Probe-dependent negative allosteric modulators of the long-chain free fatty acid receptor FFA4

Article abstract of DOI:10.1124/mol.116.107821

High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential of free fatty acid receptor 4 (FFA4) as a novel therapeutic target for the treatment of type II diabetes, the broad distribution pattern of this receptor suggests it may play a range of roles beyond glucose homeostasis in different cells and tissues. To date, a single molecule, 4-methyl- N-9H-xanthen-9-yl-benzenesulfonamide (AH-7614), has been described as an FFA4 antagonist; however, its mechanism of antagonism remains unknown. We synthesized AH-7614 and a chemical derivative and demonstrated these to be negative allosteric modulators (NAMs) of FFA4. Although these NAMs did inhibit FFA4 signaling induced by a range of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many anticancer drugs suggests that a novel close chemical analog of AH-7614 devoid of FFA4 activity, 4-methyl-N-(9H-xanthen-9-yl)benzamide (TUG-1387), will also provide a useful control compound for future studies assessing FFA4 function. Using TUG-1387 alongside AH-7614, we show that endogenous activation of FFA4 expressed by murine C3H10T1/2 mesenchymal stem cells is required for induced differentiation of these cells toward a more mature, adipocyte-like phenotype.

Full text of DOI:10.1124/mol.116.107821

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Title page
Probe-dependent negative allosteric modulators of the
long-chain free fatty acid receptor FFA4
Kenneth R. Watterson, Steffen V. F. Hansen, Brian D. Hudson, Elisa Alvarez-Curto,
Sheikh Zahir Raihan, Carlos M. G. Azevedo, Gabriel Martin,
Julia Dunlop, Stephen J. Yarwood,
Trond Ulven, and Graeme Milligan
Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems
Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow,
Glasgow, United Kingdom (KRW, BDH,EA-C, SZR, JD, SJY, GM)
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark,
Odense, Denmark (SVFH, CMGA, GM, TU)
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering
and Physical Sciences, Heriot-Watt University, Edinburgh (SJY)
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Molecular Pharmacology Fast Forward. Published on April 6, 2017 as DOI: 10.1124/mol.116.107821
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Running Title Page
Running title: FFA4 Negative Allosteric Modulators
Address correspondence to: Graeme Milligan, Centre for Translational Pharmacology,
Wolfson Link Building 253, University of Glasgow, Glasgow G128QQ, Scotland, U.K., e-
mail: Graeme.Milligan@glasgow.ac.uk. Tel (44) 141 330 5557
Manuscript Information
Text pages 35
Tables 1
Figures 9
Words in Abstract 241
Words in Introduction 706
Words in Discussion 690
References 52
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Non-standard abbreviations
aLA, a linolenic acid; BRET, bioluminescence resonance energy transfer; DMEM,
Dulbecco’s modified Eagle’s medium; eYFP, enhanced yellow fluorescent protein; FBS,
foetal bovine serum; FFA1, Free fatty acid receptor 1; FFA4, Free fatty acid receptor 4;
GPCR, G protein-coupled receptor; HBSS, Hanks’ balanced salt solution; IBMX, 3-isobutyl-
1
-methylxanthine; NAM, negative allosteric modulator, PPARγ, peroxisome proliferator
activator receptor γ,
Chemical names
AH-7614, 4-methyl-N-9H-xanthen-9-yl-benzenesulfonamide; GSK137647A, 4-methoxy-N-
(2,4,6-trimethylphenyl)-benzenesulfonamide; Merck compound A, 3-[2-chloro-5-
(trifluoromethoxy)phenyl]-3-azaspiro[5.5]undecane-9-acetic acid; TUG-770, (4-[2-[2-
(cyanomethyl)phenyl]ethynyl]-2-fluorobenzenepropanoic acid); TUG-891, 4-[(4-fluoro-4'-
methyl[1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid; TUG-1197, 2-(3-(pyridin-2-
yloxy)phenyl)-2,3-dihydrobenzo[d]isothiazole 1,1-dioxide; TUG-1387, 4-methyl-N-(9H-
xanthen-9-yl)benzamide, TUG-1506, 4-methyl-N-(9H-thioxanthen-9-
yl)benzenesulfonamide.
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Abstract
High affinity and selective antagonists that are able to block the actions of both endogenous
and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor
function and to support suggestions of therapeutic potential. Although there is great interest
in the potential of free fatty acid receptor 4 (FFA4) as a novel therapeutic target for the
treatment of type II diabetes, the broad distribution pattern of this receptor suggests it may
play a range of roles beyond glucose homeostasis in different cells and tissues. To date a
single molecule, AH-7614, has been described as an FFA4 antagonist, however it’s
mechanism of antagonism remains unknown. We synthesized AH-7614 and two chemical
derivatives and demonstrate these to be negative allosteric modulators (NAMs) of FFA4.
Although these NAMs did inhibit FFA4 signalling induced by a range of endogenous and
synthetic agonists, clear agonist probe-dependence in the nature of allosteric modulation was
apparent. Although AH-7614 did not antagonize the second long chain free fatty acid
receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing
features found in many anti-cancer drugs suggests that a novel close chemical analog of AH-
7
614 devoid of FFA4 activity, TUG-1387, will also provide a useful control compound for
future studies assessing FFA4 function. Using TUG-1387 alongside AH-7614, we show that
endogenous activation of FFA4 expressed by murine C3H10T1/2 mesenchymal stem cells is
required for induced differentiation of these cells towards a more mature, adipocyte-like
phenotype.
