Discovery of a potent and selective GPR120 agonist

Article abstract of DOI:10.1021/jm300215x

GPR120 is a receptor of unsaturated long-chain fatty acids reported to mediate GLP-1 secretion, insulin sensitization, anti-inflammatory, and anti-obesity effects and is therefore emerging as a new potential target for treatment of type 2 diabetes and metabolic diseases. Further investigation is however hindered by the lack of suitable receptor modulators. Screening of FFA1 ligands provided a lead with moderate activity on GPR120 and moderate selectivity over FFA1. Optimization led to the discovery of the first potent and selective GPR120 agonist.

Full text of DOI:10.1021/jm300215x

Brief Article
pubs.acs.org/jmc
Discovery of a Potent and Selective GPR120 Agonist
Bharat Shimpukade,† Brian D. Hudson,‡ Christine Kiel Hovgaard,† Graeme Milligan,‡
and Trond Ulven*,†
†Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
‡Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
S
* Supporting Information
ABSTRACT: GPR120 is a receptor of unsaturated long-chain fatty acids reported to mediate GLP-1 secretion, insulin
sensitization, anti-inflammatory, and anti-obesity effects and is therefore emerging as a new potential target for treatment of type
2 diabetes and metabolic diseases. Further investigation is however hindered by the lack of suitable receptor modulators.
Screening of FFA1 ligands provided a lead with moderate activity on GPR120 and moderate selectivity over FFA1. Optimization
led to the discovery of the first potent and selective GPR120 agonist.
and type 2 diabetes.7 Another study found that GPR120 is
INTRODUCTION
■
likely to mediate unsaturated fatty acid reversal of diet-induced
hypothalamic inflammation, a determining factor for develop-
ment of obesity.8 Notably, dysfunctional GPR120 was recently
found to lead to obesity in both mice and human.9 As a
consequence of these observations, GPR120 is currently
emerging as an interesting new potential target for treatment
of metabolic and inflammatory diseases such as obesity and
type 2 diabetes. Further research is required to establish
GPR120 as a new target; however, this research is currently
hindered by the lack of potent and selective receptor
modulators. Here we report the first potent and selective
GPR120 agonist.
Omega-3 (n-3) fatty acids are found to beneficially influence
the development of metabolic syndrome and type 2 diabetes.1
The G protein-coupled 7-transmembrane domain receptor
GPR120 is abundantly expressed in intestines, lung, adipose
tissue, and proinflammatory macrophages and is activated by
long-chain free fatty acids (FFAs), in particular n-3
polyunsaturated FFAs such as α-linolenic acid (1, Chart 1),
Chart 1. Current GPR120 Agonists11,12
The free fatty acid receptor 1 (FFA1 or GPR40) is another
potential target for treatment of type 2 diabetes.10 Although
GPR120 and FFA1 are phylogenetically distant, there is a close
