Discovery of the First Environment-Sensitive Fluorescent Probe for GPR120 (FFA4) Imaging

Article abstract of DOI:10.1021/acsmedchemlett.7b00023

GPR120, which is activated by long-chain free fatty acids (FFAs), has been recognized as a new attractive target for the treatment of type 2 diabetes and metabolic disease. The visualization and location of GPR120 in native cells can provide powerful information for guiding the physiological and pathological studies of GPR120. We report herein the first potent fluorescent probes that sensitively detect GPR120. We designed and synthesized a series of novel environment-sensitive probes with suitable fluorescence property, high biological activity on the GPR120, and acceptable cytotoxicity. These fluorescent probes targeting GPR120 are expected to expand the toolkit for further studies on GPR120.

Full text of DOI:10.1021/acsmedchemlett.7b00023

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Letter
Discovery of the First Environment-Sensitive
Fluorescent Probe for GPR120 (FFA4) Imaging
Jiaxiang Liu, Chengsen Tian, Tianyu Jiang, Yuqi Gao, Yubin Zhou, Minyong Li, and Lupei Du
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00023 • Publication Date (Web): 27 Mar 2017
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Discovery of the First Environment-Sensitive Fluorescent Probe for
GPR120 (FFA4) Imaging
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Jiaxiang Liu,aChengsen Tian,a,b Tianyu Jiang,aYuqi Gao,aYubin Zhou,c Minyong Li,a and Lupei Dua,*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong Univerꢀ
sity, Jinan, Shandong 250012, China. Tel./fax: +86ꢀ531ꢀ8838ꢀ2076; Eꢀmail: dulupei@sdu.edu.cn
bSchool of Chemical Engineering, Qilu Normal University, Jinan, Shandong 250200, China
cCenter for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University Health Science
Center, Houston, TX 77030, USA
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KEYWORDS. GPR120, fluorescent probes, cell imaging, subcellular localization.
ABSTRACT:GPR120, which is activated by longꢀchain free acids (FFAs), has been recognized as a new attractive target for the
treatment of type 2 diabetes and metabolic disease. The visualization and location of GPR120 in native cells can provide powerful
information for guiding the physiological and pathological studies of GPR120. We report herein the first potent fluorescent probes
that sensitively detect GPR120. We designed and synthesized a series of novel environmentꢀsensitive probes with suitable fluoresꢀ
cence property, high biological activity on the GPR120, and acceptable cytotoxicity. These fluorescent probes targeting GPR120
are expected to expand the toolkit for further studies on GPR120.
G proteinꢀcoupled receptor 120 (GPR120), as the prominent
members of G proteinꢀcoupled receptors (GPCRs), is abunꢀ
dantly expressed in various types of organs, tissues, and cells,
which mediate GLPꢀ1 secretion, insulin sensitization, antiꢀ
bright fluorescence in low polarity and high viscosity solvents
or when bound to hydrophobic domains in cells (Scheme 2).13ꢀ
15 Regarding GPR120, there has been thus far no report on the
detection of GPR120 on the cell surface with small molecule
fluorescent probes. Given that small molecule fluorescent
probes exhibit high sensitivity, selectivity, visualization, and
fast response, there is an urgent need for developing a convenꢀ
ient fluorescent ligand toolbox to trace GPR120 for underꢀ
standing the physiological and pathological studies.
1 2
,
inflammatory, and antiꢀobesity effects. It should be noted
that GLPꢀ1, a potent incretin hormone, has been reported to
play a pivotal role in insulin sensitivity, appetite, and gastric
emptying.3 Thus, GPR120 activated by longꢀchain free fatty
acids (FFAs) has been recognized as a new potential target for
treatment of type 2 diabetes and obesity.4 5 So far, a series of
,
potent agonists have been discovered for the treatment and
diagnosis of GPR120ꢀrelated diseases, such as GW9508,
TUGꢀ891, and NCG21 (Scheme 1).6 GW9508, a smallꢀ
molecule agonist of the fatty acid receptors GPR40, also posꢀ
sesses moderate GPR120 agonist activity.7 TUGꢀ891 was reꢀ
cently discovered as a potent and selective agonist for
GPR120.8 NCG21, derived from PPARγ ligands, turned out to
be a potent and selective GPR120 agonist.9 We have previously
reported a series of excellent agonists for GPR120 based on
pharmacophore modeling and virtual screening.10 However,
we still face many challenges on investigating the physiologiꢀ
cal and pharmacological functions of GPR120. The similarity
between GW9508 and TUGꢀ891 sparkled our interest in exꢀ
ploring fluorescent probes to detect GPR120. Here we report
the first potent environmentꢀsensitive fluorescent probe on
GPR120.
