Structural insights into the differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor

Article abstract of DOI:10.1371/journal.pone.0213552

Lactisole, an inhibitor of the human sweet taste receptor, has a 2-phenoxypropionic acid skeleton and has been shown to interact with the transmembrane domain of the T1R3 subunit (T1R3-TMD) of the receptor. Another inhibitor, 2,4-DP, which shares the same molecular skeleton as lactisole, was confirmed to be approximately 10-fold more potent in its inhibitory activity than lactisole; however the structural basis of their inhibitory mechanisms against the receptor remains to be elucidated. Crystal structures of the TMD of metabotropic glutamate receptors, which along with T1Rs are categorized as class C G-protein coupled receptors, have recently been reported and made it possible to create an accurate structural model for T1R3-TMD. In this study, the detailed structural mechanism underlying sweet taste inhibition was characterized by comparing the action of lactisole on T1R3-TMD with that of 2,4-DP. We first performed a series of experiments using cultured cells expressing the sweet taste receptor with mutations and examined the interactions with these inhibitors. Based on the results, we next performed docking simulations and then applied molecular dynamics-based energy minimization. Our analyses clearly revealed that the (S)-isomers of both lactisole and 2,4-DP, interacted with the same seven residues in T1R3-TMD and that the inhibitory potencies of those inhibitors were mainly due to stabilizing interactions mediated via their carboxyl groups in the vertical dimension of the ligand pocket of T1R3-TMD. In addition, 2,4-DP engaged in a hydrophobic interaction mediated by its o-Cl group, and this interaction may be chiefly responsible for the higher inhibitory potency of 2,4-DP.

Full text of DOI:10.1371/journal.pone.0213552

RESEARCH ARTICLE
Structural insights into the differences among
lactisole derivatives in inhibitory mechanisms
against the human sweet taste receptor
Tomoya Nakagita 1,2, Akiko Ishida3, Takumi Matsuya1, Takuya Kobayashi2,
ID
Masataka Narukawa1, Takatsugu Hirokawa4,5, Makoto Hashimoto3, Takumi Misaka1*
1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Tokyo, Japan, 2 Department of Cell Biology, Graduate School of Medicine, Kyoto
University, Kyoto, Japan, 3 Graduate School of Agriculture, Hokkaido University, Sapporo, Japan,
4 Molecular Profiling Research Center for Drug Discovery, National Institutes of Advanced Industrial Science
and Technology, Tokyo, Japan, 5 Department of Chemical Biology, Faculty of Medicine, University of
Tsukuba, Ibaraki, Japan
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a1111111111
a1111111111
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* amisaka@mail.ecc.u-tokyo.ac.jp
Abstract
OPEN ACCESS
Lactisole, an inhibitor of the human sweet taste receptor, has a 2-phenoxypropionic acid
skeleton and has been shown to interact with the transmembrane domain of the T1R3 sub-
unit (T1R3-TMD) of the receptor. Another inhibitor, 2,4-DP, which shares the same molecu-
lar skeleton as lactisole, was confirmed to be approximately 10-fold more potent in its
inhibitory activity than lactisole; however the structural basis of their inhibitory mechanisms
against the receptor remains to be elucidated. Crystal structures of the TMD of metabotropic
glutamate receptors, which along with T1Rs are categorized as class C G-protein coupled
receptors, have recently been reported and made it possible to create an accurate structural
model for T1R3-TMD. In this study, the detailed structural mechanism underlying sweet
taste inhibition was characterized by comparing the action of lactisole on T1R3-TMD with
that of 2,4-DP. We first performed a series of experiments using cultured cells expressing
the sweet taste receptor with mutations and examined the interactions with these inhibitors.
Based on the results, we next performed docking simulations and then applied molecular
dynamics-based energy minimization. Our analyses clearly revealed that the (S)-isomers of
both lactisole and 2,4-DP, interacted with the same seven residues in T1R3-TMD and that
the inhibitory potencies of those inhibitors were mainly due to stabilizing interactions medi-
ated via their carboxyl groups in the vertical dimension of the ligand pocket of T1R3-TMD. In
addition, 2,4-DP engaged in a hydrophobic interaction mediated by its o-Cl group, and this
interaction may be chiefly responsible for the higher inhibitory potency of 2,4-DP.
Citation: Nakagita T, Ishida A, Matsuya T,
Kobayashi T, Narukawa M, Hirokawa T, et al.
(2019) Structural insights into the differences
among lactisole derivatives in inhibitory
mechanisms against the human sweet taste
receptor. PLoS ONE 14(3): e0213552. https://doi.
org/10.1371/journal.pone.0213552
Editor: Maik Behrens, Leibniz-Institute for Food
Systems Biology at the TU Munich, GERMANY
Received: November 28, 2018
Accepted: February 23, 2019
Published: March 18, 2019
Copyright: © 2019 Nakagita et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Funding: This research was funded by Council for
Science, Technology and Innovation (CSTI),
Technologies for creating next-generation
agriculture, forestry and fisheries Cross-ministerial
Strategic Innovation Promotion Program (SIP), the
Basis for Supporting the Platform Project for
Supporting Drug Discovery and Life Science
Introduction
In humans, the taste receptor type 1 family (T1Rs), which are classified as class C G-protein
coupled receptors (GPCRs), make a major contribution to the recognition of major nutrients
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
Research from AMED (Japan Agency for Medical
Research and Development grants
such as sugars and amino acids, which represent major energy sources in human diets, in
our oral cavity [1]. The sugar-recognizing sweet taste receptor, which consists of a heterodimer
of T1R2 and T1R3 [2–6], has been shown to contain multiple ligand-binding sites in its struc-
ture [7]. Low-molecular-weight sweeteners, such as sugar, sucralose, aspartame, saccharin,
and acesulfame K, are recognized within the extracellular Venus flytrap domain of T1R2
(T1R2-VFTD), which is considered to be an orthosteric binding site [8,9]. In our previous
study, in which we sought to elucidate the mode of action between the human sweet taste
receptor and those individual sweeteners, we established cell lines that stably express each
of the point mutants for the sweet taste receptor together with a chimeric G-protein
(Gα16gust44) and performed a series of mutational analyses that successfully revealed
suitable candidates for residues in T1R2-VFTD that interact with such sweeteners [9].