structural resemblance between the ligands capable of
recognizing and activating the two receptors. The few
GPR120 agonists described hitherto, including FFAs, require
micromolar concentrations and have poor or no selectivity over
FFA1.6 Briscoe and co-workers described the potent FFA1
agonist GW9508 (2, Chart 1), which also possesses moderate
GPR120 agonist activity.11 Suzuki and co-workers reported
GPR120 agonists derived from PPARγ ligands. NCG21 (3)
came out as the most potent and selective analogue, 16-fold
selective over FFA1.12,13 The natural product grifolic acid and
its methyl ether have also been reported to act as selective
partial agonists on GPR120 in concentrations above 30 μM.14
We recently described the structure−activity relationship
(SAR) studies of a dihydrocinnamic acid series on FFA1.15
The similarity between these compounds and GW9508 sparked
our interest in exploring the activity of the FFA1 series on
GPR120.15
eicosapentaenoic acid (EPA), and docosahexaenoic acid
(DHA).2−6 The receptor has been reported to mediate FFA
promoted release of glucagon-like peptide-1 (GLP-1), an
incretin hormone which increases glucose-stimulated insulin
secretion, insulin sensitivity, and β-cell mass and reduces
appetite and gastric emptying.2 Recently, GPR120 was also
found to mediate insulin-sensitizing and anti-inflammatory
effects of the n-3 fatty acids EPA and DHA.3 It is notable in
relation to this that chronic low-grade inflammation is
implicated in the pathogenesis of obesity, insulin resistance,
Received: February 16, 2012
Published: April 23, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
Table 1. Screening of FFA1 Agonists on GPR120
a
a
b
compd
R
X
GPR120 (BRET) pEC50 (efficacy)
FFA1 (BRET) pEC50 (efficacy)
select
1
(Chart 1)
3-OPh
(Chart 1)
(ref 14)
H
5.49 0.14 (99)
5.85 0.05 (102)
5.62 0.10 (96)
5.65 0.05 (100)
4.93 0.04 (105)
4.24 0.07 (104)
5.27 0.08 (47)
5.76 0.04 (95)
6.32 0.06 (90)
5.51 0.03 (104)
4.68 0.03 (102)
5.89 0.03 (98)
5.17 0.05 (98)
6.25 0.10 (94)
4.78 0.08 (63)
5.35 0.08 (44)
4.23 0.11 (93)
5.62 0.04 (23)
5.32 0.04 (93)
6.43 0.10 (95)
4.29 0.09 (97)
5.06 0.06 (79)
5.20 0.02 (53)
5.54 0.30 (88)
7.55 0.15 (86)
5.01 0.23 (97)
7.52 0.03 (100)
6.26 0.09 (90)
5.94 0.33 (99)
6.78 0.18 (108)
6.34 0.19 (114)
7.12 0.01 (93)
6.62 0.30 (87)
6.13 0.06 (96)
7.04 0.18 (109)
6.70 0.07 (112)
7.20 0.16 (90)
6.81 0.21 (120)
7.01 0.10 (88)
6.66 0.26 (118)
6.17 0.07 (110)
7.08 0.09 (112)
5.00 0.13 (99)
5.32 0.15 (53)
7.78 0.30 (86)
7.89 0.06 (109)
−0.05
−1.70
0.61
2
NH
3
4
−1.87
−1.33
−1.70
−1.51
−0.58
−0.80
−1.11
−1.45
−1.15
−1.53
−0.95
−2.03
−1.66
−2.43
−0.52
−1.76
1.43
5
O
6
H
NH
O
7
4-Br
8
2-Br
O
9
3-Br
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
3-Br
NH
O
3-CN
3-CF3
3-CF3
3-OPh
4-OPh
4-OPh
4-Ph
O
NH
O
O
NH
NH
O
3-Ph
3-Ph
NH
O
2-Ph
2-Ph
NH
O
−1.03
−2.72
−2.69
3-(2-MeC6H4)
3-(2-MeC6H4)
NH
a
b
Efficacy is relative to 4. Calculated as the difference between pEC50 for GPR120 and FFA1.