Scheme 1. Current GPR120 agonists.
In general, a smallꢀmolecule fluorescent probe for a specific
biotarget contains two parts: a fluorophore group that can tag
the target with fluorescent properties, as well as a pharmacoꢀ
phore moiety that can recognize the biotarget through the reꢀ
ceptorꢀligand interaction.16 The essential components of TUGꢀ
891 and GW9508 were chosen as the pharmacophore moiety
for its high potency to GPR120. In addition, naphthalimide
and coumarin groups are selected as fluorophore group due to
their favorable fluorescent properties, which were linked to the
pharmacophore moiety by an aliphatic spacer. Subsequently, a
series of fluorescent probes were well designed and syntheꢀ
sized as small molecule fluorescent probes for GPR120
(Scheme 2).
During the past decade, fluorescent GPCR ligands have
been widely applied to localize the distribution of receptors
and enable realꢀtime monitoring of processes triggered by
ligandꢀreceptor interactions, such as internalization, traffickꢀ
ing, sequestration, and recycling.11 12 Environmentꢀsensitive
,
fluorescent ligands are very sensitive to the change in the
physicochemical properties of the surrounding environment,
which exhibit weak fluorescence in aqueous solution but emit
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Scheme 2.The strategy of smallꢀmolecule fluorescent probes for GPR120.
Scheme 3. The synthetic schemes of fluorescent probes.
The two synthetic routes of fluorescent probes were shown
in Scheme 3. Briefly, coupling the pharmacophore moiety with
fluorophore group under HATU/DIPEA condition yielded our
key ester intermediates. All final probes were obtained via
ester hydrolysis. Further synthesis details can be found in the
Supporting Information.
spectroscopy was evident (Table1). The results showed that
their fluorescence emissions were quenched in methanol and
PBS buffer, and the quantum yields in PBS are low. In particuꢀ
lar, when the polarity of a solvent decreases, fluorescence inꢀ
tensity increases significantly. It indicated that the bright fluoꢀ
rescence is released when these probes bind to GPR120 active
site as the hydrophobic domain with low polarity. Herein, we
discover these compounds could serve as environmentꢀ
sensitive fluorescent probes for GPR120.
Table 1. Photophysical parameters of synthesized probes.
Фa/%
EA
Comp
d
λmax/nm
λex/nm
λem/nm
PBS
DCM
32.4
147.2 39.0
83.9 69.2
Table 2. Cytotoxicity results of synthesized probes.
L1
L2
L3
L4
L5
L6
L7
430
435
430
424
429
434
430
415
435
415
415
415
435
420
535
545
535
490
470
475
475
4.0
1.1
2.6
10.8
IC50 (µM)
Compd
HEK293
>100
>100
HTꢀ29
>100
>100
>100
92±2
28±5
>100
STCꢀ1
>100
L1
L2
L3
L4
L5
L6
L7
20.1 159.0 244.7
11.6 174.5 275.0
93±3
50±0.7
55±0.7
75±1
>100
>100
6.7
8.6
162.5 252.6
192.6 316.1
49±3
41±4
>100
a Fluorescence quantum yields of these probes were calculated in
reference to fluorescein in 0.1 M NaOH as a standard (Φ = 0.92).
>100
>100
>100
The absorption and fluorescence spectroscopy of these
probes were measured in methanol, ethyl acetate, dichloroꢀ
methane and PBS buffer with Thermo Varioskan microplate
reader (Figures S1ꢀ3). In addition, the fluorescence quantum
yields of these probes were measured in a different solvent of
ethyl acetate, dichloromethane, and PBS buffer. The influence
of solvents on fluorescence quantum yield and fluorescence
It is important that the reasonable probes should possess low
cytotoxicity while labeling the GPR120. Additionally, the cyꢀ
totoxicity of these probes was evaluated by an MTT cytotoxiꢀ
city assay with STCꢀ1, HTꢀ29 and HEKꢀ293 cells. As shown
in Table 2, all compounds had acceptable cytotoxicity that
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were biocompatiable to cells. Overall, our results demonstrated GPR120 was reported to be localized on the cell surface using
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that these fluorescent probes have acceptable cell toxicity for
use in labeling and imaging of GPR120 in living cells at the
nanomolar concentration.