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Molecular Pharmacology Fast Forward. Published on April 6, 2017 as DOI: 10.1124/mol.116.107821
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Introduction
Free fatty acid receptor 4 (FFA4) (Davenport et al., 2013, Milligan et al., 2017), which is also
still frequently designated GPR120 (Hirasawa et al., 2005), was identified in 2005 as a G
protein-coupled receptor (GPCR) responsive to long-chain free fatty acids (Hirasawa et al.,
2
005). Since then, not least because essential and other polyunsaturated w-3 fatty acids with
health promoting properties are among the most potent fatty acid activators of this receptor
Hirasawa et al., 2005, Christiansen et al., 2015), there has been strong interest in better
understanding the biological functions of FFA4 and in its potential as a therapeutic target
Liu et al., 2015, Li et al., 2016). Because reported effects of FFA4 activation include
(
(
regulation of glucose homeostasis (Oh et al., 2014, Azevedo et al., 2016), the regulation of
release of incretins and satiety-regulating hormones (Hirasawa et al., 2005), modulation of
insulin sensitivity (Oh et al., 2010, Oh et al., 2014, Azevedo et al., 2016) and potential effects
on weight gain (Ichimura et al., 2012, Mo et al., 2013, Azevedo et al., 2016), a large focus of
studies on FFA4 has centred on the possibility that agonists of this receptor might be useful
anti-diabetic agents (Zhang and Leung, 2014, Cornall et al., 2014, Moran et al., 2016).
However, it is clear that the expression pattern of FFA4, which includes high levels in the
lung, in macrophage populations, and in various cancer cell types (Houthuijzen, 2016,
Milligan et al., 2017), indicates a broader set of roles and potential for therapeutic
interventions targeting this receptor (Milligan et al., 2016). This has resulted in screening for,
and the identification of, a number of series of FFA4 agonist ligands (Milligan et al., 2015,
Milligan et al., 2017), although chemical diversity within these series has, until recently (e.g.
Sparks et al., 2014, Azevedo et al., 2016), remained rather limited and based on carboxylate-
containing ligands that resemble synthetic fatty acids. FFA4 has been shown to be a potential
regulator of adipocyte development and differentiation (Gotoh et al., 2007), potentially via
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increasing expression of the adipogenic transcription factor, peroxisome proliferator activator
receptor γ (PPARγ) (Hasan et al., 2015, Song et al., 2016). Moreover, adipocyte
differentiation from bone marrow mesenchymal stem cells has also been suggested to be
promoted by FFA4 (Gao et al., 2015).
A major challenge in studying all aspects of both in vivo and ex vivo regulation of cell
and tissue function by FFA4 is that the endogenous fatty acid ligands that are able to activate
the receptor are present ubiquitously. Furthermore, being bound extensively by plasma
proteins, the concentrations and disposition of ‘free’ fatty acids is often challenging to define
(
Ulven and Christiansen, 2015), as is the extent of receptor activation in situ that can be
attributed to such systemic, circulating fatty acids. As such, alongside various ‘knock-down’
and ‘knock-out’ strategies (Alvarez-Curto et al., 2016), well characterized and selective
blockers of FFA4 function would be highly valuable tool compounds to further unravel
biological roles of FFA4. To date, a single FFA4 ‘antagonist’ has been described. Compound
3
9 (Sparks et al., 2014) (now designated AH-7614 by commercial vendors) was shown to
block effects of both a fatty acid and a synthetic FFA4 agonist in FFA4-transfected U2OS
human osteosarcoma cells and to antagonize both FFA4-mediated insulin secretion from
mouse insulinoma MIN6 cells and the secretion of GLP-1 from the human intestinal cell line
NCI-H716 (Sparks et al., 2014).
Herein we examine the antagonistic properties of AH-7614, and a small number of
chemical derivatives, at FFA4. This demonstrated that AH-7614 is able to inhibit FFA4-
mediated signals induced by a range of distinct fatty acids and synthetic FFA4 agonist ligands
across all endpoints tested. Analysis of the mechanism of antagonism indicates that these
compounds are negative allosteric modulators (NAMs) of FFA4 function, which display
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‘
probe-dependence’ in the nature of their inhibitory properties depending on the specific
agonists they are inhibiting. This work also identified a close chemical derivative of AH-7614
that lacks antagonistic activity at FFA4, which given the current lack of knowledge about
potential off-target effects of AH-7614, will be a useful tool in defining FFA4 specific
biological effects. We have demonstrated the utility of this approach in assessing the role of
FFA4 in adipogenesis of murine C3H10T1/2 mesenchymal stem cells.