Scheme 1. Synthesis of ortho-Biphenyl Ligands
close to two orders of magnitude. All 4-benzyloxy- and 4-
benzylaminodihydrocinnamic acids turned out to also activate
GPR120 (Table 1). The SAR on GPR120 of compounds with
no or small substituents on the terminal phenyl followed the
trends previously observed for FFA1: a central O-linker is
preferable over an NH-linker (5/6, 9/10, 12/13) and meta-
substituents on the terminal ring appeared to be preferred (7−
9), with nonpolar substituents performing better (9 and 12 vs
11). In contrast to FFA1, a general preference of the O-linker
compounds for GPR120 was still observed with larger
substituents on the terminal ring, i.e., biphenyl and
phenoxyphenyl systems (e.g., 14 vs 2, 18 vs 19), although
the trend was less consistent. Several compounds behaved as
RESULTS AND DISCUSSION
■
Compounds were screened on GPR120 transfected HEK cells
in a β-arrestin 2 interaction bioluminescence resonance energy
transfer (BRET) assay and counterscreened on FFA1 in an
equivalent assay. α-Linolenic acid (1), GW9508 (2), and
NCG21 (3) exhibited values in the BRET assay of both
GPR120 and FFA1, in agreement with those reported
previously in calcium assays (Chart 1).2,11,12
A diverse selection of 4-benzyloxy- and 4-benzylaminodihy-
drocinnamic acids and the alkyne FFA1 agonist 4-(2-
toluylethynyl)dihydrocinnamic acid (TUG-424, 4)16 were
included in the screen. 4 exhibited full agonist activity on
GPR120 in the micromolar range and a selectivity for FFA1 of
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Brief Article
Table 2. Optimization of the ortho-Biphenyl Ligands
a
a
b
compd
R1
R2
GPR120 (BRET) pEC50 (efficacy)
FFA1 (BRET) pEC50 (efficacy)
select
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
2′-Me
3′-Me
4′-Me
4′-F
5.15 0.11(86)
5.71 0.05 (97)
6.75 0.05 (96)
6.23 0.15 (100)
6.51 0.03 (103)
6.53 0.07 (98)
5.69 0.04 (97)
6.72 0.11 (100)
5.66 0.06 (83)
5.36 0.04 (80)
6.63 0.22 (88)
6.62 0.13 (98)
6.04 0.03 (105)
6.29 0.05 (102)
6.96 0.03 (99)
7.36 0.15 (104)
6.22 0.15 (60)
6.28 0.09 (93)
6.20 0.14 (107)
6.03 0.20 (116)
6.29 0.18 (99)
6.07 0.19 (103)
5.94 0.21 (97)
5.69 0.03 (101)
5.35 0.37 (59)
6.20 0.28 (53)
5.45 0.20 (97)
6.22 0.12 (91)
6.96 0.25 (51)
6.39 0.16 (88)
6.02 0.22 (58)
4.19 0.97
−1.06
−0.56
0.54
H
H
H
0.20
H
4′-Cl
4′-Br
4′-CF3
4′-OMe
4′-OEt
4′-OCF3
4′-SMe
H
0.21
H
0.46
H
−0.25
1.02
H
H
0.31
H
−0.85
1.18
H
4-Cl
5-Cl
4-Cl
4-F
4-F
0.40
H
−0.92
−0.09
0.94
4′-Me
H
4′-Me
3.17
a
b
Efficacy is relative to 4. Calculated as the difference between pEC50 for GPR120 and FFA1.
partial agonists on GPR120 with efficacy down to 23% in the
case of 18. No clear structural determinants of partial agonism
were observed except that all carried substituents in the meta-
or para-position of the benzyl group. The potent and selective
FFA1 agonist 23 (TUG-469) turned out to be a micromolar
partial agonist on GPR120, slightly more potent than the
corresponding O-linked analogue 22 but still 500-fold selective
for FFA1.15 All compounds were 10−1000-fold less potent on
GPR120 than on FFA1 with one notable exception: the ortho-
biphenyl analogue 20 exhibited pEC50 = 6.43 for GPR120 with
27-fold selectivity over FFA1. The corresponding NH-linked 21
was >100-fold less active. Thus, 20 was selected for further
optimization.
produced a considerable increase in potency. Gratifyingly,
when the 4-fluoro was combined with 4′-methyl (43), a
significant further increase in potency up to pEC50 = 7.36 was
observed. Equally notable was the low activity on FFA1,
resulting in excellent selectivity for GPR120 over FFA1. Potent
full agonist activity on GPR120 and appreciable selectivity over
FFA1 was confirmed in the calcium assay (GPR120: pEC50
=
7.02 0.09, 114%. FFA1: pEC50 = 5.30 0.04, 121%.).
