high degree of homology with
MCH1 receptors. These findings demonstrate that MQ1 is a
highly selective NAM for MCH1 receptors.
Discussion
The results presented here demonstrate that MQ1 is an
Figure 7
antagonist, which inhibits multiple signalling pathways of
MCH1 receptors. In addition, MQ1 is a reversible antagonist
that dissociates slowly from the receptor. Furthermore, it was
demonstrated that MQ1 is a NAM of the MCH1 receptor.
Effect of antagonists on concentration-dependent activity of MCH in
the cAMP assay. Concentration-dependent antagonism by (A) MQ1
and (B) MQ2 in the presence of increasing concentrations of MCH was
measured in the cAMP assay. Activity is plotted as percentage of
maximal MCH response. Data points are the mean SD of four values
from a representative experiment of three separate experiments.
Inhibition of multiple signalling pathways
MQ1 inhibited Gαi and β-arrestin signalling with IC50 values
of 5.7 nM (95% CI: 2.7–12 nM) and 1.7 nM (95% CI: 1.4–
2.0 nM), respectively, consistent with its IC50 value of 2.2 nM
(95% CI: 1.8–2.7 nM) in the [125I]-MCH-(4-19) binding assay.
Compared with the results from these three assays, MQ1 had
a slightly larger IC50 of 31 nM (95% CI: 18–51 nM) in the
the PathHunter β-arrestin recruitment assay following tran-
sient transfection. Substitution of most of these individual
residues did not show a significant change in the pEC50 or
pIC50 value compared with the wild type (P > 0.05, Supporting
British Journal of Pharmacology (2014) 171 1287–1298 1293

T Sakurai et al.
BJP
Table 3
Emax values of MCH at different concentrations of MQ1 and MQ2 in the cAMP assay
MQ1
Control (nM)
100
10
100
300
1000
3000
99 1.8
98 1.5
95 2.6
92 0.91**
88 0.91***
MQ2
Control (nM)
100
100
300
1000
3000
96 1.8
89 1.9
85 2.5**
75 0.81**
Emax values of MCH with MQ1 and MQ2 were assessed with the cAMP assay. All values are expressed as mean SEM of four values from
a representative experiment of three separate experiments. Statistical comparison was performed using ANOVA with a Dunnett’s test. (** P <
0.01, *** P < 0.001 vs. Emax control values).
that the pharmacologically relevant signalling pathway of
MCH1 receptors is not clearly understood and that it even
remains uncertain whether or not MCH1 receptors activate
multiple signalling pathways in vivo, inhibition of all the
signalling pathways (including the β-arrestin pathway) that
have been detected in recombinant overexpression systems
could be important for a compound to exhibit the expected
pharmacological effects in vivo. Hence, the finding that MQ1
can inhibit multiple signalling pathways might increase the
likelihood of it exhibiting efficacy in vivo. Moreover, the
importance of understanding the relationship between dif-
ferent signalling pathways and their physiological conse-
Figure 8
quences in drug discovery programmes is becoming more
Effect of MQ1 on the dissociation of [125I]-MCH from MCH1 recep-
strongly appreciated as recent studies have identified a wide
tors. Human MCH1 receptor membrane fractions were incubated
variety of compounds that have differential effects on various
with 50 pM of [125I]-MCH-(4-19) for 2 h before adding an excess
signalling pathways (Violin and Lefkowitz, 2007; Drake et al.,
2008; Gesty-Palmer et al., 2009; Kenakin, 2009; Reiter et al.,
2012). Thus, our efforts to determine the potency of MQ1 for
each signalling pathway could be important with regard to
elucidating the physiologically relevant signalling pathway
for MCH1 receptors in the drug discovery process.
(1 μM) of unlabelled MCH-(4-19) to initiate dissociation of [125I]-
MCH in the absence or presence of MQ1 , MQ2 and MCH-(1-19).
Each condition contains 1% DMDO. Initial binding and the plateau
of specific binding were set at 100 and 0% respectively. Data points
are the mean SD of three values from a representative experiment
of three separate experiments.
calcium flux assay, presumably because that assay was con-
ducted at hemi-equilibrium. This hypothesis was supported
by the finding that pre-incubation for 60 min increased the
potency of MQ1 to 5.2 nM (95% CI: 2.1–13 nM) in the
calcium flux assay (data not shown), which was similar to
the results of the other assays. Therefore, when tested at
equilibrium, MQ1 exhibited equal inhibitory activity in all of
the cell-based functional assays that we performed, demon-
strating its ability to inhibit multiple signalling pathways.