Another binding site in the sweet taste receptor was found in the transmembrane domain
of T1R3 (T1R3-TMD). This domain has been shown to be characterized by features that
enable its interaction with both sweeteners and sweet taste inhibitors [10–13]. It has been
shown that sweeteners such as cyclamate and neohesperidin dihydrochalcone (NHDC) both
interact with T1R3-TMD, revealing that they are recognized as sweet by themselves (in other
words, they actually act as agonists), and act as positive allosteric modulators (PAMs) of
orthosteric ligands; they are therefore termed ago-PAMs [14–18].
JP18am0101079 to TK and JP180101114 to TH),
MEXT as “Priority Issue on Post-K computer”
(hp160213 to TH). The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Sweet taste inhibitors, such as lactisole, also interact with T1R3-TMD but act as negative
allosteric modulators (NAMs). Lactisole was originally isolated from roasted coffee beans [19].
Because the molecule contains a lactic acid structure, lactisole has naturally occurring optical
isomers. In general, there is more L-lactic acid [(S)-lactic acid] than D-lactic acid in plants, so
the (S)-isomer of lactisole is predominant in roasted coffee beans [20]. Several other inhibitors
of sweet taste receptors have also been characterized in previous publications. Another sweet
taste inhibitor, 2,4-DP (2-(2,4-dichlorophenoxy)propionic acid), has a 2-phenoxypropionic
acid skeleton similar to that of lactisole [21]. Although 2,4-DP also interacts with T1R3-TMD
and exhibits approximately 10-fold higher inhibitory potency than lactisole, the structural
basis for this difference in potency has not been elucidated.
In addition, a previous publication proposed an interaction model between T1R3-TMD
and lactisole based on the crystal structure of rhodopsin, a representative class A GPCR [11].
However, the amino acid sequence identity between rhodopsin and T1R3-TMD is quite low,
making it unlikely that the constructed structural model for T1R3-TMD accurately describes
the detailed structural mechanisms of lactisole-T1R3 interactions. Recently, the crystal struc-
tures of the transmembrane domain of metabotropic glutamate receptors (mGluRs), which
along with T1Rs are categorized as class C GPCRs, were solved [22,23]. These structures made
it possible to create an accurate interaction model of T1R3-TMD.
In this study, we sought to characterize the similarities and differences between the modes
of action of lactisole and 2,4-DP by creating docking models with T1R3-TMD. We reasoned
that common features are likely to be important for inhibitory activity, and distinct features
are likely to be responsible for differences in inhibitory potency among those inhibitors.
Materials and methods
Cell-based assay
Construction of cell lines stably expressing the sweet taste receptor. The previously
reported method to construct a cell line stably expressing the human sweet taste receptor
[14,24] was also used in this study. The genes encoding T1R2, T1R3, and Gα16gust44 were
incorporated into a modified version of the pcDNA5/FRT vector (Thermo Fisher Scientific,
Waltham, MA, U.S.A.). Following the Flp-In System protocol (Thermo Fisher Scientific), this
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
construct was transfected into Flp-In 293 cells. Each cell line stably expressing a variant of the
sweet taste receptor was generated in the same manner. Nucleotide mutations were introduced
into the expression construct by PCR-based mutations. The residues to be mutated were pri-
marily chosen based on previous studies [10–12].
Measurement of cellular responses. Measurements of cellular responses were performed
as described previously [14,24]. Cells stably expressing the sweet taste receptor were seeded in
96-well plates and incubated for an additional 23 hours. The cells were washed with assay
buffer and loaded using the FRIPR Calcium 4 Assay Kit (Molecular Devices, San Jose, CA,
U.S.A.). Measurement was performed on FlexStation 3 (Molecular Devices) after the samples
were incubated at 37 ˚C for 1 hour. The temperature of the FlexStation 3 was also maintained
at 37 ˚C.
Data analysis. All data were fitted to Hill’s equation, which was drawn using Clampfit 9.2
(Molecular Devices, Palo Alto, CA, USA), and EC50 and IC50 values were calculated from a
dose-response curve.
Computer simulation
Creation of the homology model of T1R3-TMD. All calculations were conducted using
Schro¨dinger Suite 2017–1 (Schro¨dinger, LLC) and performed under the OPLS3 force field.
First, the crystal structure of mGluR1-TMD (Protein Data Bank ID code: 4OR2) was prepared,
then the T1R3-TMD homology model was created with reference to alignments of mGluR1,
mGluR5, and T1Rs. The result of alignment is shown in S1 Fig (according to GPCRdb (http://
gpcrdb.org), and the sequence identities vs T1R3 were mGluR1, 20%; mGluR5, 18%; T1R1,
27%; and T1R2, 22% respectively. (As a side note, sequence identities vs class A GPCRs were
quite low: rhodopsin, 9% and β2-adrenoceptor, 6%, respectively. (S1B Fig)). The alignments
and calculation to create the model were performed with Prime (Schro¨dinger). Insertion gaps
were considered using both structures of mGluR1 (4OR2) and mGluR5 (4OO9). All residues
shown in this study are labeled with superscript characters according to generic GPCR residue
numbers [25].