Critically in light of the importance of rodent models and cell-
lines, 43 was also found to be highly potent on murine GPR120
(pEC50 = 7.77
0.09, 75% efficacy). The compound was
confirmed to be devoid of activity on the short-chain FFA
receptors FFA2 and FFA3.
Analogues of the ortho-biphenyl ligand 20 were prepared by
coupling of 24a−d with 25 to form 2-bromo- or 2-iodobenzyl
intermediates 26a−d, followed by Suzuki−Miyaura cross-
coupling and ester hydrolysis to provide the target compounds
(Scheme 1). Introduction of methyl groups in the 2′- or 3′-
position of the terminal phenyl ring (28, 29) resulted in
reduced potency, whereas a methyl substituent in the 4′-
position (30) doubled the potency (Table 2). Focusing on the
4′-position, various small substituents (31−38), including
halogens and alkoxys, were investigated. Most substituents
produced GPR120 agonists with pEC50 > 6, but the larger
substituents (36, 37) tended to give somewhat lower potency.
Although the 4′-OMe substituent (35) produced an equipotent
compound, none were more potent than 30. Most compounds
exhibited a moderate selectivity for GPR120.
A model of GPR120 in complex with 43 was constructed
using the crystal structure of a nanobody-stabilized active state
of the β2-adrenoceptor (PDB code 3P0G)19 as template
(Figure 1). The model shows that the carboxylic acid residue is
We then turned our attention to the proximal ring of the
biphenyl system. Introduction of a 4-chloro substituent (39)
boosted potency, whereas a 5-chloro (40) produced a moderate
drop relative to 20. The combination of 4-chloro and 4′-methyl
(41) gave a compound with lower potency than both parent
compounds and the unsubstituted 20, demonstrating that the
effects of substituents in different parts of the biphenyl system
are not necessarily additive. A 4-fluoro substituent (42)
Figure 1. Model of 43 in complex with hGPR120. The shown surface
is defined by M118, T119, G122, N215, W277, I280, I281, V307, and
F311. Residues are labeled with sequence number and with Schwartz−
Baldwin17 and Ballesteros−Weinstein18 notations given as superscript.
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Journal of Medicinal Chemistry
Brief Article
chromatography (SiO2, EtOAc:petroleum ether, 1:9); Rf = 0.43
(SiO2, EtOAc:petroleum ether, 1:4). H NMR (400 MHz, CDCl3) δ
engaged in an electrostatic and double hydrogen bond
interaction with Arg99. The phenylpropionate moiety interacts
with Phe304 on the upper side and Phe311 on the bottom side.
The narrow lipophilic pocket around the biphenyl moiety is
defined by Met118, Thr119, Gly122, Phe211, Asn215, Ile280,
Ile281, and Trp277. The model thus provides a rationale for the
preference of GPR120 for the ortho-biphenyl moiety and
explains the restricted tolerability for substitution around this
moiety such as the lower activity observed with the slightly
longer substituted biphenyl system of 41 compared to 43. The
interaction with Arg99 is confirmed by the complete lack of β-
arrestin recruitment upon addition of 43 to cells expressing the
GPR120 R99Q mutant.