HEK293 cells stably (and also transiently) expressing the
GPR120ꢀGFP fusion protein.5 In order to extend the applicaꢀ
tion of fluorescent probes, HEK293 cells were firstly chosen
for fluorescence imaging. The imaging results showed that its
fluorescence mainly distributed on the cell surface (Figure1A).
Furthermore, it is a known fact that GPR120 are highly exꢀ
These fluorescent probes were evaluated on GPR120 transꢀ
fected HEK293 cells by βꢀarrestin 2 interaction bioluminesꢀ
cence resonance energy transfer (BRET) assay.17 In this assay,
TUGꢀ891 was chosen as the positive control. The results reꢀ
vealed that these probes have a high activity for the GPR120
and are close to the positive control (Table3). When the fluorꢀ
ophore group is changed from naphthalimide to coumarin, the
activity to GPR120 has no obvious effect. In all fluorescent
probes, probe L3 with a linker of six carbon atoms showed the
best activity, while the other probes, which have the spacer
shorter than six carbon atoms, displayed lower and similar
activity to GPR120. It indicated that the probes with a longer
carbon chain as the linker would possess better activity.
pressed in HTꢀ29 and STCꢀ1 cells.18 19 After successful stainꢀ
,
ing of GPR120 with fluorescent probes in HEK293 cells, we
tested these probes on HTꢀ29 and STCꢀ1 cells that endogeꢀ
nously express GPR120. In the case of HTꢀ29 cells, we found
the strong fluorescence aggregation mainly distributed on the
cell surface, as well as slight decoration on plasma membrane
(Figure1B). These probes exhibited the distribution of fluoresꢀ
cence, in agreement with those reported previously using a
GPR120ꢀspecific antibody.20 An observation that is further
supported by fluorescent probes label GPR120 in STCꢀ1 cells
that endogenously express GPR120. As shown in Figure 1C,
its fluorescence was not merely partitioning in the cell memꢀ
brane and are highly consistent with Thompson’s conclusion
using 14Cꢀlabelled dodecanoic acid (one type of FFA) to probe
the site of fatty acid action.21 Accordingly, we choose PCꢀ3
cells as a negative control which expresses lower levels of
GPR120 mRNA.22 The results showed that feeble fluorescence
was detected in negative PCꢀ3 cells (Figure1D). These findꢀ
ings confirm that these probes can label the target for visualiꢀ
zation in GPR120 overexpressing cells. The other six probes
present similar results in fluorescence imaging (see Supporting
Information).
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Table 3. Bioactivity of synthesized probes onGPR120.
b
Compd
TUG-891
L1
GPR120 (BRET)a pEC50 (% Emax
7.17±0.02 (100)
5.89±0.03 (110)
5.90±0.02 (98)
)
L2
L3
L4
L5
L6
6.62±0.05 (108)
5.65±0.01 (111)
5.88±0.02 (115)
6.01±0.06 (106)
5.84±0.04 (135)
L7
a GPR120 (BRET) pEC50 determined using βꢀarrestin 2 interaction
b
bioluminescence resonance energy transfer (BRET) assay. Emax
expressed as % of TUGꢀ891.
The fatty acid receptors GPR120 (FFA4) and GPR40
(FFA1), which bind the same broad group of fatty acids, have
very similar pharmacological properties. The GPR120 agonists
described hitherto have poor or no selectivity overGPR40 reꢀ
ceptor. Therefore, further investigation has been done to detect
whether the potent compound L3 has the selectivity. As a reꢀ
sult, compound L3 has a weak activity for the GPR40 in this
calcium assay and the [Ca2+]i response is low (Figure S4). We
next examined the medium chain length fatty acid receptor
GPR84 and the short chain length fatty acid receptor GPR43,
which would be standard for an new chemical series targrting a
member of this receptor family. In brief, the result revealed
that the compound L3 also has a weak [Ca2+]i response for the
GPR84 and the GPR43. Moreover, the pEC50 value of probe
L3 for GPR120 was 6.72±0.18, which is close to that of TUGꢀ
891 (pEC50=7.26±0.04) in this calcium assay (Figure S4). Theꢀ
se probes displayed the similar molecular mechanism to the
TUGꢀ891 at GPR120 across various assay endpoints, including
βꢀarrestin 2 recruitment and stimulation of Ca2+ mobilization.