Ligand docking. Five ligands, (S)-lactisole, (R)-lactisole, (S)-2,4-DP, (R)-2,4-DP, and (S)-
2-phenoxypropionic acid were calculated in advance, with proper form and ionic charge,
using LigPrep (Schro¨dinger). The ligands were then docked to the ligand pocket surrounded
by residues identified by mutational analysis. Docking simulations were performed using
Glide (Schro¨dinger) in Standard Precision-mode. After docking, we recalculated the ligand
pocket configuration using the Protein Preparation function in Maestro.
Molecular dynamics-based energy minimization. (S)-lactisole, (R)-lactisole, (S)-2,4-DP,
and (R)-2,4-DP were subjected to four independent molecular dynamics (MD) simulations
using Desmond (Schro¨dinger). The initial docking models were placed into a large POPC
bilayer, and TIP3P water molecules were solvated with 0.15 M NaCl. After minimization and
relaxation of the model, the production MD phase was performed for 100 ns in the isother-
mal-isobaric (NPT) ensemble at 300 K and 1 bar using a Langevin thermostat. Long-range
electrostatic interactions were computed using the smooth particle mesh Ewald method. The
obtained trajectory was processed utilizing the AMBER11 tool ptraj [26] for RMSD calcula-
tions and PMSF for protein and ligand RMSDs. Representative structures of each ligand were
chosen from 10,000 trajectory coordinates (last 100 ns) with a k-means clustering algorithm
(k = 1) using ptraj. The time course of protein-RMSD (red) and ligand-RMSD (blue) were
shown in S2 Fig. Since the RMSD of each model reached a plateau by 100 ns (S2B Fig), the
structures of 100 ns were chosen to apply heavy-atom-restrained minimization and they were
regarded as an MD-minimized model.
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
Synthesis
(S)-lactisole. Step 1. Synthesis of methyl (S)-2-(4-methoxyphenoxy)propanoate [(S)-
2a]. To 4-methoxyphenol 1a (201 mg, 1.62 mmol) in dry CH2Cl2 (14.9 mL), methyl D-(+)-lac-
tate (253 mg, 2.43 mmol) and PPh3 (718 mg, 2.74 mmol) were added at 0 ˚C. After the reaction
mixture was stirred for 10 min at 0 ˚C, DEAD (486 mg, 2.79 mmol) was slowly added at the
same temperature. The reaction mixture was stirred overnight at room temperature and then
partitioned between water and CH2Cl2. The organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated. The residue was purified by column chromatography
(ethyl acetate/n-hexane, 1:9) to yield (S)-2a (305 mg, 89%). 1H NMR (270 MHz, CDCl3): δ =
6.81 (s, 4H), 4.67 (q, J = 6.7 Hz, 1H), 3.73 (s, 6H), 1.58 (d, J = 6.9 Hz, 3H) ppm, 13C NMR (67.5
MHz, CDCl3): δ = 172.7, 154.4, 151.5, 116.4, 114.5, 73.4, 55.4, 52.0, 18.4 ppm.
Step 2. Synthesis of (S)-lactisole [(S)-3a]. Methyl (S)-2-(4-methoxyphenoxy)propanoate
(S)-2a (193 mg, 0.92 mmol) was dissolved in MeOH (17 mL) and H2O (2 mL), and then
K2CO3 (399 mg, 2.89 mmol) was added. The reaction mixture was stirred at reflux for 2 hours,
cooled to room temperature, and then partitioned between ethyl acetate and water. The water
layer was acidified with 1 M HCl aq. and extracted with ethyl acetate. The organic layer was
washed with H2O and brine and then dried over MgSO4, filtered, and concentrated to yield
(S)-3a (148 mg, 82%). 1H NMR (270 MHz, CDCl3): δ = 6.77 (d, J = 3.0 Hz, 4H), 4.62 (q, J = 6.9
Hz, 1H), 3.69 (s, 3H), 1.56 (d, J = 6.9 Hz, 3H) ppm, 13C NMR (67.5 MHz, CDCl3): δ = 177.8,
154.7, 151.2, 116.7, 114.8, 73.2, 55.7, 18.4 ppm.
(R)-lactisole. Step 1. Synthesis of methyl (R)-2-(4-methoxyphenoxy)propanoate [(R)-
2a]. The similar treatment of 4-methoxyphenol 1a (208 mg, 1.68 mmol) and methyl L-(-)-lac-
tate (262 mg, 2.52 mmol) as that just described gave (R)-2a (334 mg, 95%). 1H NMR (270 MHz,
CDCl3): δ = 6.80 (s, 4H), 4.67 (q, J = 6.8 Hz, 1H), 3.72 (s, 6H), 1.58 (d, J = 6.9 Hz, 3H) ppm, 13
NMR (67.5 MHz, CDCl3): δ = 172.7, 154.4, 151.5, 116.3, 114.5, 73.4, 55.4, 52.0, 18.4 ppm.
C
Step 2. Synthesis of (R)-lactisole [(R)-3a]. The similar treatment of methyl (S)-2-(4-meth-
oxyphenoxy)propanoate (S)-2a (250 mg, 1.19 mmol) as that just described gave (R)-3a (209
mg, 89%). 1H NMR (270 MHz, CDCl3): δ = 6.77 (s, 4H), 4.62 (q, J = 6.8 Hz, 1H), 3.69 (s, 3H),
1.56 (d, J = 6.6 Hz, 3H) ppm, 13C NMR (67.5 MHz, CDCl3): δ = 178.1, 154.6, 151.3, 116.6,
114.7, 73.1, 55.6, 18.4 ppm.