So far, several MCH1 receptor antagonists have been
reported to inhibit Gαq and/or Gαi signalling (Borowsky et al.,
2002; Takekawa et al., 2002; David et al., 2007). However, to
the best of our knowledge, there has been no report about
an MCH1 receptor antagonist with the ability to inhibit
β-arrestin signalling. In addition to playing a key role in
internalization and subsequent desensitization of receptors,
β-arrestin has been found to be involved in more diverse
signalling processes than was previously appreciated (Xiao
et al., 2010). This study shows that MQ1 inhibits β-arrestin
signalling, although it is not clear whether the compound
inhibits β-arrestin signalling directly or blocks G-protein cou-
pling, and as a result inhibits β-arrestin binding. Considering
Slow dissociation
We showed that the inhibitory effect of MQ1 increased over
time in both the cell-based PathHunter β-arrestin recruitment
assay (Figure 2A) and the cell-free [125I]-MCH-(4-19) binding
assay (Figure 2B). Although it has to be emphasized that the
system may not have reached re-equilibrium among the three
molecules (MCH, MQ1 and MCH1 receptor), the changes in
the IC50 values with different pre-incubation times suggest
that MQ1 is a time-dependent inhibitor. In addition, the
inhibitory effect of MQ1 was still observed after washout
(Figure 4). These results suggest that MQ1 undergoes slow
dissociation from the MCH1 receptor. It is generally consid-
ered that slow dissociation of an antagonist contributes to
extending the receptor residence time and prolongs its
effects, resulting in maximal antagonist activity in vivo
(Copeland et al., 2006; Brinkerhoff et al., 2008; Copeland,
2010). For example, it has been reported that candesartan, a
slowly dissociating angiotensin AT1 receptor antagonist, has a
stronger antihypertensive effect than a more rapidly dissoci-
ating antagonist (Hansson, 2001; Van Liefde and Vauquelin,
2009), while the pharmacodynamics of the μ-opioid receptor
antagonist buprenorphine have been attributed to its slow
1294 British Journal of Pharmacology (2014) 171 1287–1298

Slowly dissociating NAM of the MCH1 receptor
BJP
dissociation from the receptor (Yassen et al., 2006). Therefore,
detailed evaluation of slow dissociation kinetics in order to
accurately understand structure–activity relationships (SAR)
may be important for maximizing the in vivo efficacy of
compounds during the chemical optimization process. This
concept motivated us to apply the affinity selection-MS-based
equilibrium binding assay as a dissociation assay that enabled
us to directly determine the Koff values of the test compounds.
We found that MQ1 dissociated from the receptor with a Koff
value of 0.53 h−1 (95% CI: 0.48–0.58 h−1) and its dissociation
was five times slower than that of MQ2, in good agreement
with the results of the washout experiments. It is noteworthy
that subtle differences of the chemical structure resulted in
such a significant difference of dissociation kinetics. This
finding highlights the importance of understanding SAR as
part of a drug discovery program. This kinetic analysis
method using affinity selection-MS that we have devised
should also be applicable to the development of other com-
pounds with slow dissociation kinetics. Although a large
number of MCH1 receptor antagonists have already been
identified by various pharmaceutical companies, this is the
first report, to our knowledge, that provides clear evidence of
(Promega, Tokyo, Japan). The results confirmed that there
was no change in cell viability, indicating that MQ1 was not
cytotoxic in this assay (data not shown).
Taken together, these findings suggested that MQ1 could
be a NAM that reduces the efficiency of the receptor, resulting
in insurmountable antagonism. To confirm that MQ1 had an
allosteric interaction with the MCH1 receptor, we performed
kinetic studies using radiolabelled MCH. The rate constants
that govern the association (Kon) and dissociation (Koff) of
ligands are sensitive indicators of the interaction of each
ligand with a particular receptor conformation. Therefore, a
change in receptor conformation induced by an allosteric
modulator would theoretically be expected to lead to changes
in orthosteric ligand association and/or dissociation proper-
ties (Christopoulos and Kenakin, 2002). Our kinetic studies
revealed that MQ1 altered the rate of dissociation, whereas a
competitive peptide agonist had no such effect, a finding that
confirmed the allosteric interaction of MQ1 with MCH1
receptors. Interestingly, the dissociation rate of MCH from
the receptor was reduced by MQ1 (Figure 8). Several NAMs
have already been reported to display this property, for
example, Ellis and Seidenberg showed that some NAMs for
muscarinic acetylcholine receptors decreased the dissociation
rate of orthosteric radioligands while still reducing binding
affinity (Ellis and Seidenberg, 1992). Similar to these com-
pounds, MQ1 is likely to be a NAM that induces a change in
the receptor conformation, which results in the slowing of
both dissociation and association.
a
compound that slowly dissociates from the receptor.
