Development and Characterization of a Fluorescent Tracer for the Free Fatty Acid Receptor 2 (FFA2/GPR43)

Article abstract of DOI:10.1021/acs.jmedchem.7b00338

The free fatty acid receptor 2 (FFA2/GPR43) is considered a potential target for treatment of metabolic and inflammatory diseases. Here we describe the development of the first fluorescent tracer for FFA2 intended as a tool for assessment of thermodynamic and kinetic binding parameters of unlabeled ligands. Starting with a known azetidine FFA2 antagonist, we used a carboxylic acid moiety known not to be critical for receptor interaction as attachment point for a nitrobenzoxadiazole (NBD) fluorophore. This led to the development of 4 (TUG-1609), a fluorescent tracer for FFA2 with favorable spectroscopic properties and high affinity, as determined by bioluminescence resonance energy transfer (BRET)-based saturation and kinetic binding experiments, as well as a high specific to nonspecific BRET binding signal. A BRET-based competition binding assay with 4 was also established and used to determine binding constants and kinetics of unlabeled ligands.

Full text of DOI:10.1021/acs.jmedchem.7b00338

Article
Development and characterization of a fluorescent
tracer for the free fatty acid receptor 2 (FFA2/GPR43)
Anders Højgaard Hansen, Eugenia Sergeev, Sunil K. Pandey, Brian
D. Hudson, Elisabeth Christiansen, Graeme Milligan, and Trond Ulven
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017
Downloaded from http://pubs.acs.org on June 1, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Development and characterization of a fluorescent
tracer for the free fatty acid receptor 2
(FFA2/GPR43)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Anders Højgaard Hansen†,§, Eugenia Sergeev‡,§, Sunil K. Pandey†, Brian D. Hudson‡, Elisabeth
Christiansen†, Graeme Milligan‡ and Trond Ulven†*
†Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej
55, DKꢀ5230 Odense M, Denmark
‡Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology,
College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ,
Scotland, United Kingdom
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 28
1
2
3
4
5
6
ABSTRACT
7
8
9
The free fatty acid receptor 2 (FFA2/GPR43) is considered a potential target for treatment of
metabolic and inflammatory diseases. Here we describe the development of the first fluorescent
tracer for FFA2 intended as a tool for assessment of thermodynamic and kinetic binding
parameters of unlabeled ligands. Starting with a known azetidine FFA2 antagonist, we used a
carboxylic acid moiety known not to be critical for receptor interaction as attachment point for a
nitrobenzoxadiazole (NBD) fluorophore. This led to the development of 4 (TUGꢀ1609), a
fluorescent tracer for FFA2 with favorable spectroscopic properties and high affinity, as
determined by bioluminescence resonance energy transfer (BRET)ꢀbased saturation and kinetic
binding experiments, as well as a high specific to nonꢀspecific BRET binding signal. A BRETꢀ
based competition binding assay with 4 was also established and used to determine binding
constants and kinetics of unlabeled ligands.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 3 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
INTRODUCTION
7
8
9
The free fatty acid receptor 2 (FFA2, also known as GPR43) is a 7ꢀtransmembrane receptor
activated by shortꢀchain fatty acids (SCFAs) produced endogenously during colonic fermentation
of dietary fiber by the gut microbiota.1ꢀ5 The receptor is expressed in various cell types such as
adipocytes and pancreatic βꢀcells.6,7 FFA2 is also expressed in immune cells, including
neutrophils, eosinophils, B lymphocytes, and peripheral blood mononuclear cells, and has been
shown to mediate SCFAꢀpromoted chemotaxis in neutrophils.8ꢀ11 In recent years FFA2 has
attracted much attention as a possible target for treatment of obesity,12 inflammation,9,13,14 and
metabolic diseases.5,15 It has however frequently been unclear if the preferred mode of action
was agonism or antagonism.3 It has been shown that the loss of FFA2 and the related SCFA
receptor FFA3 (GPR41) leads to increased insulin secretion and improved glucose tolerance,
indicating a therapeutic potential of antagonists against metabolic diseases.7 The role of the
receptor in chemotaxis of neutrophils has also suggested antiꢀinflammatory potential of
antagonists. For example, the FFA2 antagonist GLPG0974 (1, Chart 1) has been demonstrated to
inhibit human neutrophil recruitment in vitro and in vivo, suggesting that the compound could
represent a potential therapeutic target for treatment of inflammatory diseases.13,16 Clinical trials
with 1 for treatment of ulcerative colitis indeed showed decreased neutrophil infiltration although
no significant improvement of symptoms was observed in the patients.17 To fully assess the
therapeutic potential of FFA2, high quality tool compounds are needed for further investigations
of FFA2 pharmacology and function.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fluorescent tracer molecules have been established as useful tools for realꢀtime monitoring of
receptorꢀligand interactions and offer several advantages over conventional radioactive tracers.18ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 28
1
2
21
3
4
The small, greenꢀemitting fluorophore 4ꢀaminoꢀ7ꢀnitrobenzoxadiazole (NBD) has previously
5
6
7
8
been employed for labeling small molecules, lipids and proteins and has been applied in
competition binding assays and for imaging of living cells.22ꢀ25 The fluorescence of NBD is
almost completely quenched in polar and protic solvents, whereas high fluorescence activity is
preserved in nonꢀpolar environments, which can amplify signal to noise ratio for the bound
NBD.26 Bioluminescence resonance energy transfer (BRET) between a receptor tagged at its Nꢀ
terminus with Nanoluciferase (NLuc) and a fluorescent ligand is useful for realꢀtime interaction
monitoring, also between GPCRs and small molecules.27 We have recently taken advantage of
the properties of NBD by developing two free fatty acid receptor 1 (FFA1) fluorescent tracers as
well as a BRET competition binding assay to NLuc tagged FFA1 and demonstrated its
usefulness for investigating the binding of natural and synthetic ligands to the FFA1 receptor.21
NBD was selected due to the almost perfect overlap between its absorption band and the
emission band of NLuc. NBD is known to be prone to photobleaching.28 However, the avoidance
of an external light source in the BRET assay minimizes this problem. Recognizing the need for
a similar fluorescent tracer and assay for FFA2, we aimed to follow an equivalent strategy for
this receptor. We here report on the design, synthesis and characterization of the first fluorescent
tracer for FFA2 and the use of the tracer in a BRET competition binding assay. The carboxylic
acid chain of 1 was originally introduced to tackle ADME issues in the compound series.13
Although the carboxylate contributes somewhat to potency of the compound and has been
demonstrated to depend on the presence of at least one of the two orthosteric arginine residues,
the group is not required for the activity of the compound.29 The closely related azetidine
analogue 2 as well as its methyl ester (3) are also efficient FFA2 antagonists.13,29 As the
carboxylic acid group appeared to be dispensable for the affinity of the compounds, we decided
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 5 of 28
Journal of Medicinal Chemistry
1
2
3
4
to explore this group as attachment point for an NBD fluorophore in the design of the proposed
5
6
FFA2 fluorescent tracer 4 (TUGꢀ1609, Chart 1).
7
8
9
S
S
R1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CF3
O
( )
n
O
NO2
N
O
N
O
N
N
OR2
N
N
N
H
H
O
O
N
O
4
1: (GLPG0974): R1 = 3-Cl, R2 = H, n=0
2: R1 = 4-CF3, R2 = H, n=1
linker
NBD-probe
3: R1 = 4-CF3, R2 = Me, n=1
Chart 1. Design strategy for an antagonistꢀbased FFA2 fluorescent tracer.
RESULTS AND DISCUSSION
Synthesis
The fluorescent tracer 4 was synthesized in 13 steps from commercial starting materials.
Azetidine 5 was synthesized according to the procedures described by Pizzonero and coꢀworkers
(Scheme 1).13 Benzothieneꢀ3ꢀacetic acid
6
was activated with bis(2ꢀoxoꢀ3ꢀ
oxazolidinyl)phosphinic chloride (BOPꢀCl) and coupled with 5 to give 3. Methyl ester 3 was
hydrolyzed to carboxylic acid 2, activated using BOPꢀCl and coupled to monoꢀBocꢀprotected
1,3ꢀdiaminopropane to give 7. Bocꢀdeprotection afforded the crude amine, which was reacted
directly with NBDꢀCl in a nucleophilic aromatic substitution to give the fluorescent 4 in 47%
yield after purification by preparative HPLC.21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 28
1
2
Scheme 1. Synthesis of 4.a
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aReagents and conditions: (i) Et3N, BOPꢀCl, DCM, 80 °C, 25 h, 40%; (ii) LiOH, THF, water,
rt, 96%; (iii) a): Et3N, BOPꢀCl, DCM, 50°C, 3 h; b): Et3N, tertꢀbutyl (3ꢀaminopropyl)carbamate,
30 min at 50 °C, then rt overnight, 46%; (iv) TFA, DCM, rt; (v) NBDꢀCl, NaHCO3, MeOH, 50
°C, 4 h, 38% over 2 steps.
Characterization of Fluorescence Properties
Absorption and fluorescence spectra of 4 were recorded in PBS7.4 and in nꢀoctanol, with the
latter solvent emulating the environment of a predominately hydrophobic receptor binding site or
membrane.30 A shift of 30 nm was observed when comparing the maximum excitation of 4 in nꢀ
octanol (λmax = 462 nm) with that in PBS buffer (λmax = 492 nm, spectrum not shown). The
extinction coefficient (ε) of 4 at its maximum excitation wavelength was determined (ε462 nm
=
12,550 Mꢀ1 cmꢀ1) from absorption spectra recorded in nꢀoctanol at increasing concentrations of 4
(Table 1). Fluorescent tracer 4 exhibits maximum emission at 525 nm with a Stokes shift of 63
nm and a quantum yield (Φ) of 0.62 in nꢀoctanol (Table 1 and Figure 1A), whereas the
fluorescence was almost absent in PBS (Figure 1B). Overall, 4 demonstrated properties
comparable to the values observed for the recently reported NBDꢀbased FFA1 tracers.21
ACS Paragon Plus Environment