(S)-2,4-DP. Step 1. Synthesis of methyl (S)-2-(2,4-dichlorophenoxy)propanoate [(S)-
2b]. The similar treatment of 2,4-dichlorophenol 1b (338 mg, 2.07 mmol) and methyl D-
(+)-lactate (324 mg, 3.11 mmol) as that just described gave (S)-2b (491 mg, 95%). 1H NMR
(270 MHz, CDCl3): δ = 7.30 (d, J = 2.6 Hz, 1H), 7.05 (dd, J = 8.2, 2.6 Hz, 1H), 6.70 (d, J = 8.9
Hz, 1H), 4.65 (q, J = 6.8 Hz, 1H), 3.68 (s, 3H), 1.59 (d, J = 6.6 Hz, 3H) ppm, 13C NMR (67.5
MHz, CDCl3): δ = 171.7, 152.2, 130.2, 127.5, 127.1, 124.8, 116.1, 74.3, 52.4, 18.4 ppm.
Step 2. Synthesis of (S)-2,4-DP [(S)-3b]. The similar treatment of methyl (S)-2-(2,4-
dichlorophenoxy)propanoate (S)-2b (397 mg, 1.60 mmol) as that just described gave (S)-3b
(266 mg, 71%). 1H NMR (270 MHz, CDCl3): δ = 10.63 (s, 1H), 7.39 (d, J = 2.3 Hz, 1H), 7.16
(dd, J = 8.7, 2.5 Hz, 1H), 6.83 (d, J = 8.9 Hz, 1H), 4.77 (q, J = 6.9 Hz, 1H), 1.72 (d, J = 6.9 Hz,
3H) ppm, 13C NMR (67.5 MHz, CDCl3): δ = 176.8, 151.8, 130.4, 127.6, 125.0, 116.5, 74.9,
18.2 ppm.
(R)-2,4-DP. Step 1. Synthesis of methyl (R)-2-(2,4-dichlorophenoxy)propanoate [(R)-
2b]. The similar treatment of 2,4-dichlorophenol 1b (346 mg, 2.12 mmol) and methyl L-
(-)-lactate (331 mg, 3.18 mmol) as that just described gave (R)-2b (524 mg, 99%). 1H NMR
(270 MHz, CDCl3): δ = 7.32 (d, J = 2.6 Hz, 1H), 7.14 (dd, J = 8.7, 2.5 Hz, 1H), 6.78 (d, J = 8.9
Hz, 1H), 4.73 (q, J = 6.8 Hz, 1H), 3.76 (s, 3H), 1.67 (d, J = 6.9 Hz, 3H) ppm, 13C NMR (67.5
MHz, CDCl3): δ = 171.7, 152.2, 130.3, 127.5, 127.1, 124.8, 116.2, 74.4, 52.4, 18.4 ppm.
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
Step 2. Synthesis of (R)-2,4-DP [(R)-3b]. The similar treatment of methyl (R)-2-(2,4-
dichlorophenoxy)propanoate (R)-3b (438 mg, 1.76 mmol) as that just described gave (R)-3b
(232 mg, 62%). 1H NMR (270 MHz, CDCl3): δ = 10.97 (s, 1H), 7.31 (d, J = 2.6 Hz, 1H), 7.08
(dd, J = 8.7, 2.5 Hz, 1H), 6.75 (d, J = 8.9 Hz, 1H), 4.69 (q, J = 6.8 Hz, 1H), 1.64 (d, J = 6.9 Hz,
3H) ppm, 13C NMR (67.5 MHz, CDCl3): δ = 177.1, 151.9, 130.4, 127.6, 127.6, 125.0, 116.4,
73.9, 18.2 ppm.
Results
Measurement of the inhibitory activities of ( )-lactisole and ( )-2,4-DP
against the human sweet taste receptor with point mutants in T1R3-TMD
Here, we performed a series of cellular experiments on cells stably expressing each point
mutant of the human sweet taste receptor to characterize candidate residues in T1R3-TMD
that may be involved in the interaction between the inhibitors and the receptor. After the
introduction of PCR-based mutations into an expression construct suitable for stable expres-
sion of the human sweet taste receptor [9,14,24], we successfully constructed more than 30 cell
lines that stably express different receptors, each with a single point mutation in T1R3-TMD
(S3 and S4 Figs). To confirm the responsiveness of the cell lines expressing each of the mutant
receptors, we first measured the cellular responses to aspartame, an orthosteric agonist that
interacts with T1R2-VFTD. Using individual dose-dependent curves, the EC50 values for each
cell line were calculated, indicating the functionalities of the cell lines used in this study (S1
Table and S3A Fig).
Next, we measured the inhibitory activities of ( )-lactisole [( )-2-(4-methoxyphenoxy)-
propionic acid] and ( )-2,4-DP [( )-2-(2,4-dichlorophenoxy)propionic acid] (Fig 1A) on the
cellular responses of the receptor-expressing cell lines in response to aspartame application (S1
Table and S3B Fig). For this purpose, we measured the cellular responses to the application of
mixtures containing both 1 mM aspartame and eight different concentrations of inhibitors
(Fig 1B, S3B and S5 Figs). The IC50 values of the inhibitors for the wild type (WT) receptor-
expressing cells were found to be 65 μM for ( )-lactisole and 6.2 μM for ( )-2,4-DP, respec-
tively, confirming that the inhibitory activity of ( )-2,4-DP was approximately 10-fold more
potent than that of ( )-lactisole under our experimental conditions (Fig 1B and 1C).
From the results of a series of cellular experiments on all cell lines expressing mutant recep-
tors, the IC50 ratio (i.e., IC50 for the mutant divided by IC50 for the WT) was calculated (S1
Table, also summarized in Fig 1C). Large reductions in the inhibitory activities of both ( )-lac-
tisole and ( )-2,4-DP were confirmed for residues highlighted in orange or magenta, which
were therefore considered to be candidates for involvement in the interaction between the
inhibitors and the receptor (Fig 1C). Despite the difference in inhibitory potency between
( )-lactisole and ( )-2,4-DP, reductions in the inhibitory activities of both were observed for
same seven mutants (Q637E3.33, H641A3.37, A733V5.43, H734N5.44, F778A6.53, Q794N7.32 and
C801Q7.39) compared to the WT (Fig 1C). Moreover, the H641A3.37 and Q794N7.32 mutants
both nearly abolished the inhibitory activity (S5 Fig). These findings suggested that lactisole
and 2,4-DP shared the same binding mode with T1R3-TMD and that H6413.37 and Q7947.32 in
TMD are particularly important residues for interaction with these ligands.