Materials and Methods
Materials and compounds
Unless otherwise stated, all biochemicals and reagents were from Sigma-Aldrich Co. Tissue
culture reagents and buffers were from Life Technologies Inc. TUG-891 [3-(4-((4-fluoro-4’-
methyl-[1,1’-biphenyl]-2-yl)methoxy)phenyl)-propanoic acid] was synthesized as described
previously (Shimpukade et al., 2012). Compound 34 (TUG-1197) (2-(3-(pyridin-2-
yloxy)phenyl)-2,3-dihydrobenzo[d]isothiazole 1,1-dioxide) was synthesized as described by
Azevedo et al., (2016). GSK137647A (4-methoxy-N-(2,4,6-trimethylphenyl)-
benzenesulfonamide) was synthesized according to Sparks et al., (2014). TUG-770 (4-[2-[2-
(cyanomethyl)phenyl]ethynyl]-2-fluorobenzenepropanoic acid) was synthesized as described
previously (Christiansen et al., 2013), Cpd A (3-[2-chloro-5-(trifluoromethoxy)phenyl]-3-
azaspiro[5.5]undecane-9-acetic acid) was synthesized essentially as described by Chelliah et
al., (2014). AH-7614/compound 39 (4-methyl-N-9H-xanthen-9-yl-benzenesulfonamide) was
synthesized as follows: Xanthone (100 mg, 0.5 mmol) was dissolved in methanol (10 mL),
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sodium borohydride (96 mg, 2.6 mmol) was added portion-wise over 20 minutes and the
reaction stirred at room temperature for 24 hours. The reaction was partitioned between ethyl
acetate and water. The two phases were separated and the aqueous phase was extracted twice
with ethyl acetate. The organic phases were combined, washed with brine, dried over
anhydrous sodium sulfate, filtered and concentrated. The crude product was used directly in
the next step without further purification.. The crude was used directly in the next step
without further purification. To the crude mixture was added p-toluenesulfonamide (95 mg,
0
.56 mmol) followed by acetic acid (5 mL). The reaction was allowed to stir for 2 hours at
room temperature. The reaction was partitioned between water and ethyl acetate. The two
phases were separated and the aqueous phase was extracted twice with ethyl acetate. The
organic phases were combined, washed with brine, dried over anhydrous sodium sulfate,
filtered and concentrated. The crude product was purified by silica gel flash chromatography
(ethyl acetate/petroleum ether 1:4) to give the desired compound as a white solid (136 mg,
o
1
7
7
8
1
1
3
1
3
7% over two steps): mp 194.1-195.5 C; H NMR (400 MHz, CDCl ) 7.83 – 7.75 (m, 2H),
.33 (d, J = 8.0 Hz, 2H), 7.30 – 7.23 (m, 2H), 7.15 (dd, J = 7.8, 1.4 Hz, 2H), 7.08 (dd, J =
.3, 1.0 Hz, 2H), 6.99 (td, J = 7.7, 1.2 Hz, 2H), 5.77 (d, J = 8.6 Hz, 1H), 4.91 (d, J = 8.6 Hz,
1
3
H), 2.47 (s, 3H); C NMR (100 MHz, CDCl
3
): 151.3, 143.6, 138.6, 129.8, 129.5, 127.2,
+
23.6, 120.4, 116.7, 49.2, 21.6; HRMS (ESI) m/z: calculated C20
H17NaO
3
S [M+Na ]:
74.0821; found: 374.0804; HPLC 99.7 %. Data agrees with that of Liu et al. (2013). TUG-
387, 4-methyl-N-(9H-xanthen-9-yl)benzamide, was synthesized as follows: 4-
Methylbenzamide (205 mg, 1.5 mmol) and 9H-xanthen-9-ol (150 mg, 0.76 mmol) were
suspended in acetic acid (1.5 mL) and heated to 90°C. The compounds went in to solution
after 1 min and a few min later a white solid started to precipitate. The reaction was heated
overnight and cooled to room temperature, filtered and washed with acetic acid (0.5 mL),
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then washed with cold ethyl acetate (3 mL). Subsequent drying gave the product as a white
1
solid (180 mg, 75%): mp 229.8-231.6 °C; t
400 MHz, d
Hz, 1H), 7.37 – 7.31 (m, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.20 – 7.11 (m, 1H), 6.55 (d, J = 8.6
R
= 12.88 min (HPLC purity: 98.7%); H NMR
(
6
-DMSO) δ 9.36 (d, J = 8.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.40 (d, J = 7.6
1
3
Hz, 1H), 2.34 (s, 1H); C NMR (101 MHz, d
6
-DMSO) δ 165.7, 150.6, 141.3, 131.1, 130.0,
1
28.9, 128.8, 127.5, 123.4, 121.9, 116.1, 43.0, 20.9; ESI-HRMS calcd for C21H17NnaO
2
+
[
M+Na ] 338.1151, found 338.1163. Data agrees with that of Patrick and Dolan (1973).
TUG-1506, (4-methyl-N-(9H-thioxanthen-9-yl)benzenesulfonamide), was synthesized as
follows: A 250 mL flask was charged with thioxanthone (2.0 g, 9.4 mmol), THF (30 mL)
and methanol (10 mL). The solution was stirred at room temperature and NaBH
4
(1.07 g,
2
8.3 mmol) was added slowly. The initially yellow reaction mixture slowly turned orange.
After 45 min the solution was diluted with water and extracted three times with ethyl acetate.
The combined organic phases were washed with brine, dried over sodium sulfate and
concentrated to give the crude alcohol as an orange solid (2.02 g) that was used immediately
1
in the next step: H NMR (400 MHz, d
6
-DMSO) δ 7.70 (d, J = 7.6 Hz, 2H), 7.50 (dd, J = 7.7,
0
1
1
.9 Hz, 2H), 7.36 (td, J = 7.5, 1.2 Hz, 2H), 7.28 (td, J = 7.5, 1.0 Hz, 2H), 6.40 (d, J = 5.8 Hz,
1
3
H), 5.19 (d, J = 5.7 Hz, 1H); C NMR (101 MHz, d
6
-DMSO) δ 139.0, 130.8, 126.8, 126.6,
26.4, 125.3, 69.3. Data agrees with that of Cozzi and Zoli (2008). 9H-Thioxanthen-9-ol
(2.02 g, 9.4 mmol) and p-toluenesulfonamide (2.22 g, 10.4 mmol) were dissolved in acetic
acid (30 mL) and stirred at room temperature overnight. The resulting precipitate was isolated
by filtration and washed first with cold acetic acid and then cold petroleum ether to afford the
product as a white solid (2.2 g, 64%): mp 158.7-163.9 °C; t
R
= 12.94 min (HPLC purity:
1
9
1
5
9.0%); R
f
=0.40 (EtOAc:PE, 1:1); H NMR (400 MHz, d
6
-DMSO) δ 8.76 (d, J = 8.4 Hz,
H), 7.65 (d, J = 8.3 Hz, 2H), 7.50 – 7.46 (m, 2H), 7.37 – 7.28 (m, 4H), 7.28 – 7.19 (m, 4H),
1
3
6
.23 (d, J = 8.4 Hz, 1H), 2.35 (s, 3H); C NMR (101 MHz, d -DMSO) δ 142.4, 138.8, 134.9,
9
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1
32.2, 129.3, 127.3, 126.8, 126.7, 126.5, 126.3, 56.5, 20.9; ESI-HRMS calcd for
+
C
20
H
17NNaO
2
S
2
[M+Na ] 390.0593, found 390.0585. Data agrees with that of Tamura et al.,
(1977).