Page 7 of 28
Journal of Medicinal Chemistry
1
2
Table 1. Absorption and fluorescence properties of 4. a
3
4
5
6
7
nꢀOctanol
PBS buffer (pH 7.4)
8
9
b
Φb
λex λem
[nm] [nm]
SS
ε462 nm
λex λem
[nm] [nm]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[nm] [Mꢀ1 cmꢀ1]
462 525 63
12,550 ± 276 0.62 ± 0.012 492 557
aSS, Stokes shift; Φ, quantum yield; ε, extinction coefficient. Values are reported ± standard
error (SE).
b
A
B
4×1005
3×1005
2×1005
1×1005
0
462 nm 525 nm
1.0
0.8
0.6
0.4
0.2
0.0
557 nm
400
500
600
700
400
500
600
700
Wavelength [nm]
Wavelength [nm]
Figure 1. Absorption and fluorescence properties of 4. A) Normalized absorption (dotted line)
and fluorescence (solid line) spectra in nꢀoctanol. B) Effect of solvent on fluorescence activity of
4 in nꢀoctanol (black curve) and PBS buffer (blue curve, maximum indicated by arrow).
Functional Characterization of 4 on FFA2
Tracer 4 was found to act as an antagonist of propionate (C3)ꢀmediated elevation of inositol
monophosphate (IP1) levels in FlpꢀInTM TꢀRExTM 293 cells induced to express a form of human
FFA2 that had enhanced Yellow Fluorescent Protein linked in frame to the intracellular Cꢀ
terminal tail of the receptor (hFFA2ꢀeYFP) (Figure 2A). The ability of 4 to inhibit the effects of
an EC80 concentration of C3 was similar to that of the related clinically tested FFA2 antagonist 1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 28
1
2
3
4
5
6
7
8
9
and analogue 2 (Figure 2A, Table 2). Furthermore, in membrane preparations derived from these
cells, increasing concentrations of 4 were able to prevent specific binding of [3H]ꢀ1 to the
hFFA2ꢀeYFP construct with affinity akin to that of unlabeled 1 and 2 (Figure 2B, Table 2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A
B
100
75
50
25
0
150
100
50
1
2
4
1
2
4
0
-10
-9
-8
-7
-6
-5
-4
-10
-9
-8
-7
-6
-5
-4
log [compound] M
log [compound] M
Figure 2. (A) Functional characterization of 4 against 1 and 2 in IP1 accumulation assay. (B)
Displacement of [3H]ꢀ1 using 1, 2 and 4. Error bars represent SE, n=3.
Table 2. Characterization of antagonist properties and affinities of unlabeled carboxylic acid 1,
methyl ester 2 and fluorescent tracer 4 as antagonist in functional IP1 and radioligand
displacement assays.
Cmpd
pIC50 (IP1)a
6.94 ± 0.04
7.08 ± 0.05
6.57 ± 0.10
pKi (Displacement)b
7.76 ± 0.11
1
2
4
7.64 ± 0.08
7.31 ± 0.06
aThe ability of 4 to inhibit IP1 accumulation at EC80 concentration of propionate. Ki was
b
determined in [3H]ꢀ1 displacement assays. Values are means ± SE, n=3.
ACS Paragon Plus Environment