Simulation of binding modes between lactisole/2,4-DP and the homology
model of T1R3-TMD
To explain the result of mutational analyses, we next performed docking simulations and MD-
based energy minimizations with the constructed homology model to investigate in detail the
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
Fig 1. Lactisole and 2,4-DP as sweet taste inhibitors. (A) Structural formulas of ( )-lactisole and ( )-2,4-DP. (B) Dose-response curve showing
the inhibition of the cellular responses of cells stably expressing the WT sweet taste receptor by ( )-lactisole and ( )-2,4-DP. Each inhibitor was
added to 1 mM aspartame. The results for ( )-lactisole are shown in blue, and those for ( )-2,4-DP are shown in green. Data are shown as the
mean SEM (n = 4). (C) Summary of mutational analysis for inhibitors (S.E.: standard error of each IC50). The results are summarized in S1 Table.
Each ratio was calculated by dividing the value for the mutant by that for the WT. Mutants with IC50 ratios < 1/2 are colored in blue, � 10 in
orange, and � 50 or N.D. in magenta, respectively. N.D., not determined since inhibitory activity was almost entirely eliminated.
https://doi.org/10.1371/journal.pone.0213552.g001
differences in the binding configuration between lactisole and 2,4-DP with T1R3-TMD. Prior
to MD, a structural homology model of T1R3-TMD was created based on the crystal structure
of mGluR1 (PDB ID: 4OR2). A whole view of the homology model and a view around the resi-
dues extracted by mutational analyses are shown in Fig 2A. The seven residues are shown by
colored stick illustrations. Q6373.33, A7335.43, H7345.44, F7786.53 and C8017.39 are colored
orange, and H6413.37 and Q7947.32 are colored magenta. All seven residues are positioned near
each other in this structural model and seem to constitute a hydrophobic pocket in the upper
region of the transmembrane domain. Moreover, the side chains of two residues, H6413.37 and
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
Fig 2. MD Simulations and their pre-simulations for lactisole and 2,4-DP. (A) T1R3-TMD homology model created based
on mGluR1 TMD (PDB ID: 4OR2). An enlarged view of the candidate ligand pocket is shown in the right window. Residues
extracted by cellular analyses are indicated by colored stick illustrations. (B) Docking modes of (S)-lactisole (shown in blue)
and (R)-lactisole (shown in pink) are shown in the left figure. (S)-2,4-DP (green) and (R)-2,4-DP (gray) are shown on the
right. (C) MD minimizations (100 ns) were performed for each ligand from the docking models. The two left panels show the
results for isomers of lactisole (upper panel: (S)-lactisole and lower panel: (R)-lactisole). The two right panels show the results
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
for isomers of 2,4-DP (upper panel: (S)-2,4-DP and lower panel: (R)-2,4-DP). The estimated interactions between residues
and the ligands are shown by dotted lines in the illustrations after MD: hydrogen bonds are shown in yellow, ionic bonds in
magenta, and hydrophobic π-π interactions in cyan.
https://doi.org/10.1371/journal.pone.0213552.g002
Q7947.32, which were indicated to be the most important for the interaction between the inhib-
itors and the receptor, face each other in the model, suggesting that the common chemical
structure of the inhibitors would be positioned between the side chains of H6413.37 and
Q7947.32 and that this interaction is critical for their inhibitory activities.
Next, docking simulations of lactisole and 2,4-DP were carried out in the space of a ligand
pocket constructed of the seven residues, showing reasonable positions for these ligands. Since
both lactisole and 2,4-DP are chiral and since the cellular analysis in this study was carried out
with commercially available racemates, both the (S)- and (R)-isomers of both ligands were
simulated (Fig 2B). To verify the orientation of the ligands, we also utilized the information for
another ligand, ( )-2-phenoxypropionic acid (( )-2-PP). 2-PP is a structural analog of lactisole
with no methoxy group on its benzene moiety (S1 Table). When the inhibitory activities of
( )-lactisole and ( )-2-PP against each mutant were compared, the results for the C801Q7.39
mutant showed a clear difference. Lactisole had less inhibitory activity toward the C801Q7.39
mutant, whereas ( )-2-PP had almost the same activity as toward the WT (S1 Table). Since
Cys8017.39 oriented to the p-methoxy group direction of (S)-lactisole, the docking results the
ligands would be considered to be docked correctly (Fig 2B).
However, because the docking position of each ligand was almost the same, we couldn’t
ascertain which parts of particular isomers are important for their inhibitory activities. To
identify this, we applied MD simulations with the docking models for 100 ns, and allowed the
ligands to converge to the positions where they are energetically stable (Fig 2C). As a result,
(S)-lactisole had three interactions which comprised ionic bonds with H6413.37, F7786.53 and
Q7947.32, whereas (R)-lactisole had an ionic interaction with H6413.37 and a hydrogen bond
with Q7947.32 in a different manner than (S)-lactisole. Similarly (S)-2,4-DP had four interac-
tions with H6413.37, H7345.44, F7786.53 and Q7947.32, while (R)-2,4-DP only had one ionic
interaction with H6413.37. In summary, only the (S)-isomers of both lactisole and 2,4-DP
could maintain the salt bridges between the carboxyl group and H6413.37, and also maintain a
H-bond between the carboxyl group and Q7947.32; in contrast, the (R)-isomers could only
maintain the interaction between the carboxyl group and H6413.37 (Fig 2C). Based on these
results, we concluded that the (S)-isomers of lactisole and 2,4-DP are predominantly responsi-
ble for their inhibitory activities. Further information from 80 ns to 100 ns of MD simulations
are shown in S6 Fig. The data show the frequency of each residue’s contact with the ligands
during these 20 ns. Because each (R)-isomer had more interaction via water compared to (S)-
isomers, (S)-isomers were thought to be more stable during simulations. In particular, 2,4-DP
had four specific interactions as shown Fig 2C.