1
7.34 (dd, J = 9.7, 2.7 Hz, 1H), 7.26 (dd, J = 8.3, 5.9 Hz, 1H), 7.21 (s,
4H), 7.10−7.02 (m, 3H), 6.80−6.73 (m, 2H), 4.89 (s, 2H), 3.66 (s,
3H), 2.87 (t, J = 7.8 Hz, 2H), 2.58 (t, J = 7.8 Hz, 2H), 2.39 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 173.3, 163.4, 161.0, 156.8, 137.2,
137.2, 136.7, 136.68, 136.5, 133.0, 131.6, 131.5, 129.2, 129.1, 129.0,
115.3, 115.1, 114.9, 114.8, 114.6, 67.6, 51.6, 35.9, 30.1, 21.1. HRMS
calcd for C22H24FO3 (M+), 379.1704; found, 379.1683. Step 2:
Following general procedure C, the methyl ester from step 1 (75 mg,
0.20 mmol) was hydrolyzed to give 68 mg (94%) of a thick syrup. 1H
NMR (400 MHz, CDCl3) δ 7.34 (dd, J = 9.7, 2.7 Hz, 1H), 7.28−7.24
(m, 1H), 7.21 (s, 4H), 7.11−7.02 (m, 3H), 6.78 (d, J = 8.6 Hz, 2H),
4.89 (s, 2H), 2.88 (t, J = 7.7 Hz, 2H), 2.63 (t, J = 7.7 Hz, 2H), 2.39 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 178.0, 163.5, 161.0, 156.9, 137.2,
137.2, 136.7, 136.6, 136.6, 132.7, 131.6, 131.5, 129.2, 129.1, 129.0,
115.3, 115.1, 114.9, 114.8, 114.6, 67.6, 35.6, 29.7, 21.1. HRMS calcd
for C23H21FO3Na (M + Na+), 387.1367; found, 387.1349.
Biological Assays. BRET β-Qrrestin 2 Interaction Assay. The
BRET assay was performed as described previously.20 Briefly, plasmids
encoding either GPR120 or FFA1 fused at their C-terminal to
enhanced yellow fluorescent protein were cotransfected into HEK 293
cells with a plasmid encoding β-arrestin 2 fused to Renilla luciferase.
Cells were distributed into white 96-well plates 24 h post-transfection
and then maintained in culture for another 24 h prior to their use. To
conduct the assay, cells were first washed in Hank’s Balanced Salt
Solution and then the Renilla luciferase substrate coelenterazine h (5
μM), and the ligand of interest were added. Cells were incubated at 37
°C for either 5 min (GPR120) or 30 min (FFA1) before luminescence
at 535 and 475 nm was measured using a Pherastar FS. The ratio of
luminescence at 535/475 nm was then used to calculate the BRET
response. To test the activity of 43 on GPR120 R99Q in the BRET
assay, this point mutation was incorporated into the wild-type
GPR120-enhanced yellow flourescent protein construct using the
Quickchange method (Stratagene).
Calcium Asssay. Calcium assays were carried out on Flp-In T-
REx293 cell lines, generated to inducibly express either GPR120 or
FFA1 upon treatment with doxycycline. One day prior to conducting
the experiment, cells were seeded at 50000/well in black clear-bottom
96-well microplates. Cells were allowed to adhere for 3−4 h before the
addition of 100 ng/mL doxycycline to induce receptor expression. The
following day, cells were incubated in culture medium containing the
calcium sensitive dye Fura2-AM (3 μM) for 45 min. Cells were then
washed three times and then allowed to equilibrate for 15 min in
Hank’s Balanced Salt Solution prior to conducting the assay. Fura2
fluorescent emission was measured at 510 nm following excitation at
both 340 and 380 nm during the course of the experiment using a
Flexstation plate reader (Molecular Devices). Calcium responses were
then measured as the difference between 340/380 ratios before and
after the addition of test compounds.
CONCLUSION
■
In conclusion, by screening representatives from our collection
of FFA1 agonists on GPR120 and optimizing the most
promising hit, we have identified the first potent and selective
GPR120 agonist (43). The compound has high potency on
both the human and murine receptor and will enable studies to
further explore GPR120 as a target for the treatment of type 2
diabetes, obesity, and other metabolic diseases.
EXPERIMENTAL SECTION
■
Chemistry. All commercial starting materials and solvents were
used without further purification unless otherwise stated. THF was
freshly distilled from sodium/benzophenone. Acetone and N,N-
diisopropylethyl amine (DIPEA) were dried over 4 Å sieves.
Purification by flash chromatography was carried out using silica gel
60 (0.040−0.063 mm, Merck). 1H and 13C NMR spectra were
calibrated relative to TMS internal standard or residual solvent peak.