Page 9 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
Characterization of 4 as an FFA2 Tracer Molecule
7
8
9
A saturation binding experiment was performed by adding varying concentrations of 4 to
membrane preparations generated from FlpꢀInTM TꢀRExTM 293 cells induced to express hFFA2ꢀ
eYFP. This allowed BRET (Figure 3A) between NLuc and the NBD domain of bound 4.
Subtraction of signal obtained when the same concentrations of 4 were coꢀincubated with 50 µM
1 was used to define nonꢀspecific binding of 4 (Figure 3A) and to generate a monophasic binding
curve with an estimated Kd for 4 of 65.1 ± 1.8 nM (Figure 3B). Following addition of 100 nM 4
to membrane preparations and waiting 2 hours to allow binding to reach equilibrium, washꢀout
of unbound 4 allowed the dissociation of 4 to be monitored under conditions of infinite dilution
by the reduction of specific BRET signal over time (Figure 3C). The tendency of the signal to
extrapolate below zero in Figure 3C may be due to depletion of substrate for NLuc at the end of
the longer measurements. Time courses of development of specific BRET signals following
addition of different concentrations of 4 showed the estimated ligand association rate to increase
with ligand concentration as predicted by mass action (Figure 3D). These experiments allowed
the determination of a dissociation rate koff of 0.0249 ± 0.0009 minꢀ1 and an association rate kon
of 368,000 ± 29300 Mꢀ1 minꢀ1 for tracer 4. Using these constants to independently calculate the
affinity of 4 yielded a Kd of 67.7 nM, a value very close to that obtained from the saturation
binding experiment.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 28
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. (A) BRETꢀbased saturation binding experiment of tracer 4 bound to NLuc tagged
FFA2 (total binding signal); BRET between 4 and NLuc tagged FFA2 recorded using nonꢀ
fluorescent 1 at high concentration (10 µM) (nonꢀspecific binding signal). (B) Monophasic
binding curve for 4 obtained by subtracting total binding signal from nonꢀspecific binding signal
of 4. (C) Dissociation of 4 monitored under conditions of infinite dilution (asymptote at ꢀ0.17 ±
0.02). (D) Time courses measuring the association of 4 to NLuc tagged FFA2 at varying
concentrations of 4. Error bars represent SE, n=3.
To compare the usefulness of 4 with that of the radiolabeled FFA2 antagonist [3H]ꢀ1, we
employed the BRET signal produced by 4 bound specifically to NLuc tagged FFA2 in the
presence of varying concentrations of either 1 or the FFA2 antagonist CATPB (8) as a means to
ACS Paragon Plus Environment

Page 11 of 28
Journal of Medicinal Chemistry
1
2
concomitantly measure both ‘on’ and ‘off’ rates of these unlabeled ligands (Table 3).31
Antagonist 8 is structurally different from 1 but is known to bind to the same site at the FFA2
receptor.29,32 Previous studies using [3H]ꢀ1 indicated that although these two compounds have
similar affinity at human FFA2, 8 is relatively ‘fast onꢀfast off’ whilst 1 displays both slower
association and slower dissociation kinetics.29 These features were reproduced and confirmed
when employing 4 in such binding kinetic analyses (Figure 4). The rate constants corresponded
to dissociation constants of 46.9 nM and 18.9 nM for 1 (Figure 4A) and 8 (Figure 4B),
respectively (Table 3). No problems related to bleaching or dayꢀtoꢀday variability was noticed
using the BRET assay when care was taken to protect the compound from unnecessary exposure
to light.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A
B
8
1
0.4
0.3
0.2
0.1
0.0
0.4
0.3
0.2
0.1
0.0
0
0
1 x Ki
3 x Ki
10 x Ki
1 x Ki
3 x Ki
10 x Ki
0
20
40
60
0
20
40
60
Time (min)
Time (min)
Figure 4. Application of 4 in binding kinetic analyses of unlabeled ligands; (A) 1, (B) 8. Error
bars represent SE, n=3.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 28
1
2
3
4
5
6
Table 3. Kinetic properties for fluorescent tracer 4 and unlabeled ligands 1 and 8.
7
8
9
Cmpd
4a
koff (minꢀ1)
kon (Mꢀ1 minꢀ1)
Kd (nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.0249 ± 0.0009
0.0099 ± 0.0014
0.0205 ± 0.0010
368,000 ± 29,300
211,000 ± 4,780
1,083,000 ± 59,800
67.7 ± 5.9
46.9 ± 6.7
18.9 ± 1.4
1b
8b
aRate constants and dissociation constant of 4 determined in BRETꢀbased saturation binding
assay. Rate constant and dissociation constants for unlabeled ligands 1 and 8 determined using
tracer 4 in BRETꢀbased competition binding assay. Values are means ± SE, n=3.
b
Importantly, when varying concentrations of 4 were coꢀadded with a range of concentrations of
1, analysis of the binding of 4, as reported by the specific BRET signal, was consistent with
competition between the ligands to bind to the receptor (Figure 5A). Higher concentrations of 4
required increasing concentrations of 1 to compete for the receptor binding site (Figure 5A,
Table 4). Equivalent conclusions were reached for 8 (Figure 5B) and provided an estimated
affinity for human FFA2 of 14.5 nM, almost identical to the value determined previously when
employing [3H]ꢀ1.29 Notably, affinity values for 1 and 8 determined using 4 in competition
binding experiments (Table 4) were found to be internally consistent with dissociation constants
obtained when using 4 in binding kinetic analyses (Table 3). To be useful as a ligand with which
to screen novel chemical ligands for affinity at human FFA2, it is important that employing 4
should result in ligand SAR as anticipated from other studies. It is known that replacement of the
carboxylate moiety of 8 by methyl ester (9) reduces affinity at hFFA2 by more than 10 fold.29
Herein, using 4 this expectation was again fulfilled (Figure 5C) with 9 displaying 23.6 fold lower
affinity for human FFA2, when compared to 8.
ACS Paragon Plus Environment