The results of docking simulations and MD minimizations for (S)-lactisole and (S)-2,4-DP
are superimposed in Fig 3A. Although the docking poses of these compounds were almost
identical, the positions of the two ligands were slightly different after 100 ns of simulation (Fig
3A right and S7 Fig). (S)-lactisole was closer than (S)-2,4-DP to TM7, and correspondingly,
(S)-2,4-DP was located closer than (S)-lactisole to TM3. Moreover, focusing on the ortho-
position of the aromatic ring of each ligand (Fig 3B), (S)-2,4-DP has an o-Cl group that
engaged in van der Waals interactions with S6403.36, L6443.40, and L7987.36 (Fig 3B, right). In
contrast, lactisole has no such functional group and was positioned farther from these residues
than 2,4-DP, with interactions that appeared to be weaker (Fig 3B, left). To summarize, the
mutational analyses and docking simulations revealed that the same residues were responsible
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
Fig 3. Comparison of the results of MD minimizations for (S)-lactisole and (S)-2,4-DP. (A) Results of the MD minimization of (S)-lactisole
(shown in blue) and (S)-2,4-DP (shown in green). In the superimposed view after MD minimization, the (S)-lactisole model is shown by thin lines,
and the (S)-2,4-DP model is shown by thick lines. (B) Van der Waals interaction areas between T1R3-TMD and (S)-lactisole or (S)-2,4-DP are
shown.
https://doi.org/10.1371/journal.pone.0213552.g003
for interaction with lactisole and 2,4-DP; however, the MD minimization revealed a slight dif-
ference in position involving the o-Cl group of (S)-2,4-DP.
To confirm whether (S)-lactisole or (S)-2,4-DP shifted farther from the appropriate position
to act as an inhibitor, we superimposed the (S)-2,4-DP minimized model with the crystal struc-
tures of mGluR-TMDs (mGluR1 4OR2, mGluR5 4OO9, 5CGC, 5CGD, 6FFI, and 6FFH)
[22,23,27,28]. This analysis showed that the position of (S)-2,4-DP was similar to the ligands in
other receptors (S8 Fig). This result suggested that (S)-2,4-DP, but not (S)-lactisole, stayed in
the correct position to act as an inhibitor and could therefore act as a more potent inhibitor.
Comparison of the inhibitory activities of the (S)-isomer and (R)-isomer
MD minimization suggested that the (S)-isomers of both ligands ((S)-lactisole and (S)-2,4-DP)
interacted with residues of T1R3-TMD. To verify whether this assumption was correct, we
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
Fig 4. Sweet taste inhibitory activity of (S)-, (R)-lactisole and (S)-, (R)-2,4-DP. (A) Flow chart of the synthesis of each isomer of lactisole and
2,4-DP. (B) Cellular response to 1 mM aspartame plus the indicated concentration of each compound against the cells expressing the WT sweet
taste receptor. The left panel shows the results for the lactisole isomers: (S)-lactisole (blue) and (R)-lactisole (pink). The right panel shows the results
for the 2,4-DP isomers: (S)-2,4-DP (green), and (R)-2,4-DP (gray). Data are shown as the mean SEM (n = 4).
https://doi.org/10.1371/journal.pone.0213552.g004
synthesized four ligands, (S)-lactisole, (R)-lactisole, (S)-2,4-DP, and (R)-2,4-DP, according to
the flow chart shown in Fig 4A. The (S)-isomers of both lactisole and 2,4-DP showed potent
inhibitory activities with the following IC50 values: 20 μM for (S)-lactisole, 2.6 μM for (S)-
2,4-DP, and 45 μM for (R)-2,4-DP (Fig 4B). (R)-2,4-DP was � 10-fold less effective than
(S)-2,4-DP, and (R)-lactisole exerted almost no inhibition at the measured concentration (Fig
4B). This result strongly supports the idea that the (S)-isomers interact more strongly with
T1R3-TMD, confirming the accuracy of the MD-minimized models constructed in this study.
Discussion
In this study, we assessed the differences between the interaction modes of lactisole and
2,4-DP using mutational analyses and MD-based energy minimization. Consequently, it was
necessary to evaluate the homology model and the validity of the simulation based on compar-
ison between the results of the simulation and experimentally obtained data.
Homology model
The homology model was created based on the structure of mGluR1-TMD (PDB ID: 4OR2)
according to the alignment shown in S1 Fig. Although the alignment was calculated by Prime
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
independently, it was matched with generic GPCR numbers and the same numbered residues
of mGluR1 were replaced with residues of T1R3. Because the sequence identity of the TMD
between T1R3 and mGluR1 was much higher than that between T1R3 and rhodopsin, this
model was thought to be more accurate than that based on rhodopsin. The comparison of
´
these models was also performed by Cheron et al. [29].