Whereas some targets are reported to cause adverse side
effects due to prolonged occupancy (Copeland et al., 2006;
Bryant et al., 2008; Tummino and Copeland, 2008), it is
thought that the unique property of MQ1 would be beneficial
with respect to prolongation of pharmacodynamic efficacy
in vivo.
We also performed mutation analysis to predict the
binding site of MQ1 using the PathHunter β-arrestin recruit-
ment assay. One of the advantages of using this assay is
that the expression level of transfected receptors does not
affect the potency of MCH, as there is a linear relationship
between occupancy and effect in the PathHunter β-arrestin
recruitment assay (Nickolls et al., 2011). To select residues
for substitution, we used information obtained from the
corticotropin-releasing factor receptor 1 crystal structure
because it is the only crystal structure of a GPCR available in
complex with a NAM, and because it clearly shows the allos-
teric site (Hollenstein et al., 2013). The potency of MCH at
most of the mutant receptors is unaltered, suggesting that
these mutations do not affect the overall receptor conforma-
tion and MCH binding to the MCH1 receptor, whereas
alanine 136 and histidine 147, which are predicted to be
within TM helix 3, might be involved in the binding of MQ1.
These data further support the hypothesis that MQ1 allosteri-
cally binds to the MCH1 receptor when compared to MCH.
Finally, we demonstrated that MQ1 is a highly selective
NAM for MCH1 receptors, because it had no obvious effect on
other molecular targets with a high level of homology,
including GPCRs. Allosteric modulators are generally consid-
ered to display considerable selectivity, presumably because
many receptors exhibit greater divergence of sequence
homology at allosteric sites compared with orthosteric sites
(Christopoulos and Kenakin, 2002; Kenakin and Miller,
2010). Thus, the very high selectivity of MQ1 might be due to
its binding to an allosteric site on the MCH1 receptor.
To date, a competitive antagonist (Borowsky et al., 2002),
orthosteric insurmountable antagonist (David et al., 2007)
and non-competitive antagonists (Chaki et al., 2005) for
MCH1 receptors have been reported. However, to the best
Negative allosteric modulation
While studying the mode of the inhibition induced by MQ1,
we observed insurmountable antagonism. In general, the fol-
lowing molecular mechanisms have been suggested to con-
tribute to insurmountable antagonism: assessment of assay
results at hemi-equilibrium, irreversible non-competitive
inhibition, cytotoxicity of the test compound and negative
allosteric modulation of the receptor (Ojima et al., 2011).
Firstly, to exclude the possibility that the assay was con-
ducted at hemi-equilibrium, we incubated the cells with MQ1
and MCH for 8 h in our experiments. We did so because we
considered that the system would reach equilibrium within
this period, based on the results of previous experiments with
different pre-incubation times (Figure 2). This hypothesis was
supported by our observations that the potency of MCH
obtained at each concentration of MQ1 did not change
between 4 and 8 h of incubation (Supporting Information
Table S1), indicating that the system reached equilibrium
within 4 h. Therefore, we considered that the insurmount-
able antagonism exhibited by MQ1 was not due to perform-
ing the assay at hemi-equilibrium.
Secondly, we investigated the possibility that MQ1 was an
irreversible non-competitive antagonist by using an equilib-
rium binding assay with affinity selection-MS. We found that
bound MQ1 was displaced from the membrane fraction by
the structurally related MQ2, suggesting that the binding of
MQ1 was reversible (Figure 3).
Thirdly, to examine whether the reduction of the
maximal response was due to cytotoxicity of MQ1, we evalu-
ated cell viability after 8 h of incubation with the compound
by using a CellTiter-Glo Luminescent Cell Viability assay
British Journal of Pharmacology (2014) 171 1287–1298 1295

T Sakurai et al.
BJP
ATC0065 and ATC0175: nonpeptidic and orally active melanin-
concentrating hormone receptor 1 antagonists. J Pharmacol Exp
Ther 313: 831–839.
of our knowledge, there has been no report about a low
molecular weight compound that clearly exhibits an allos-
teric interaction with the MCH1 receptor. The findings of this
study are of importance because this was the first demonstra-
tion that there is an allosteric site of the MCH1 receptor to
which a low molecular weight compound can bind. In addi-
tion, the allosteric site to which MQ1 binds could be useful in
the drug discovery process for MCH1 receptor blockers,
because this compound exerts preferable antagonistic effects,
such as inhibition of multiple signalling pathways, slow dis-
sociation from the receptor and high selectivity. Thus, we
expect that our findings will help to accelerate the discovery
of MCH1 receptor blockers that can be developed as anti-
obesity agents.