Page 13 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A
B
C
[4]
[4]
[4]
0.5
0.4
0.3
0.2
0.1
0.0
0.5
0.5
30 nM
30 nM
30 nM
0.4
0.3
0.2
0.1
0.0
0.4
0.3
0.2
0.1
0.0
100 nM
300 nM
100 nM
300 nM
100 nM
300 nM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-10
-9
-8
-7
-6
-5
-4
-10
-9
-8
-7
-6
-5
-4
-10
-9
-8
-7
-6
-5
-4
log [1] M
log [8] M
log [9] M
Figure 5. Application of 4 in competition binding experiments of unlabeled ligands (A) 1, (B) 8,
(C) 9. Error bars represent SE, n=3.
Table 4. Competition binding affinities of selected FFA2 antagonists.
1
8
9
pKi
7.16 ± 0.06
7.84 ± 0.04
14.5
6.42 ± 0.03
380
Affinitya
Ki (nM) 69.2
a Values for pKi are means ± SE, n=3.
CONCLUSION
By taking advantage of SAR information on known antagonists for FFA2, the fluorescent
tracer molecule 4 was developed. Tracer 4 contains an NBD fluorescent probe and exhibits
desirable spectroscopic properties, including a Stokes shift of 63 nm, low fluorescence activity in
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 28
1
2
3
4
5
6
7
8
9
aqueous solution, and a high quantum yield in nonꢀpolar environments. Using an NLuc construct
built into the extracellular Nꢀterminal domain of the FFA2 receptor and BRETꢀbased binding
experiments, tracer 4 was found to exhibit a Kd of 65 nM and a low nonꢀspecific binding signal.
The BRET binding assay with 4 was further applied to determine thermodynamic and kinetic
binding parameters of unlabeled FFA2 ligands, resulting in values comparable to those obtained
previously using a radiolabeled ligand. Compound 4 is the first fluorescent tracer for FFA2 and
is expected to be useful tool for further studies of the receptor as well as for characterization of
FFA2 ligands.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
Synthesis
Commercial staring materials and solvents were used without further purification, unless
otherwise stated. THF was freshly distilled from sodium/benzophenone, and MeOH and DCM
were dried over 3 Å sieves. Petroleum ether (PE) refers to alkanes with pb 60 – 80 °C. TLC was
performed on TLC silica gel 60 F254 plates and visualized at 254 or 365 nm or by staining with
ninhydrin or KMnO4 stains. Purification by flash chromatography was carried out using silica gel
1
60 (0.040ꢀ0.063 mm, Merck). H and 13C NMR spectra were recorded at 400 and 101 MHz,
respectively, on a Bruker Avance III 400 at 300 K. Rotamer peaks have been assigned by
asterisk (*). Integrals reported as H represent sum of rotamers, whereas integrals reported as H’
and H’’ represent specific rotamers where H’ + H’’ = H for the relevant signal. Highꢀresolution
mass spectra (HRMS) were obtained on a Bruker micrOTOFꢀQ II (ESI). Purity was determined
13
by HPLC and confirmed by inspection of NMR spectra (1H and C NMR). HPLC analysis was
performed using a Gemini C18 column (5 ꢁm, 4.6x150 mm); flow: 1 mL/min; 10% MeCN in
ACS Paragon Plus Environment

Page 15 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
water (0ꢀ1 min), 10ꢀ100% MeCN in water (1ꢀ10 min), 100% MeCN (11ꢀ15 min), with both
solvents containing 0.1% HCOOH as modifier; UV detection at 254 nm. All test compounds
were of ≥95% purity unless otherwise stated.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compounds 1, 8 and 9 were synthesized according to published procedures.13,33
4-(1-(2-(Benzo[b]thiophen-3-yl)acetyl)-2-methyl-N-(4-(trifluoromethyl)benzyl)azetidine-
2-carboxamido)butanoic acid (2). Compound 3 (50.0 mg, 92 ꢁmol) dissolved in THF (1 mL)
was added 0.6 M aqueous LiOH (0.45 mL) and stirred for 3.5 h at rt, whereafter the reaction
mixture was acidified with 1 M HCl until pH 2ꢀ3 and extracted with EtOAc (x3). The extract
was washed with brine, dried over Na2SO4, and concentrated in vacuo to give 2 as a sticky oil
(46.9 mg, 96%); 1H NMR (400 MHz, CDCl3) δ 7.93 – 7.51 (m, 4H), 7.49 – 7.13 (m, 5H), 4.93 –
4.26 (m, 2H), 4.09 – 2.95 (m, 6H), 2.61 – 2.43 (m, 1H), 2.40 – 2.12 (m, 3H), 2.00 – 1.70 (m,
13
5H); C NMR (101 MHz, CDCl3) δ 172.6, 170.2, 140.31, 140.25, 139.3, 138.8, 130.1, 129.9,
128.0, 127.0, 125.8, 124.6, 124.3, 124.1, 124.0, 122.9, 122.8, 122.2, 122.0, 121.8, 71.3, 46.7,
46.2, 43.3, 34.3, 33.6, 30.5, 29.8, 29.5, 29.0, 25.3, 23.7; ESIꢀHRMS calcd for C27H27F3N2NaO4S
1
(M + Na+) 555.1536, found 555.1544; HPLC: tR = 11.80 min, 97.1% pure. H NMR in overall
agreement with literature.13
Methyl
4-(1-(2-(benzo[b]thiophen-3-yl)acetyl)-2-methyl-N-(4-(trifluoromethyl)benzyl)-
azetidine-2-carboxamido)butanoate (3). In a dry microwave vial carboxylic acid 6 (57.4 mg,
0.30 mmol) and amine 5 (93.4 mg, 0.23 mmol) were suspended in dry DCM (0.45 mL). To the
stirred suspension were added Et3N (175 ꢁL, 1.26 mmol) and BOPꢀCl (88.5 mg, 0.35 mmol),
and the vial was heated at 80 °C for 25 h. The reaction was cooled to rt, vented, water was added,
and the mixture extracted with DCM (x5). The extract was dried over Na2SO4 and concentrated
in vacuo. The product was obtained after flash chromatography (EtOAc:PE 4:1 ꢀ 100% EtOAc)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 28
1
2
1
3
4
as a pale yellow sticky oil (50.2 mg, 40%): Rf = 0.26 (EtOAc); H NMR (400 MHz, CDCl3) δ
5
6
7.95 – 7.53 (m, 4H), 7.50 – 7.12 (m, 5H), 4.94 – 4.26 (m, 2H), 4.10 – 2.96 (m, 6H), 3.66* (s,
7
8
9
13
3H’), 3.63 (s, 3H’’), 2.63 – 2.46 (m, 1H), 2.43 – 2.14 (m, 3H), 2.00 – 1.71 (m, 5H); C NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(101 MHz, CDCl3) δ 172.6, 172.0, 169.7, 141.4, 140.3, 139.3, 138.8, 130.1, 128.0, 126.9, 126.3,
125.8, 124.5, 124.3, 124.1, 124.0, 123.8, 122.9, 122.8, 122.1, 121.8, 71.0, 51.9, 51.8, 46.7, 46.3,
43.1, 34.4, 33.7, 31.3, 30.7, 29.4, 29.1, 23.8, 23.1; ESIꢀHRMS calcd for C28H29F3N2NaO4S (M +
Na+) 569.1692, found 569.1714.
1-(2-(Benzo[b]thiophen-3-yl)acetyl)-2-methyl-N-(4-((3-((7-nitrobenzo[c][1,2,5]oxadiazol-
4-yl)amino)propyl)amino)-4-oxobutyl)-N-(4-(trifluoromethyl)benzyl)azetidine-2-
carboxamide (4). To a preꢀdried microwave vial at rt, the Bocꢀprotected amine 7 (23.9 mg, 35
ꢁmol) was dissolved in dry DCM (70 ꢁL) followed by dropwise addition of TFA (66 ꢁL, 0.87
mmol). Upon full deprotection indicated by TLC the reaction mixture was diluted with DCM,
added NaHCO3 (sat), extracted with DCM (x5), the organic phases dried over Na2SO4, and
concentrated in vacuo. The crude amine (17 mg) was used in the next step without further
purification.
To a preꢀdried microwave vial under argon were added crude amine (17 mg), NBDꢀCl (17.3
mg, 87 ꢁmol), NaHCO3 (9.8 mg, 117 ꢁmol) and the reactants were dissolved in dry MeOH (0.7
mL). The vial was capped, protected from light, and stirred at 50 °C for 4 h. The reaction was
cooled to rt, concentrated under a flow of N2, redissolved in a mixture of EtOAc (2 mL) and
water (2 mL), extracted with EtOAc (x3), washed with brine, and concentrated in vacuo. The
crude product was purified on preparative HPLC using a Phenomenex Luna C18 5 µm column
(250×21.2 mm), with gradient elution of 0.05% TFA/MeCN and 0.05% TFA/H2O at a flow rate
of 20 mL/min. HPLC fractions were combined an concentrated under reduced pressure and the
ACS Paragon Plus Environment