In conclusion, compound L3 is a potent and selectiveGPR120
agonist.
Figure 1. Fluorescence microscopic imaging of mammalian cells
incubated with probe L3. All cells are incubated with L3 at 37 °C
for 5 min and washed immediately, and the exposure time reꢀ
mained the same among all groups. The background was adjusted
by Image J software. Performed in Zeiss Axio Observer A1, GFP
channel, objective lens, 63×.(A) Representative images of
HEK293 cells incubated with L3 (20 nM); (B) Image of HTꢀ29
cells incubated with L3 (20 nM); (C) Image of STCꢀ1 cells incuꢀ
bated with L3 (20 nM); (D) Image of PCꢀ3 cells incubated with
L3 (20 nM).
As a result, these cells endogenously expressing GPR120
could be labeled by fluorescent probes at the nanomolar conꢀ
centration(Figure 1), which would be a new milestone for loꢀ
cating the position of GPR120 with fluorescent probes. Subseꢀ
quently, we want to define whether the observed fluorescence
actually represents specific binding. For this to be the case it
should be outcompeted by ligands from distinct chemical seꢀ
ries that also have affinity for GPR120. The HEK293 cell staꢀ
bly expressing the GPR120 protein was used and the selective
diarylsulfonamide GPR120 agonist GSK137647A was chose
as a negative control, which might make a good choice for a
structurally orthogonal GPR120 active agonist to figure out
this issue. These results exhibited that the fluorescence intensiꢀ
Overall, appropriate fluorescent properties, high bioactivity
to GPR120 and acceptable cell toxicity were observed, and
thus, laid a solid foundation for the following fluorescence
imaging in living cells. Thus, cell imaging potential was furꢀ
ther evaluated. So far, few reports are available on GPR120
subcellular localization in native cells. Our preliminary study
with fluorescent probes indicated the varying distribution of
GPR120 in different cells. In subcellular localization, the
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The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
ty was significantly decreased by incubating the cells with
GSK137647A (30 ꢁM) together with probe L3 and indicated
that the labeling with probe L3 can be outcompeted by a non
fluorescent GPR120 ligand (Figure 2). The implication of a
development of a labeled GPR120 agonist indicated that these
probes can be used as labeling tools for binding to this receptor
and be expected to guide the physiological and pathological
studies of GPR120. Moreover, we expect that these probes
could be employed as fluorescent competitive substrates in the
GPR120 ligand activity screening.
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ACKNOWLEDGMENT
The present work was supported by the Major Project of Science
and Technology of Shandong Province (No. 2015ZDJS04001).
Our cell imaging work was performed at the Microscopy Characꢀ
terization Facility, Shandong University
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ABBREVIATIONS
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GPR120, G proteinꢀcoupled receptor 120; FFA4, free fatty acid
receptor 4 (GPR120); FFA, free fatty acid; GPCRs, G protein
coupled receptors; GLPꢀ1,glucagonꢀlikepeptideꢀ1; BRET, bioluꢀ
minescence resonance energy transfer.
Figure 2. Fluorescence microscopic imaging of mammalian cells
incubated with probe L3. All cells are incubated with L3 at 37 °C
for 15 min and washed immediately, and the exposure time reꢀ
mained the same among all groups. The background was adjusted
by Image J software. Performed in Zeiss Axio Observer A1, GFP
channel, objective lens, 63×. (A) Representative images of
HEK293 cells stably expressing the GPR120 protein incubated
with L3 (200 nM); (B) Representative images of HEK293 cells
stably expressing the GPR120 protein incubated with
GSK137647A (30 ꢁM) and probe L3 (200 nM).
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Corresponding Author
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Suzuki, T.; Igari, S.; Hirasawa, A.; Hata, M.; Ishiguro,
M.; Fujieda, H.; Itoh, Y.; Hirano, T.; Nakagawa, H.; Ogura, M.;
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proteinꢀcoupled receptor 120ꢀselective agonists derived from
PPARgamma agonists. J. Med. Chem. 2008,51 (23), 7640ꢀ4.
*Lupei
Du,
Tel./fax:
+86ꢀ531ꢀ8838ꢀ2006;
Eꢀmail:
dulupei@sdu.edu.cn.
Author Contributions
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Li, A.; Yang, D.; Zhu, M.; Tsai, K. C.; Xiao, K. H.; Yu,
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