Seeing the model, all seven residues identified in our mutational analysis (Fig 1C) were
located near one another (Fig 2A). Furthermore, because H6413.37 and Q7947.32, the two most
important residues, were facing each other, these two residues are strongly suggested to inter-
act with inhibitors (Fig 2A). Four residues out of the seven, Q6373.33, H6413.37, A7335.43 and
F7786.53, were previously reported as residues that interacted with lactisole [11], whereas
H7345.44, Q7947.32 and C8017.39 were newly discovered in this study. A previous study reported
that R72345.51 interacts with the carboxyl group of lactisole [11], whereas in our hands, the
IC50 of R723A45.51 did not differ significantly relative to the WT receptor (S1 Table). This dif-
ference could be explained in at least two ways. First, in the previous study, each mutant was
measured at only three concentrations, preventing the influence of lactisole in R723A45.51
from being accurately evaluated. In contrast, in this study, the response of each mutant was
measured at eight inhibitor concentrations, and the IC50 values obtained should be more accu-
rate. The second possibility involves Q7947.32, another important residue. In a previous study,
the Q794A7.32 mutant was reported to be nonfunctional [11]. Based on this observation, we
created the new Q794N7.32 mutant, revealing that Q7947.32 was an important residue for inter-
action with lactisole and 2,4-DP (S5 Fig).
Ligand docking
H6413.37 and Q7947.32, which face each other, were considered to be the most important resi-
dues for the binding of both lactisole and 2,4-DP (Fig 3A). In both docking models, these resi-
dues formed salt bridges via the carboxyl groups of the ligands, and the docking poses are very
reasonable. In addition, regarding the C801Q7.39 mutation, C8017.39 lies in the deep part of the
ligand pocket (Fig 2A). In this study, we replaced this residue with a bulkier residue, Gln, to fill
the space where the ligand should interact. Indeed, the inhibitory activity of both ( )-lactisole
and ( )-2,4-DP, was greatly reduced by this mutation (Fig 1C). On the other hand, the IC50
of ( )-2-PP, in which the p-position methoxy group was removed from lactisole, was not cru-
cially affected by the C801Q7.39 mutation (S1 Table and S5 Fig). Based on these findings, we
expected the p-position groups of lactisole and 2,4-DP to be oriented toward the deep part of
the ligand pocket (Fig 2B).
MD-based energy minimization
The simulation results differed greatly between the (S)- and (R)-isomers for both lactisole and
2,4-DP, and both ligands exhibited stronger receptor-ligand interactions in the (S)-isomer-
minimized models (Fig 2C). To confirm this result, we synthesized all four isomers and com-
pared their inhibitory activities (Fig 4). Given that the inhibitory activities of each isomer were
consistent with the simulation results, we concluded that the results strongly supported the
accuracy of the MD-minimized models in this study.
Furthermore, we compared some ligands in crystal structures of class C GPCRs (PDB ID:
4OR2 (mGluR1), and 4OO9, 5CGC, 5CGD, 6FFH and 6FFI (mGluR5)) with the (S)-2,4-DP-
minimized model (S8 Fig). The residues involved in receptor-ligand interactions, such as
those at positions 5.44, 6.53, and 7.39, were present at the same position. This would indicate
that MD minimization was performed appropriately. In addition, these residues are very
important for ligands to act as NAMs in class C GPCRs [22,23,27,28]. The interaction with
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
T8157.32 was also important for the ligand FITM to function as an NAM with mGluR1 [23].
Similarly, the 7.32 position in T1R3, Q7947.32, is important for the function of 2,4-DP as an
NAM with T1R3 (Fig 1C). Based on these observations, we conclude that the results of our
simulation would be reasonable.
Next, we examined the commonalities and differences between the inhibitory mode of
action of lactisole and 2,4-DP. Both ligands have lactic acid skeletons (Fig 1A), and their (S)-
isomers interact more strongly with T1R3-TMD (Fig 2B and 2C). Moreover, both ligands
interact with the same residues (Fig 3A). This result might be related to the fact that lactisole is
found in coffee beans primarily as the (S)-isomer [20].
In contrast, one difference between the two compounds involved the behaviors of the (R)-
isomers. (R)-2,4-DP had some inhibitory activity, whereas (R)-lactisole had barely any (Fig
4B). Thus, the inhibitory activity of lactisole was dependent mainly on the carboxyl group,
whereas 2,4-DP interacted with T1R3-TMD via two moieties, the carboxyl group and the aro-
matic ring with two Cl groups. The MD-minimized results of (S)-lactisole and (S)-2,4-DP (Fig
3A) show clearly that (S)-lactisole was closer to TM7 than 2,4-DP. Thus, while (S)-lactisole was
stabilized in the vertical dimension of the ligand pocket via its carboxyl group, there was not
sufficient interaction in the horizontal dimension. In contrast, in (S)-2,4-DP, the o-position Cl
group engaged in hydrophobic interactions with S6403.36, L6443.40, and L7987.36 in the hori-
zontal dimension (Fig 3B). In addition, in the mutational analysis, only ( )-2,4-DP had a
lower IC50 for the L798I7.36 mutant (Fig 1C). This result provides further evidence that the o-
position Cl group is involved in the interaction with L7987.36
.
Based on the above, we concluded that the difference in potency between (S)-lactisole and
(S)-2,4-DP was governed by their stability in the ligand pocket of T1R3-TMD: both ligands
were stabilized in the vertical dimension by the common 2-phenoxypropionic acid skeleton,
but only (S)-2,4-DP was stabilized in the horizontal dimension via its o-Cl group (Fig 3A and
3B).
In the experiments described above, we succeeded in clarifying the difference between lacti-
sole and 2,4-DP using interaction models (Fig 3). However, since these models were only sim-
ulated using low-molecular weight NAMs, it is difficult to explain the interaction mechanism
of larger NAMs such as gymnemic acid [13]. Indeed, three residues in T1R3-TMD were
reported to be quite important for gymnemic acids: H6413.37, A7335.43, and R725ECL2 in extra-
cellular loop 2 (ECL2) [13]. Thus, although H6413.37and A7335.43 were commonly important
for binding of both lactisole and 2,4-DP, gymnemic acid has another interaction mode extend-
ing from the ligand pocket to the vicinity of ECL2.