Page 17 of 28
Journal of Medicinal Chemistry
1
2
3
4
remaining aqueous phase extracted with EtOAc (x5). The organic phases were combined,
5
6
washed with brine and concentrated in vacuo to give 4 as a red sticky oil (10.1 mg, 38% over 2
7
8
9
1
steps): Rf = 0.32 (DCM [10% MeOH]); H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 8.4 Hz, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8.01 – 7.92 (m, 1H), 7.84 – 7.77 (m, 1H), 7.72 – 7.67 (m, 1H), 7.63 (d, J = 7.7 Hz, 2H), 7.42 –
7.36 (m, 1H), 7.36 – 7.29 (m, 2H), 7.28 – 7.24 (m, 1H), 7.23 (br s, 1H), 6.01 (d, J = 8.4 Hz, 1H),
4.67 – 4.58 (m, 1H), 4.36 – 4.25 (m, 1H), 4.21 – 3.99 (m, 3H), 3.68 – 3.63 (m, 2H), 3.51 – 3.22
(m, 6H), 2.58 – 2.45 (m, 1H), 2.39 – 2.17 (m, 4H), 1.94 – 1.73 (m, 4H), 1.70 – 1.55 (m, 2H);
ESIꢀHRMS calcd for C36H37F3N7O6S (M + H+) 752.2473, found 752.2502; HPLC: tR = 12.29
min, 99.9% pure.
tert-Butyl-(3-(4-(1-(2-(benzo[b]thiophen-3-yl)acetyl)-2-methyl-N-(4-(trifluoromethyl)-
benzyl)azetidine-2-carboxamido)butanamido)propyl)carbamate (7). The crude carboxylic
acid 2 (40.0 mg, 75 ꢁmol) in dry DCM (150 ꢁL) was added Et3N (20 ꢁL, 0.15 mmol) followed
by BOPꢀCl (23.9 mg, 94 ꢁmol), and the reaction was heated slowly to 50 °C. After 3 h,
additional Et3N (25 ꢁL, 0.19 mmol) was added followed by tertꢀbutyl (3ꢀaminopropyl)carbamate
(17.3 mg, 1.0 mmol). The reaction was stirred at 50 °C for 30 min, cooled to rt, and stirred over
night before concentration in vacuo and purification of the crude product by flash
chromatography (DCM [5% MeOH]) to yield the desired product as clear sticky oil (23.9 mg,
46%): Rf = 0.12 (DCM [5% MeOH]); 1H NMR (400 MHz, CDCl3) δ 7.93 – 7.51 (m, 4H), 7.49 –
7.13 (m, 5H), 7.24 – 7.11 (m, 1H), 6.79 – 6.40 (m, 1H), 4.92 – 4.49 (m, 2H), 4.47 – 4.27 (m,
1H), 4.24 – 3.84 (m, 3H), 3.78 – 3.38 (m, 2H), 3.26 – 3.16 (m, 2H), 3.14 – 3.00 (m, 2H), 2.58 –
2.41 (m, 1H), 2.38 – 2.20 (m, 1H), 1.94 – 1.73 (m, 5H), 1.71 – 1.48 (m, 4H), 1.43* (s, 9H’), 1.42
(s, 9H’’); ESIꢀHRMS calcd for C35H43F3N4NaO5S (M + Na+) 711.2798, found 711.2786.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 28
1
2
3
4
Characterization of absorption and fluorescence properties of 4
5
6
7
8
Single concentration absorption and fluorescence spectra of fluorescent tracer 4 were recorded
at 8.82 ꢀM. Extinction coefficient for 4 in nꢀoctanol was recorded in duplicates using a
FLUOstar Omega Microplate Reader (BMG labtech). For fluorescent quantum yield
determination, fluorescence spectra for 4 were recorded in duplicates using a ChronosFD timeꢀ
resolved spectrofluorometer coupled to PMT detectors. Coumarin 153 (Cꢀ153) and 4 were
excited at a fixed excitation wavelength of 402 nm, and Cꢀ153 in EtOH was used as an internal
standard for determination of quantum yield (ΦCꢀ153 = 0.546, λex = 402 nm).34 All experiments
were performed at 25 °C. Data analysis was performed using Graphpad Prism v5.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IP1 accumulation assay
All IP1 experiments were performed using FlpꢀInTM TꢀRExTM 293 cells able to express receptor
constructs of interest in an inducible manner. Experiments were carried out using a homogeneous
timeꢀresolved FRETꢀbased detection kit (CisꢀBio Bioassays, Codolet, France) according to the
manufacturer’s protocol. Cells were induced to express the receptor of interest by treatment for
24 h with 100 ng/mL doxycycline and plated at 7500 cells/well in lowꢀvolume 384ꢀwell plates.
The ability of C3 to induce accumulation of IP1 was assessed following a coꢀincubation for 2
hours with C3, which was preceded by a 15ꢀmin preꢀincubation with antagonist to allow for
equilibration.
Radioligand displacement assay
All receptor radioligand binding experiments using [3H]ꢀ1 were conducted in glass tubes, in
binding buffer (50 mM TrisꢀHCl, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4).
ACS Paragon Plus Environment