Reactions were routinely monitored by TLC using Merck silica gel
F254 plates. HPLC analysis was performed using a Dionex 120 C18
column (5 μ, 4.6 mm × 150 mm); flow, 1 mL/min; 10% acetonitrile in
water (0−1 min), 10−100% acetonitrile in water (1−10 min), 100%
acetonitrile (11−15 min), with both solvents containing 0.05% TFA as
modifier; UV detection at 254 nm. High-resolution mass spectra
(HRMS) were obtained on a Bruker micrOTOF-Q II (ESI). Melting
points were measured on a Buchi melting point apparatus and are
̈
uncorrected. Purity was determined by HPLC and confirmed by
NMR. All test compounds were of >95% purity.
General Procedure A (Williamson Ether Synthesis). A mixture of
benzyl chloride (1 equiv), phenol (1.1 equiv), potassium carbonate
(2.0 equiv), potassium iodide (0.5 equiv), and anhydrous acetone (5
mL/mmol) was heated at reflux. After consumption of the benzyl
chloride, the reaction mixture was cooled, filtered, and concentrated,
and the residue was purified by flash chromatography.
General Procedure B (Suzuki Cross-Coupling). A flask charged
with aryl halide (1.0 equiv), boronic acid (1.1 equiv), potassium
carbonate (1.2 equiv), acetonitrile (6.4 mL/mmol), water (2.1 mL/
mmol), and Pd(PPh3)4 (5 mol %) was evacuated, backfilled with argon
three times, and heated at 75 °C until consumption of the aryl halide.
The reaction mixture was concentrated, diluted with water, and
extracted with EtOAc. The combined organic phases were washed
with brine, dried over Na2SO4, concentrated, and purified by flash
chromatography.
General Procedure C (Ester Hydrolysis). To the ester (1 equiv)
dissolved in THF (5 mL/mmol ester) was added LiOH (2−3 equiv)
in H2O (2 mL/mmol ester). The reaction was stirred at room
temperature until complete consumption of the starting material as
indicated by TLC, typically after 2−4 h. The reaction was acidified
with 1 M HCl until pH <1 and extracted with EtOAc. The combined
extracts were washed with brine, dried over Na2SO4, and concentrated.
3-(4-((4-Fluoro-4′-methyl-[1,1′-biphenyl]-2-yl)methoxy)phenyl)-
propanoic Acid (43). Step 1: Following general procedure B, 26d (97
mg, 0.26 mmol) was coupled with p-tolylboronic acid (43 mg, 0.32
mmol) to give 83 mg (83%) of a colorless oil after flash
Counterscreen on FFA2 and FFA3. The activity of 43 was tested on
FFA2 and FFA3 in the [35S]GTPγS assay as described previously.21
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis and compound characterization, procedures for
biological testing and molecular modeling. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +45 6550 2568 . E-mail: ulven@sdu.dk.
Notes
The authors declare no competing financial interest.
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Journal of Medicinal Chemistry
Brief Article
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ACKNOWLEDGMENTS
■
We thank the Danish Council for Independent Research|
Technology and Production (grant 09-070364), the Danish
Council for Strategic Research (grant 11-116196), and the
Canadian Institutes of Health Research (fellowship to B.D.H.)
for financial support.
ABBREVIATIONS USED
■
BRET, bioluminescence resonance energy transfer; DHA, all-
cis-4,7,10,13,16,19-docosahexaenoic acid; DIPEA, diisopropyle-
thylamine; EPA, all-cis-5,8,11,14,17-eicosapentaenoic acid; FFA,
free fatty acid; FFA1, free fatty acid receptor 1 (GPR40); FFA2,
free fatty acid receptor 2 (GPR43); FFA3, free fatty acid
receptor 3 (GPR41); GLP-1, glucagon-like peptide-1
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Selective Agonists for the Free Fatty Acid Receptor 1 (FFA1/GPR40),
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