At any rate, it may be possible to propose a design for further high-affinity ligands using the
minimized model created in this study. In concrete terms, comparison of the ligand positions
in the mGluR crystal structures with those of (S)-2,4-DP in S8 Fig revealed that the other
mGluR ligands were extended in the direction of the two Cl groups in 2,4-DP. On the other
hand, in the L798I7.36 mutant, 2,4-DP was a more potent inhibitor (Fig 1C), and there was
space for (S)-lactisole to slide away (Fig 3B). These data showed that a more potent sweet taste
inhibitor could be created by designing a derivative with larger hydrophobic functional groups
in both the o- and p-directions of (S)-2,4-DP.
Supporting information
S1 Table. Summary of the results of cellular experimental analyses for each cell line that
stably expressed the wild-type (WT) or a point mutant of the human sweet taste receptor.
The EC50 values for aspartame and the IC50 values for ( )-lactisole, ( )-2,4-DP, and ( )-2-PP
(2-phenoxypropionic acid) are indicated (S.E.: standard error of each EC50 or IC50). Each ratio
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
was calculated by dividing the value for the mutant by that for the WT. Mutants with
ratios < 1/2 are colored in blue, � 5 in gray, � 10 in orange, and � 50 or N.D. in magenta,
respectively. N.D., not determined since the inhibitory activity was almost completely elimi-
nated. References upon which we created mutations are indicated by superscripts. Some of
them were modified to other mutations because they had been reported as inactive or hyperac-
tive mutations.
(TIF)
S1 Fig. The Alignment of mGluR1, 5 and T1R1, 2 and 3. (A)The alignment of the TMD
regions of five receptors: mGluR1, mGluR5, T1R1, T1R2 and T1R3. Each area surrounded by
a green line indicates transmembrane (TM) regions. (B) Sequence identities of each receptor
are shown in the upper right of the table, while sequence similarities of each receptor are
shown in the lower left of the table. It should be noted that rhodopsin and β2-adrenoceptor
(β2-AR) are categorized as class A GPCRs.
(TIF)
S2 Fig. Time course plots of protein-RMSD and ligand-RMSD. (A) Each RMSD of four MD
simulations is shown. Protein RMSD is shown in blue, and ligand RMSD is shown in red.
Upper left: is the one of (S)-lactisole. Lower left: (R)-lactisole. Upper right: (S)-2,4-DP. Lower
right: (R)-2,4-DP. (B) The time course during 80 ns to 100 ns.
(TIF)
S3 Fig. Plots of cellular experimental analyses for each cell line that stably expressed the
wild-type (WT) or a point mutant of the human sweet taste receptor. (A) Dose response
curves against aspartame of the all utilized mutants. Data are shown as the mean SEM
(n = 3–4). (B) Dose response curves against 1 mM aspartame and each concentration of lacti-
sole. Data are expressed as the mean SEM (n = 4).
(TIF)
S4 Fig. Snake plot of T1R3-TMD. Residues mutated in this study are highlighted. Residues
thought to interact with both lactisole and 2,4-DP, based on the cellular analyses in this study,
are colored in orange and magenta.
(TIF)
S5 Fig. Sweet taste inhibitory activity of three ligands in each mutant in which the inhibi-
tory activities were largely reduced. Dose-response curves of WT and seven mutant receptors
for three ligands are shown: ( )-lactisole (blue), ( )-2,4-DP (green), and ( )-2-PP (red). Data
are shown as the mean SEM (n = 4).
(TIF)
S6 Fig. The residue-ligand contact frequencies during MD-simulations. The contact fre-
quency during 80 ns– 100 ns is shown. Hydrophilic interactions are shown in violet and
hydrophobic interactions are shown in green. The percentages shown on line denote contact
frequency: contacts with >50% frequency are shown in Fig 2C. The thick lines surrounding
each ligand indicate the type of adjacent molecule: Blue indicates hydrophilic molecules
including water and green indicates hydrophobic molecules.
(TIF)
S7 Fig. Comparison of pre- and post-MD simulations. Superimposed view of docking-
model and MD-simulated model. The docking model is shown in thin lines and the MD-simu-
lated model is shown in thick lines. (A): (S)-lactisole model. (B): (S)-2,4-DP model.
(TIF)
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Differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor
S8 Fig. Superimposed view of (S)-2,4-DP model and other class C GPCR structures. Com-
parison of the (S)-2,4-DP minimized model and crystal structure of other class C GPCRs. MD-
minimized models for (S)-2,4-DP (green) and six other ligands (FITM from 4OR2, shown in
light blue; mavoglurant from 4OO9, shown in orange; Compound14 from 5CGC, shown in
violet; HTL14242 (Compound25) from 5CGD, shown in magenta; MMPEP from 6FFI, shown
in dark gray, and fenobam from 6FFH, shown in light gray) are superimposed.
(TIF)
S1 Appendix. The models created in this study. Four models are available by downloading
S1 Appendix.zip. This file contains (S)-lactisole_dock.pdb, (S)-lactisole_MD.pdb, (S)-2,4-
DP_dock.pdb, and (S)-2,4-DP_MD.pdb.
(ZIP)
Author Contributions
Conceptualization: Tomoya Nakagita, Takumi Misaka.
Data curation: Tomoya Nakagita, Akiko Ishida, Takumi Matsuya, Takatsugu Hirokawa.
Formal analysis: Tomoya Nakagita.
Funding acquisition: Takuya Kobayashi, Takatsugu Hirokawa.
Methodology: Takumi Misaka.
Project administration: Takumi Misaka.
Resources: Makoto Hashimoto.
Software: Takatsugu Hirokawa.
Supervision: Takumi Misaka.
Validation: Tomoya Nakagita, Takatsugu Hirokawa.
Visualization: Takuya Kobayashi, Makoto Hashimoto.
Writing – original draft: Tomoya Nakagita.
Writing – review & editing: Masataka Narukawa, Takumi Misaka.
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