Page 19 of 28
Journal of Medicinal Chemistry
1
2
Membrane protein was generated from FlpꢀInTM TꢀRExTM 293 cells induced to express the
receptor construct of interest with 100 ng/mL doxycycline. For [3H]ꢀ1 competition binding
assays, the radioligand at approximately Kd concentration and varying concentrations of
unlabeled ligand of choice were coꢀadded to 5 µg of membrane protein. Nonꢀspecific binding of
the radioligand was determined in the presence of 10 µM 8. After an incubation period of 2 h at
25 °C, bound and free [3H]ꢀ1 were separated by rapid vacuum filtration through GF/C glass
filters using a 24ꢀwell Brandel cell harvester (Alpha Biotech, Glasgow, UK), and unbound
radioligand was washed from filters by three washes with iceꢀcold PBS. After drying (3–12 h), 3
mL of Ultima GoldTM XR (PerkinElmer Life Sciences) was added to each sample vial, and
radioactivity was quantified by liquid scintillation spectrometry. Aliquots of [3H]ꢀ1 were also
quantified to define the concentration of [3H]ꢀ1 added per tube. Data were fitted to a oneꢀsite
model using Graphpad Prism v6 and used to calculate pKi values based on a Kd value for [3H]ꢀ
1 of 7.5 nM.16
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Equilibrium BRET binding assay
The NLucꢀhFFA2 construct and cell line were developed as described by Christiansen et al.20
NLucꢀhFFA2 FlpꢀInTM TꢀRExTM 293 cells were induced to express the receptor construct by
treating with 100 ng/mL doxycycline for 24 h. Cells were then harvested and used to make total
cell membrane preparations. Membranes were suspended in binding buffer and transferred into a
white 96ꢀwell plate at 5 ꢁg membrane protein per well. Membranes were then coꢀincubated with
the indicated concentration of 4 (and 1 for nonꢀspecific binding measurements; or competing
ligand for competition experiments) for 2 h at 30 °C. Following incubation, the NLuc substrate,
NanoꢀGlo (Promega) was added to a final 1:800 dilution. Membranes were incubated a further 5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 28
1
2
3
4
5
6
7
8
9
min before the bioluminescent emission at 460 and 545 nm was measured using a Clariostar
plate reader (BMG labtech). Total and nonꢀspecific saturation binding data were then globally fit
to a oneꢀsite binding model using Graphpad Prism v6. Competition binding experiments were fit
to a oneꢀsite model and used to calculate pKi values based on a Kd value for 4 of 65.1 nM.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Kinetic BRET binding assay
For kinetic BRET binding assays, cell membranes were generated as for the equilibrium
binding assay and again distributed in white 96ꢀwell microplates (2.5 ꢁg membrane
protein/well). The NanoꢀGlo substrate was then added (1:800 final dilution) before incubation
for 5 min at 30 °C. Plates were then inserted into a Clariostar plate reader, with temperature set
to 30 °C and set to read emission at 460 and 545 nm at 90 s intervals. For association
experiments, 4 was manually added to the plate after 60 s to a final concentration of 100, 300,
500 or 1000 nM. Readings were continued at 90 s intervals for the indicated time period. For
dissociation experiments the reaction was preꢀincubated for 2 h at 30 °C followed by two washes
with binding buffer, performed via centrifugation at 14,000 rpm at 4 °C for 15 min. The pellet
was then resuspended in binding buffer at 37 °C and transferred into a white 96ꢀwell microplate
at 90 µL/well and standard kinetic binding assay procedure as described above was followed. In
all kinetic experiments parallel wells were assessed in which 1 had been preꢀadded, and these
were used to subtract the nonꢀspecific binding signal for 4. Kinetic binding data were then fit to
oneꢀphase association or dissociation models using Graphpad Prism v6 to obtain estimates of koff,
kon, and Kd.
ACS Paragon Plus Environment