Cell culture
HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
TM
2
supplemented with 10% (v/v) fetal bovine serum (FBS) at 37°C and 5% (v/v) CO . Flp-In
TM
T-REx 293 cell lines, generated to inducibly express various tagged versions of human
FFA4 (hFFA4), human FFA1 (hFFA1) or murine FFA4 (mFFA4) following treatment with
doxycycline were maintained in DMEM supplemented with 10% (v/v) FBS, 100 U/mL
penicillin, 100 µg/mL streptomycin, 200µg/mL hygromycin B. Human brain astrocytoma
1
321N1 cells stably transfected with hFFA1 were grown in DMEM supplemented with 10%
(v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 400 µg/mL G418. To study
adipogenesis, murine C3H10T1/2 cells (Tang et al., 2004) were grown in DMEM containing
o
1
5
3
0% (v/v) FBS and 100 U/mL penicillin, 100 µg/mL streptomycin in a 37 C incubator with
% (v/v) CO
2
and induced to differentiate in 10 cm dishes with IID (100 nM insulin, 500 µM
-isobutyl-1-methylxanthine (IBMX) and 10 nM dexamethasone) for 5 days in the
presence/absence of either vehicle (0.1% (v/v) DMSO), 10µM AH-7614 or 10µM TUG-
387.
1
Plasmids
Plasmids encoding either the human (short isoform) or mouse FFA4 receptors with enhanced
yellow fluorescent protein (eYFP) or the related fluorescent protein mVenus fused to their C
terminal and incorporating a N-terminal FLAG epitope tag in the pcDNA5 FRT/TO
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expression vector were generated as previously described (Hudson et al., 2013, Butcher et al.,
014, Prihandoko et al., 2016, Alvarez-Curto et al., 2016).
2
b-Arrestin-2 interaction assay
β-Arrestin-2 recruitment to either human or mouse isoforms of FFA4 was measured using a
bioluminescence resonance energy transfer (BRET)-based approach. HEK293T cells were
co-transfected with eYFP-tagged forms of each receptor in a 4:1 ratio with a β-arrestin-2
Renilla luciferase (Rluc) plasmid using polyethylenimine. Cells were then transferred into
white 96-well plates at 24 h post-transfection. At 48 h post-transfection, cells were washed
with Hanks’ balanced salt solution (HBSS) and then incubated in fresh HBSS prior to
conducting the assay. Cells were pre-incubated for 15 mins with HBSS supplemented either
with vehicle (1% (v/v) DMSO), AH-7614, TUG-1506, or TUG-1387. Cells were incubated
with 2.5 µM of the Rluc substrate, coelenterazine h (Nanolight Tech, Pinetop, CA) at 37°C
for 10 min and then stimulated with various FFA4 agonists for a further 5 min at 37°C.
BRET resulting from receptor-β-arrestin-2 interaction was then determined by measuring the
ratio of luminescence at 535 and 475 nm using a Pherastar FS fitted with the BRET1 optic
module (BMG Labtech, Aylesbury, UK).
2
+
Ca mobilization
TM
TM
Calcium assays were carried out on either Flp-In T-REx 293 cell lines, generated to
inducibly express hFFA4 upon treatment with doxycycline, or 1321N1 cells stably expressing
the hFFA1 receptor. One day prior to conducting the experiment, cells were seeded at 50000
TM
TM
cells/well (Flp-In REx 293) or 25000 cells/well (1321N1) in poly-D-lysine coated black
clear-bottom 96-well microplates. Cells were allowed to adhere for 3-4 h before the addition
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TM
TM
of 100 ng/mL doxycycline to induce receptor expression in the case of Flp-In T-REx
2
93 cell lines. 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 HBSS 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,
Brambleside, UK). Calcium responses were then measured as the difference between 340/380
ratios before and after the addition of the relevant compounds. For antagonism, the cells
were pre-incubated for 15 minutes prior to agonist addition with HBSS supplemented with
either vehicle (1% (v/v) DMSO), AH-7614 or TUG-1387 (10 µM) prior to the addition of
either 50 µM aLA, 500 nM TUG-891 or 13 nM TUG-770.
High content imaging quantitative internalization assay
TM
TM
hFFA4-mVenus Flp-In T-REx 293 cells were plated 75000 cells per well in black with
clear bottom 96-well plates. Cells were allowed to adhere for 3 to 6 hours before the addition
of doxycycline (100 ng/mL) to induce receptor expression. After an overnight incubation,
culture medium was replaced with serum-free DMEM containing the ligand to be assessed
and incubated at 37°C for 30 min before fixation with 4% paraformaldehyde. After washing
with phosphate-buffered saline, cell nuclei were stained for 30 minutes with Hoechst33342.