Chambers J, Ames RS, Bergsma D, Muir A, Fitzgerald LR, Hervieu G
et al. (1999). Melanin-concentrating hormone is the cognate ligand
for the orphan G-protein-coupled receptor SLC-1. Nature 400:
261–265.
Chen Y, Hu C, Hsu CK, Zhang Q, Bi C, Asnicar M et al. (2002).
Targeted disruption of the melanin-concentrating hormone
receptor-1 results in hyperphagia and resistance to diet-induced
obesity. Endocrinology 143: 2469–2477.
Cheon HG (2012). Antiobesity effects of melanin-concentrating
hormone receptor 1 (MCH-R1) antagonists. Handb Exp Pharmacol
209: 383–403.
Christopoulos A, Kenakin T (2002). G protein-coupled receptor
allosterism and complexing. Pharmacol Rev 54: 323–374.
Copeland RA (2010). The dynamics of drug-target interactions:
drug-target residence time and its impact on efficacy and safety.
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Acknowledgements
The authors would like to express their gratitude to K Takami,
T Okawa and T Murata for compound preparation. We also
thank I Miyahisa, T Sameshima, M Hixon, K Okada, Y Hiro-
zane and Y Shimizu for helpful discussion of data analysis.
We would also like to acknowledge the support and encour-
agement of Y Nagisa, J Matsui and N Tarui when implement-
ing this study.
Copeland RA, Pompliano DL, Meek TD (2006). Drug-target
residence time and its implications for lead optimization. Nat Rev
Drug Discov 5: 730–739.
David DJ, Klemenhagen KC, Holick KA, Saxe MD, Mendez I,
Santarelli L et al. (2007). Efficacy of the MCHR1 antagonist
N-[3-(1-{[4-(3,4-difluorophenoxy)phenyl]methyl}(4-piperidyl))-4-
methylphenyl]-2-methylpropanamide (SNAP 94847) in mouse
models of anxiety and depression following acute and chronic
administration is independent of hippocampal neurogenesis.
J Pharmacol Exp Ther 321: 237–248.
Conflict of interest
Della-Zuana O, Presse F, Ortola C, Duhault J, Nahon JL, Levens N
(2002). Acute and chronic administration of melanin-concentrating
hormone enhances food intake and body weight in Wistar and
Sprague-Dawley rats. Int J Obes Relat Metab Disord 26: 1289–1295.
The authors state no conflict of interest.
Drake MT, Violin JD, Whalen EJ, Wisler JW, Shenoy SK, Lefkowitz
RJ (2008). beta-arrestin-biased agonism at the beta2-adrenergic
receptor. J Biol Chem 283: 5669–5676.
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Figure S1 Inhibitory effect of three MCH1 receptor antago-
nists. Concentration-dependent inhibition by MQ1 (A), MQ2
(B) and MQ3 (C) was assessed with the [125I]-MCH-(4-19)
British Journal of Pharmacology (2014) 171 1287–1298 1297

T Sakurai et al.
BJP
binding assay with 1 h (□) and 8 h (■) of incubation. Each
data point (n = 2) is plotted on the graph. Results are from
representative experiments that were performed twice.
Figure S2 Time course of inhibitory effect (pIC50) of MQ1.
CHO-K1-BAEA-hMCH1 receptor cells were incubated with
MQ1 and 10 or 100 nM MCH for 1, 2, 4, 8, 24 and 32 h of
incubation. Each data point represents pIC50 values of two
independent experiments performed in quadruplicate (n = 4).
Table S1 pEC50 values of MCH at different concentration of
MQ1 with different incubation time. EC50 values of MCH at
different concentration of MQ1 were assessed with the Path-
Hunter β-arrestin recruitment assay after incubation for 4
and 8 h. All data are represented as mean
SEM of four
values from a representative experiment of three separate
experiments. Statistical comparison of pEC50 values was per-
formed using an unpaired t-test and no values were deter-
mined different (P > 0.05).
Table S2 Effects of mutations on potency for MCH and
MQ1. Potency of MCH and the inhibitory effect of MQ1 for
MCH1 receptor mutants were assessed with the PathHunter
β-arrestin recruitment assay. Values are pEC50 and pIC50
means SEM of two independent experiments conducted in
quadruplicate (n = 4). Mutant values were compared with the
wild type using an unpaired t-test (*P < 0.05).
1298 British Journal of Pharmacology (2014) 171 1287–1298