Page 21 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
Determination of the “On” and “Off” Rates of Unlabeled Ligands through Measurement
of Competition Binding Kinetics of 4
7
8
9
The kinetic binding parameters of unlabeled ligands were determined through assessment of
the binding kinetics of 4 as described above. Here the standard procedure was followed. After 5
min preꢀincubation of membrane protein with NanoꢀGlo, 4 (100 nM) and the competing
unlabeled ligand at three different concentrations (1ꢀ, 3ꢀ, or 10ꢀfold the estimated respective Ki
concentration) were added simultaneously. Readings were then continued at 90 s intervals for 60
min. Three different concentrations of competitor were assessed to ensure that the rate
parameters calculated were independent of ligand concentration. Data were fit globally using the
kinetics of the competitive binding equation available from GraphPad Prism v6, with the kon and
koff values of 4 entered as constraints.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AUTHOR INFORMATION
Corresponding Author
*Tel: +45 6550 2568. Eꢀmail: ulven@sdu.dk
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. §These authors contributed equally.
Funding Sources
The study has been funded by the Danish Council for Strategic Research (grant 11ꢀ116196) and
the University of Southern Denmark. EC is supported by a fellowship from the Lundbeck
Foundation. BDH is supported by a fellowship from the University of Glasgow.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 28
1
2
3
4
5
6
ABBREVIATIONS
7
8
9
BOPꢀCl, bis(2ꢀoxoꢀ3ꢀoxazolidinyl)phosphinic chloride; BRET, bioluminescence resonance
energy transfer; FFA1, free fatty acid receptor 1 (GPR40); FFA2, free fatty acid receptor 2
(GPR43); FFA3, free fatty acid receptor 2 (GPR41); NBD, 4ꢀnitroꢀ7ꢀaminobenzodiazole; NLuc,
NanoLuciferase; PE, petroleum ether; PMT, photomultiplier tubes; SS, Stoke’s shift.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES
(1)
(2)
(3)
(4)
(5)
(6)
Topping, D. L.; Clifton, P. M. Shortꢀchain fatty acids and human colonic function: Roles
of resistant starch and nonstarch polysaccharides. Physiol. Rev. 2001, 81, 1031ꢀ1064.
Offermanns, S. Free fatty acid (FFA) and hydroxy carboxylic acid (HCA) receptors.
Annu. Rev. Pharmacol. Toxicol. 2014, 54, 407ꢀ434.
Ulven, T. Shortꢀchain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new
potential therapeutic targets. Front. Endocrinol. 2012, 3, 111.
Milligan, G.; Bolognini, D.; Sergeev, E. Ligands at the free fatty acid receptors 2/3
(GPR43/GPR41). Handb. Exp. Pharmacol. 2017, 236, 17ꢀ32.
Milligan, G.; Shimpukade, B.; Ulven, T.; Hudson, B. D. Complex pharmacology of free
fatty acid receptors. Chem. Rev. 2017, 117, 67ꢀ110.
Ge, H.; Li, X.; Weiszmann, J.; Wang, P.; Baribault, H.; Chen, J.ꢀL.; Tian, H.; Li, Y.
Activation of G proteinꢀcoupled receptor 43 in adipocytes leads to inhibition of lipolysis
and suppression of plasma free fatty acids. Endocrinology 2008, 149, 4519ꢀ4526.
ACS Paragon Plus Environment

Page 23 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(7)
(8)
Tang, C.; Ahmed, K.; Gille, A.; Lu, S.; Grone, H. J.; Tunaru, S.; Offermanns, S. Loss of
FFA2 and FFA3 increases insulin secretion and improves glucose tolerance in type 2
diabetes. Nat. Med. 2015, 21, 173ꢀ177.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Brown, A. J.; Goldsworthy, S. M.; Barnes, A. A.; Eilert, M. M.; Tcheang, L.; Daniels, D.;
Muir, A. I.; Wigglesworth, M. J.; Kinghorn, I.; Fraser, N. J.; Pike, N. B.; Strum, J. C.;
Steplewski, K. M.; Murdock, P. R.; Holder, J. C.; Marshall, F. H.; Szekeres, P. G.;
Wilson, S.; Ignar, D. M.; Foord, S. M.; Wise, A.; Dowell, S. J. The orphan G proteinꢀ
coupled receptors GPR41 and GPR43 are activated by propionate and other short chain
carboxylic acids. J. Biol. Chem. 2003, 278, 11312ꢀ11319.
(9)
Maslowski, K. M.; Vieira, A. T.; Ng, A.; Kranich, J.; Sierro, F.; Yu, D.; Schilter, H. C.;
Rolph, M. S.; Mackay, F.; Artis, D.; Xavier, R. J.; Teixeira, M. M.; Mackay, C. R.
Regulation of inflammatory responses by gut microbiota and chemoattractant receptor
GPR43. Nature 2009, 461, 1282ꢀ1287.
(10) Vinolo, M. A. R.; Ferguson, G. J.; Kulkarni, S.; Damoulakis, G.; Anderson, K.;
BohloolyꢀY, M.; Stephens, L.; Hawkins, P. T.; Curi, R. SCFAs induce mouse neutrophil
chemotaxis through the GPR43 receptor. PLoS One 2011, 6, e21205.
(11) Bjorkman, L.; Martensson, J.; Winther, M.; Gabl, M.; Holdfeldt, A.; Uhrbom, M.;
Bylund, J.; Hojgaard Hansen, A.; Pandey, S. K.; Ulven, T.; Forsman, H.; Dahlgren, C.
The neutrophil response induced by an agonist for free fatty acid receptor 2 (GPR43) Is
primed by tumor necrosis factor alpha and by receptor uncoupling from the cytoskeleton
but attenuated by tissue recruitment. Mol. Cell. Biol. 2016, 36, 2583ꢀ2595.
(12) Forbes, S.; Stafford, S.; Coope, G.; Heffron, H.; Real, K.; Newman, R.; Davenport, R.;
Barnes, M.; Grosse, J.; Cox, H. Selective FFA2 agonism appears to act via intestinal
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 28
1
2
3
4
5
6
PYY to reduce transit and food intake but does not improve glucose tolerance in mouse
models. Diabetes 2015, 64, 3763ꢀ3771.
7
8
9
(13) Pizzonero, M.; Dupont, S.; Babel, M.; Beaumont, S.; Bienvenu, N.; Blanque, R.; Cherel,
L.; Christophe, T.; Crescenzi, B.; De Lemos, E.; Delerive, P.; Deprez, P.; De Vos, S.;
Djata, F.; Fletcher, S.; Kopiejewski, S.; L'Ebraly, C.; Lefrancois, J. M.; Lavazais, S.;
Manioc, M.; Nelles, L.; Oste, L.; Polancec, D.; Quenehen, V.; Soulas, F.; Triballeau, N.;
van der Aar, E. M.; Vandeghinste, N.; Wakselman, E.; Brys, R.; Saniere, L. Discovery
and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2)
antagonist: From hit to clinic. J. Med. Chem. 2014, 57, 10044ꢀ10057.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(14) Ang, Z.; Ding, J. L. GPR41 and GPR43 in obesity and inflammation ꢀ protective or
causative? Front. Immunol. 2016, 7, 28.
(15) Kimura, I.; Ozawa, K.; Inoue, D.; Imamura, T.; Kimura, K.; Maeda, T.; Terasawa, K.;
Kashihara, D.; Hirano, K.; Tani, T.; Takahashi, T.; Miyauchi, S.; Shioi, G.; Inoue, H.;
Tsujimoto, G. The gut microbiota suppresses insulinꢀmediated fat accumulation via the
shortꢀchain fatty acid receptor GPR43. Nat. Commun. 2013, 4, 1829.
(16) Namour, F.; Galien, R.; Van Kaem, T.; Van der Aa, A.; Vanhoutte, F.; Beetens, J.; Van't
Klooster, G. Safety, pharmacokinetics and pharmacodynamics of GLPG0974, a potent
and selective FFA2 antagonist, in healthy male subjects. Br. J. Clin. Pharmacol. 2016,
82, 139ꢀ148.
(17) Vermeire, S.; Kojecký, V.; Knoflícek, V.; Reinisch, W.; Kaem, T. V.; Namour, F.;
Beetens, J.; Vanhoutte, F. GLPG0974, an FFA2 antagonist, in ulcerative colitis: efficacy
and safety in a multicenter proofꢀofꢀconcept study. J. Crohn's Colitis 2015, Suppl 1, S39.
ACS Paragon Plus Environment