Plates were subsequently imaged using a Cellomics Arrayscan II high content plate imager
(Thermo Fisher Scientific, Paisley, UK). Images were processed to identify internalized
mVenus, which was then normalized to cell number based on nuclei identified by
Hoechst33342 staining, to obtain a quantitative measure of hFFA4-mVenus internalization.
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HTRF-based IP1 assay
Inositol monophosphate (IP1) assays (Cisbio Bioassays, Codolet, France) were performed
according to the manufacturer’s instructions. In brief, a suspension of 7500 cells/well was
incubated with the stated concentrations of agonist for 1h in the presence of 10 mM lithium
chloride (LiCl). IP1 accumulation was subsequently measured using a Pherastar FS plate
reader (BMG Labtech, Aylesbury, UK).
RNA isolation and real-time qPCR
Total RNA was isolated from C3H10T1/2 cells using an RNEasy mini kit (Qiagen,
Manchester, UK). Following RNA isolation, RT-PCR was performed using Superscript III
(Life Technologies, Paisley, UK) and the resultant cDNA used as a template for quantitative
PCR analysis using an ABI Prism 7300 sequence detector (Applied Biosystems, Paisley,
UK). Cycling conditions were: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of
9
5°C for 15 s and 60°C for 1 min. FFA4, PPARg and Runx2 expression were then defined
−
ΔΔCt
relative to cyclophilin using the 2
method.
Western blotting
Analysis of receptor phosphorylation was performed on mFFA4-eYFP cells that were pre-
treated with either 10µM AH-7614 or TUG-1387 for 30 min after which time they were
treated with 10µM TUG-891 for 5 min. Cell lysates were prepared and size fractionated on 4-
1
2% SDS-PAGE gels and transferred to nitrocellulose membranes. Nitrocellulose membranes
containing resolved receptor proteins were incubated in Tris buffered saline LI-COR
blocking buffer and incubated subsequently with a phospho-specific antiserum produced in-
3
47
350
house that is able to recognize phosphorylated Thr and Ser residues of the receptor
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(Prihandoko et al., 2016). Membranes were air-dried and scanned using an LI-COR Odyssey
CLx Imager (Cambridge, UK).
Oil Red O staining
Untreated or differentiated C3H10T1/2 cells were fixed in 10% (v/v) formalin for 60 mins at
room temperature. The cells were washed in 60% (v/v) isopropanol and then incubated for
1
0 mins with isopropanol supplemented with 0.6% (w/v) Oil Red O. The Oil Red O solution
2
was then removed, cells washed x4 with dH O and then stored in phosphate-buffered saline
o
at 4 C until visualization using an Invitrogen™ EVOS™ FL Auto Imaging System (Life
Technologies, Paisley, UK). After removal of all liquid the Oil Red O stain was dissolved in
isopropanol and the absorbance measured at 405nm using a Pherastar FS reader (BMG
Labtech, Aylesbury, UK).
Statistical analysis
Data are presented as the mean ± S.E.M of a minimum of 3 independent experiments
performed in triplicate, unless otherwise stated in the respective figure legend. Data analysis
was carried out using GraphPad Prism software (package v5.02), with concentration-response
data fitted to three parameter sigmoidal curves. For allosteric modulation experiments, data
sets were globally fit to an operational model of allosterism described previously (Hudson et
al., 2014). In order to fit these data the t value for all allosteric modulators was constrained to
reflect the lack of detectable agonism seen within these compounds, which allowed for curve
A B
fits yielding estimates of loga, logb, logK and logK . Statistical analyses were carried out
using 1-way analysis of variance (1-way ANOVA) followed by Tukey’s post-hoc test.
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Results
We synthesized and assessed the functional activity of AH-7614 (Figure 1),
axanthene derivative of a diarylsulfonamide-based FFA4 agonist. This compound was
originally described as an antagonist of FFA4, able to block effects of both the
polyunsaturated, w-6 fatty acid, linoleic acid and the synthetic FFA4 agonist GSK137647A
(
(
Figure 1) (Sparks et al., 2014) and has since been used in a number of functional studies
Quesada-López et al., 2016, Houthuijzen et al., 2017, Villegas-Comonfort et al., 2017) but
without analysis of its mode of action. Indeed, AH-7614 inhibited, in a potent and
concentration-dependent manner, (pIC50 versus EC80 concentrations of the agonists) the
ability of either the w-3 fatty acid a linolenic acid (aLA) (pIC50 7.51 ± 0.08, n = 3) or a
synthetic ortho-biphenyl-based agonist of FFA4, TUG-891 (Figure 1) (pIC50 8.13 ± 0.08, n
2
+
=
3) (Shimpukade et al., 2012, Hudson et al., 2013, Butcher et al., 2014) to promote Ca
TM
TM
mobilization in Flp-In T-REx 293 cells induced to express hFFA4-eYFP (Figures 2A,
2
B). Similar results were obtained when measuring the ability of AH-7614 to inhibit agonist-
induced interactions of hFFA4 with b-arrestin-2 following co-expression of these two
proteins in HEK293T cells (Figures 2C, 2D). Importantly for subsequent studies, AH-7614
also functionally antagonized effects of both aLA and TUG-891 at the corresponding mouse
ortholog of the receptor (mFFA4) (Figures 2E and 2F). AH-7614 showed selectivity for
FFA4 as it was unable to antagonize activation of the other long-chain fatty acid receptor,
FFA1 (Milligan et al., 2015, Milligan et al., 2017) in studies in which the synthetic alkyne-
based FFA1 agonist TUG-770 (Christiansen et al., 2013) promoted elevation of intracellular
2
+
Ca in 1321N1 cells stably transfected to express a human FFA1 construct (Figure 2G).
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In similar EC80 inhibition experiments, AH-7614 was also effective in blocking TUG-
8
91-mediated internalization of FFA4 from the cell surface (Figure 3A) (pIC50 = 7.70 ± 0.10,
n = 3). We therefore used this internalization assay, with end point 30 minutes after addition
of agonist to facilitate achievement of ligand equilibrium, to probe the mechanism of AH-
7
614-mediated blockade of FFA4. Receptor internalization experiments were conducted in
which the ability of defined concentrations of pre-added AH-7614 to block internalization in
response to various concentrations of TUG-891 was assessed (Figure 3B). In these
experiments it was clear that the primary inhibitory effect of AH-7614 was to produce a
decrease in the maximal response to TUG-891. Although this is consistent with outcomes
2
+
reported by Sparks et al. (2014) using intracellular Ca assays, the longer time course of the
internalization studies largely eliminates potential issues of ligand hemi-equilibrium that can
greatly influence outcomes and mechanistic interpretation in assays, such as those based on
2
+
Ca mobilization, that employ much shorter end points (Charlton and Vauquelin, 2010).
These findings indicated that AH-7614 is not a competitive antagonist of FFA4. Interestingly,
even at the highest concentration of AH-7614 tested a residual response to TUG-891
remained. This response to TUG-891 was similar in the presence of 1 and 10 µM AH-7614
and, as such, the effect of AH-7614 appeared to saturate. Such an outcome is consistent with
AH-7614 functioning as a NAM of FFA4.
As it is common for small chemical changes in allosteric GPCR ligands to
significantly alter the details of their allosteric behavior (Wood et al., 2011, Hudson et al.
2
014), we next synthesized derivatives of AH-7614 and assessed their activity at FFA4. Two
key derivatives were considered. In the first the sulfonamide of AH-7614 was replaced with
an amide (TUG-1387) whilst in the second the xanthine was replaced with a thioxanthene
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(TUG-1506) (Figure 4A). These analogs were tested for their ability to antagonize EC80
concentrations of TUG-891 (Figure 4B) in a b-arrestin-2 recruitment assay. The
thioxanthene-containing TUG-1506 retained the ability to inhibit TUG-891 (pIC50 = 6.38 ±
0
.09, n=3) but with lower potency than the xanthene containing AH-7614. In contrast, the
amide-containing TUG-1387 lacked all ability to inhibit TUG-891 mediated activation of
FFA4.
As AH-7614, and likely also TUG-1506, appeared to be NAMs of FFA4 function we
characterized and quantified the nature and extent of their NAM properties against synthetic
FFA4 agonists from several distinct chemotypes. A common feature of GPCR allosteric
modulators is that they may show ‘probe dependence’, producing different allosteric effects
depending on the orthosteric agonist being modulated (May et al., 2007). Therefore, we
conducted experiments where AH-7614 (Figure 5A-D) and TUG-1506 (Figure 5E-H) were
used to antagonize b-arrestin-2 recruitment to FFA4 induced by synthetic agonists from each
of 4 distinct chemotypes: TUG-891 (Shimpukade et al., 2012), TUG-1197 (Azevedo et al.,
2
016), GSK137647A (Sparks et al., 2014), and Cpd A (Oh et al., 2014) (Figure 1). Data
from these experiments were then fit to an operational model of allosterism in order to
quantify the allosteric effect each NAM had on affinity of each orthosteric agonist (a), the
allosteric effect on efficacy of each orthosteric agonist (b), the affinity of the orthosteric
A B
agonists (K ) and the affinity of the modulator (K ) (Table 1). AH-7614 reduced the
maximal response to each agonist tested, and also produced a modest reduction in agonist
potency (Figure 5A-D). This corresponded to loga (Table 1, Figure 5I) and logb (Table 1,
Figure 5J) values that were both less than zero for each of the four agonists. However,
although the loga values were broadly similar for each agonist, the logb for GSK137647A
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was significantly lower (p<0.01) than the value obtained for either TUG-891 or Cpd A,
suggesting a subtle level of ‘probe dependence’ in AH-1764 modulation of FFA4 activity.
This can be noted in the concentration-response curves by the observation that although high
concentrations of AH-7614 eliminated measureable response to GSK137647A (Figure 5C), a
residual response was apparent for all other agonists, even at 30 µM AH-7614 (Figure 5A,
5
B and 5D). Importantly, the agonist responses were similar in the presence of between 3
and 30µM AH-7614, indicating that the inhibitory properties of AH-7614 were saturable, a
hallmark of allosteric modulators.
In examining the allosterism between TUG-1506 and the various FFA4 agonists
(
Figure 5E-H), much more striking ‘probe dependence’ was apparent. Most notably, while
TUG-1506 primarily shifted the potency of TUG-891 (Figure 5E) and Cpd A (Figure 5H)
with little effect on maximal response, for TUG-1197 and GSK137647A the opposite was
true, where TUG-1506 depressed the maximal response without effecting agonist potency.
This was reflected in the operational model curve fit parameters for these experiments, where
a large negative loga was observed for TUG-891 and Cpd A (Table 1, Figure 5K) but
instead a large negative logb was observed for TUG-1197 and GSK137647A (Table 1,
Figure 5L). This is a clear indication of the ‘probe dependence’ in the NAM properties of
TUG-1506. Interestingly, this may be related to the chemical nature of the agonists, with
TUG-891 and Cpd A both being carboxylate-based FFA4 agonists, while TUG-1197 and
GSK137647A are each sulfonamide based-agonists. Importantly, once again, in all cases the
inhibitory effects of TUG-1506 were saturable, entirely consistent with defining this
compound as a NAM at FFA4. Similar experiments were also conducted with TUG-1387
against each FFA4 agonist, although in this case, given that TUG-1387 had already been
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shown to lack activity at FFA4, only a single high concentration of TUG-1387 was used
Figure 6A- 6D). TUG-1387 did not produce a NAM effect on TUG-891, TUG-1197 or Cpd
(
A, and only a very modest effect on GSK137647A. Overall, these results are consistent
either with TUG-1387 having no affinity for FFA4, or it being a silent allosteric modulator,
that even if it does bind the receptor, does not affect orthosteric agonist function.
To further assess the potential for using AH-7614 in combination with TUG-1387 to define
biological functions of FFA4, we aimed to confirm the broader ability of AH-7614, but not
TUG-1387, to block additional FFA4 signalling pathways. We explored both FFA4-mediated
accumulation of inositol monophosphates (Figure 7A) and agonist-induced phosphorylation
3
47
350
of Thr and Ser residues within the C-terminal of FFA4- detected with previously
described phosopho-FFA4 antisera (Butcher et al., 2014, Prihandoko et al., 2016) (Figure
7
B). In each case, the agonist response was functionally inhibited by AH-7614, but was not
affected by TUG-1387. Importantly for studies on more physiologically relevant systems, we
also found that TUG-1387 lacked activity at the related fatty acid receptor FFA1 in
experiments measuring calcium release in 1321N1-hFFA1 cells (not shown).
Recently it has been suggested that FFA4 is able to act as a bi-potential regulator of
osteogenic and adipogenic differentiation of bone marrow-derived mesenchymal stem cells
(Gao et al., 2015). Furthermore, FFA4 has been proposed to be a key player in the activation
of brown adipose tissue (Quesada-Lopez et al., 2016), further reinforcing a role for FFA4 in
adipose tissue physiology. To assess further the role of FFA4 in adipogenic differentiation
and to demonstrate the utility of the FFA4 NAM, AH-7164, when used in combination with
TUG-1387 to interrogate FFA4 biology, we employed the murine mesenchymal stem cell
line C3H10T1/2 (Tang et al., 2004, Lee et al., 2016, Hashimoto et al., 2016). Here,
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differentiation towards an adipocyte phenotype was produced by maintaining the cells in the
presence of an induction mixture (IID) consisting of 100 nM insulin, 500 µM IBMX and 10
nM dexamethasone for 5 days. Oil Red O staining effectively visualized the development of
triglyceride deposits associated with adipogenesis (Figure 8A). In concert, exposure to the
IID-containing medium produced clear upregulation of mRNA for the adipogenic
development marker, peroxisome proliferator-activator receptor g (PPARg) (Figure 8B).
This effect was not produced simply by cell confluence on the tissue culture plate (Figure
8
B). In parallel, levels of Runt-related transcription factor 2 (Runx2) mRNA, a key
transcription factor associated with osteoblast differentiation (Xu et al., 2015, Yuan et al.,
016) was greatly reduced (Figure 8C). Over this time frame levels of mFFA4 mRNA were
2
also markedly increased by cell maintenance in the IID medium but, interestingly, an
equivalent upregulation of mFFA4 mRNA was also produced simply by maintaining
C3H10T1/2 cells at confluence for a 5 day period (Figure 8D). The ability of C3H10T1/2
cells to differentiate toward the adipocyte phenotype was clearly dependent upon signalling
from FFA4 because treatment of cells with AH-7614 along with the IID medium greatly
reduced visual observation of triglyceride stores as detected by Oil Red O staining and its
quantification (Figures 9A, 9B). Treatment with AH-7614 also limited IID-induced PPARg
mRNA induction (Figure 9C), without affecting the associated down-regulation of Runx2
expression (Figure 9D). In contrast, treatment of C3H10T1/2 cells with TUG-1387 during
the induction period with the IID-containing medium had no significant effect on the
development of Oil Red O staining (Figures 9A and 9B). Taken together, these data indicate
that TUG-1387, although structurally related, lacks activity at both FFA4 and the related
FFA1, and therefore indicates this compound will be a useful chemical control to confirm
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that biological effects produced by AH-7614 and related compounds such as TUG-1506 are
in fact FFA4 mediated.
Discussion
The availability of pharmacological tool compounds is central to efforts to define
functions of many cellular proteins, including GPCRs, and to assess such proteins as potential
therapeutic targets. However, for poorly- and recently-characterized receptors such tool
compounds are often lacking, described largely in the chemical/patent literature and,
therefore, often of limited availability, and/or are poorly characterized in terms of selectivity
and potential off-target effects. Within the group of GPCRs that respond to non-esterified, or
‘free’, fatty acids, to date the most broad-reaching pharmacopoeia targets the long-chain fatty
acid receptor FFA1 (Milligan et al., 2017). This reflects that FFA1 has been targeted
therapeutically and in human clinical trials to assess whether activation of this receptor might
provide an approach to treat type II diabetes (Watterson et al., 2014, Li et al., 2016,
Mohammad, 2016, Ghislain and Poitout , 2017). Although the second GPCR that is activated
by longer-chain free fatty acids, FFA4, is also frequently suggested as a potential therapeutic
target for diabetes and other metabolic disorders (Li et al., 2016, Moniri, 2016, Hansen and
Ulven, 2017), this lags behind studies on FFA1 and no synthetic ligands with activity at this
receptor have yet entered clinical studies (Milligan et al., 2017). As a result, even tool
compounds useful to assess roles of this receptor are less widely available or understood
(Hansen and Ulven, 2017). Moreover, as clear consensus is based on the therapeutic potential
of agonism of FFA4 (Li et al., 2016, Moniri, 2016), little effort has been expended to
identify, report or characterize antagonists of this receptor, despite antagonist tool compounds
being a traditional touchstone for detailed pharmacological analysis.
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The only inhibitor of FFA4 function reported to date is AH-7614 (Sparks et al., 2014).
Whilst Sparks et al., (2014) suggested that this compound would likely act as a competitive
antagonist of FFA4, the limited data presented in their report did not actually suggest such a
mode of action, which appeared to be non-competitive, at least in assays measuring
2
+
regulation of intracellular Ca levels. The studies reported here expand on this, defining AH-
7
614 as a NAM of FFA4 function. This conclusion is supported by several hallmarks of
allosterism (May et al., 2007), including that the ability of AH-7614 to inhibit FFA4 function
is saturable, and that AH-7614 displays some level of ‘probe dependence’. More striking,
however, in this regard is the chemical derivative of AH-7614, TUG-1506, described for the
first time in these studies ,which shows clear ‘probe dependence’ depending on the chemical
nature of the FFA4 agonist it is modulating. Specifically, this compound is a NAM primarily
of ligand affinity for the two carboxylate containing agonists tested, but primarily a NAM of
efficacy for the non-carboxylate, sulphonamide-based agonists. Such changes in allosteric
modulation are commonly observed with small chemical changes in allosteric ligands (Wood
et al., 2011, Hudson et al. 2014) and, indeed, the observed pattern of inhibition by both AH-
7
614 and TUG-1506 can only be explained by an allosteric mode of action.
Whilst Sparks et al., (2014) assessed more broadly the selectivity of the FFA4 agonist,
GSK137647A, described in their study, they did not extend this to AH-7614. Therefore,
having identified in the current work TUG-1387 as a close structural analog of AH-7614 that
lacks activity at FFA4, we used each of these ligands to define specific roles for FFA4.
Indeed, the FFA4 NAM AH-7614 prevents differentiation of a mouse mesenchymal stem cell
line toward an adipocyte phenotype, and does so in a manner that is consistent with blockade
of endogenous signalling via FFA4.
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We describe AH-7614 and TUG-1506 as the first allosteric ligands for the FFA4
receptor. Despite only a minor structural difference between these two compounds substantial
differences in the nature of their allosterism were observed, perhaps suggesting that further
examination of the structure activity relationship within the AH-7614 chemical series may
yield novel allosteric FFA4 ligands with diverse pharmacological properties. The fact that the
observed NAM properties within the AH-7614 series were clearly ‘probe dependent’
indicates that care must be taken when designing future studies using these ligands. Overall,
with appropriate caution, AH-7614, should prove to be a valuable tool in further unravelling
functions of the FFA4 receptor.
Authorship Contributions
Participated in Research Design Watterson, Hudson, Ulven, Yarwood, Milligan
Conducted Experiments: Watterson, Alvarez-Curto, Raihan, Hudson, Dunlop
Contributed New Reagents, Hansen, Azevedo, Martin, Ulven
Performed Data Analysis, Watterson, Alvarez-Curto, Hudson
Wrote the manuscript, Milligan, Watterson, Alvarez-Curto, Hudson
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Footnotes
These studies were supported by Biotechnology and Biological Sciences Research Council
[
grant BB/K019864/1)] and the Danish Council for Strategic Research [grant 11-116-196]
BDH, GM and TU are shareholders in Caldan Therapeutics.
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Figure Legends
Figure 1: Chemical structures of AH-7614, and range of FFA4 agonists
The chemical structures of the FFA4 antagonist AH-7614 and a range of FFA4 agonists
(
TUG-891, Cpd A, GSK137647A and TUG-1197) are shown.
Figure 2: AH-7614 is a potent and selective inhibitor of both human and murine FFA4
function
TM
TM
Flp-In T-REx 293 cells induced to express hFFA4-eYFP were pre-treated with either
vehicle (1% (v/v) DMSO) or increasing concentrations of AH-7614 for 15 mins after which
time they were treated with either 50µM aLA (A) or 500 nM TUG-891 (B). Both aLA and
TUG-891-induced calcium release that was potently inhibited in the presence of AH-7614
(aLA, pIC50 7.51 ± 0.08, n = 3, TUG-891, pIC50 8.13 ± 0.08, n = 3). HEK293T cells
transiently expressing hFFA4-eYFP and β-arrestin-2-Rluc were pre-incubated with either
vehicle (1% (v/v) DMSO) or increasing concentrations of AH-7614. The cells were then
treated with either 50 µM aLA (C) or 500 nM TUG-891 (D) and β-arrestin-2 recruitment was
subsequently determined using a BRET-based assay. Both aLA and TUG-891 promoted β-
arrestin-2 recruitment that was potently inhibited (aLA, pIC50 = 7.66 ± 0.05, n = 5, TUG-891,
pIC50 = 7.55 ± 0.07, n = 5) in the presence of increasing concentrations of AH-7614. mFFA4-
dependent β-arrestin-2 recruitment in the presence of either 50 µM aLA) (E) or 500 nM
TUG-891 (F) was also potently inhibited (aLA, pIC50 = 8.05 ± 0.08 n = 3, TUG-891, pIC50
=
7.93 ± 0.06, n = 3) by AH-7614. AH-7614 had no effect however on TUG-770 induced
calcium release in 1321N1 cells stably transfected with hFFA1 receptor, indicating that AH-
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