Page 25 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
(18) Vernall, A. J.; Hill, S. J.; Kellam, B. The evolving smallꢀmolecule fluorescentꢀconjugate
toolbox for Class A GPCRs. Br. J. Pharmacol. 2014, 171, 1073ꢀ1084.
7
8
9
(19) Ma, Z.; Du, L.; Li, M. Toward fluorescent probes for Gꢀproteinꢀcoupled receptors
(GPCRs). J. Med. Chem. 2014, 57, 8187ꢀ8203.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(20) Heisig, F.; Gollos, S.; Freudenthal, S. J.; ElꢀTayeb, A.; Iqbal, J.; Muller, C. E. Synthesis
of BODIPY derivatives substituted with various bioconjugatable linker groups: a
construction kit for fluorescent labeling of receptor ligands. J. Fluoresc. 2014, 24, 213ꢀ
230.
(21) Christiansen, E.; Hudson, B. D.; Hansen, A. H.; Milligan, G.; Ulven, T. Development and
characterization of a potent free fatty acid receptor 1 (FFA1) fluorescent tracer. J. Med.
Chem. 2016, 59, 4849ꢀ4858.
(22) Yates, A. S.; Doughty, S. W.; Kendall, D. A.; Kellam, B. Chemical modification of the
naphthoyl 3ꢀposition of JWHꢀ015: in search of a fluorescent probe to the cannabinoid
CB2 receptor. Bioorg. Med. Chem. Lett. 2005, 15, 3758ꢀ3762.
(23) Petrov, R. R.; Ferrini, M. E.; Jaffar, Z.; Thompson, C. M.; Roberts, K.; Diaz, P. Design
and evaluation of a novel fluorescent CB2 ligand as probe for receptor visualization in
immune cells. Bioorg. Med. Chem. Lett. 2011, 21, 5859ꢀ5862.
(24) Haldar, S.; Chattopadhyay, A. Application of NBDꢀlabeled lipids in membrane and cell
biology. Springer Ser. Fluoresc. 2013, 13, 37ꢀ50.
(25) Barresi, E.; Bruno, A.; Taliani, S.; Cosconati, S.; Da Pozzo, E.; Salerno, S.; Simorini, F.;
Daniele, S.; Giacomelli, C.; Marini, A. M.; La Motta, C.; Marinelli, L.; Cosimelli, B.;
Novellino, E.; Greco, G.; Da Settimo, F.; Martini, C. Deepening the topology of the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 28
1
2
3
4
5
6
translocator protein binding site by novel N,Nꢀdialkylꢀ2ꢀarylindolꢀ3ꢀylglyoxylamides. J.
Med. Chem. 2015, 58, 6081ꢀ6092.
7
8
9
(26) Lin, S.; Struve, W. S. Timeꢀresolved fluorescence of nitrobenzoxadiazoleꢀaminohexanoic
acid: effect of intermolecular hydrogenꢀbonding on nonꢀradiative decay. Photochem.
Photobiol. 1991, 54, 361ꢀ365.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(27) Stoddart, L. A.; Johnstone, E. K.; Wheal, A. J.; Goulding, J.; Robers, M. B.; Machleidt,
T.; Wood, K. V.; Hill, S. J.; Pfleger, K. D. Application of BRET to monitor ligand
binding to GPCRs. Nat. Methods 2015, 12, 661ꢀ663.
(28) Wüstner, D.; Christensen, T.; Solanko, L.; Sage, D. Photobleaching Kinetics and Timeꢀ
Integrated Emission of Fluorescent Probes in Cellular Membranes. Molecules 2014, 19,
11096.
(29) Sergeev, E.; Hansen, A. H.; Pandey, S. K.; MacKenzie, A. E.; Hudson, B. D.; Ulven, T.;
Milligan, G. Nonꢀequivalence of key positively charged residues of the free fatty acid 2
receptor in the recognition and function of agonist versus antagonist ligands. J. Biol.
Chem. 2016, 291, 303ꢀ317.
(30) Tikhonova, I.; Poerio, E. Free fatty acid receptors: structural models and elucidation of
ligand binding interactions. BMC Struct. Biol. 2015, 15, 16.
(31) Dowling, M. R.; Charlton, S. J. Quantifying the association and dissociation rates of
unlabelled antagonists at the muscarinic M3 receptor. Br. J. Pharmacol. 2006, 148, 927ꢀ
937.
(32) Brantis, C. E.; Ooms, F.; Bernard, J. Novel amino acid derivatives and their use as
GPR43 receptor modulators. PCT Int. Appl. WO2011092284 2011.
ACS Paragon Plus Environment

Page 27 of 28
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(33) Hudson, B. D.; Tikhonova, I. G.; Pandey, S. K.; Ulven, T.; Milligan, G. Extracellular
ionic locks determine variation in constitutive activity and ligand potency between
species orthologs of the free fatty acid receptors FFA2 and FFA3. J. Biol. Chem. 2012,
287, 41195ꢀ41209.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(34) Rurack, K.; Spieles, M. Fluorescence quantum yields of a series of red and nearꢀinfrared
dyes emitting at 600ꢀ1000 nm. Anal. Chem. 2011, 83, 1232ꢀ1242.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 28
1
2
3
4
TABLE OF CONTENT GRAPHICS
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment