Complex pharmacology of novel allosteric free fatty acid 3 receptor ligands

Article abstract of DOI:10.1124/mol.114.093294

Analysis of the roles of the short chain fatty acid receptor, free fatty acid 3 receptor (FFA3), has been severely limited by the low potency of its endogenous ligands, the crossover of function of these on the closely related free fatty acid 2 receptor, and a dearth of FFA3-selective synthetic ligands. From a series of hexahydroquinolone-3-carboxamides, we demonstrate that 4-(furan-2-yl)-2-methyl-5-oxo-N-(o-tolyl)-1,4,5,6,7, 8-hexahydroquinoline-3- carboxamide is a selective and moderately potent positive allosteric modular (PAM)-agonist of the FFA3 receptor. Modest chemical variations within this series resulted in compounds completely lacking activity, acting as FFA3 PAMs, or appearing to act as FFA3-negative allosteric modulators. However, the pharmacology of this series was further complicated in that certain analogs displaying overall antagonism of FFA3 function actually appeared to generate their effects via a combined positive allosteric binding cooperativity and negative allosteric effect on orthosteric ligand maximal signaling response. These studies show that various PAM-agonist and allosteric modulators of FFA3 can be identified and characterized. However, within the current chemical series, considerable care must be taken to define the pharmacological characteristics of specific compounds before useful predictions of their activity and their use in defining specific roles of FFA3 in either in vitro and in vivo settings can be made. Copyright

Full text of DOI:10.1124/mol.114.093294

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2014/05/28/mol.114.093294.DC1.html
1521-0111/86/2/200–210$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.114.093294
Mol Pharmacol 86:200–210, August 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Complex Pharmacology of Novel Allosteric Free Fatty
Acid 3 Receptor Ligands s
Brian D. Hudson, Elisabeth Christiansen, Hannah Murdoch, Laura Jenkins,
Anders Højgaard Hansen, Ole Madsen, Trond Ulven, and Graeme Milligan
Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life
Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (B.D.H., H.M., L.J., G.M.); and Department of Physics,
Chemistry, and Pharmacy, University of Southern Denmark, Odense M, Denmark (E.C., A.H.H., O.M., T.U.)
Received April 16, 2014; accepted May 28, 2014
ABSTRACT
Analysis of the roles of the short chain fatty acid receptor, free modulators. However, the pharmacology of this series was
fatty acid 3 receptor (FFA3), has been severely limited by the further complicated in that certain analogs displaying overall
low potency of its endogenous ligands, the crossover of antagonism of FFA3 function actually appeared to generate their
function of these on the closely related free fatty acid 2 receptor, effects via a combined positive allosteric binding cooperativity
and a dearth of FFA3-selective synthetic ligands. From and negative allosteric effect on orthosteric ligand maximal
a series of hexahydroquinolone-3-carboxamides, we demon- signaling response. These studies show that various PAM-agonist
strate that 4-(furan-2-yl)-2-methyl-5-oxo-N-(o-tolyl)-1,4,5,6,7, and allosteric modulators of FFA3 can be identified and charac-
8-hexahydroquinoline-3-carboxamide is a selective and moder- terized. However, within the current chemical series, considerable
ately potent positive allosteric modular (PAM)-agonist of the care must be taken to define the pharmacological characteristics of
FFA3 receptor. Modest chemical variations within this series specific compounds before useful predictions of their activity and
resulted in compounds completely lacking activity, acting as their use in defining specific roles of FFA3 in either in vitro and in
FFA3 PAMs, or appearing to act as FFA3-negative allosteric vivo settings can be made.
been derived mainly from studies of receptor knockout lines of
mice (Maslowski et al., 2009; Sina et al., 2009; Zaibi et al.,
2010; Bjursell et al., 2011; Tolhurst et al., 2012; Bellahcene
Introduction
A pair of closely related G protein–coupled receptors rec-
ognize and are activated by short chain fatty acids (SCFAs)
et al., 2013; Kim et al., 2013; Kimura et al., 2013). The reliance
produced in the body predominantly through the fermenta-
on such mouse models to define the individual functions of
tion of poorly digestible carbohydrates by the gut microbiota
FFA2 and FFA3 reflects, at least in substantial part, that both
(Brown et al., 2003; Le Poul et al., 2003; Stoddart et al., 2008b;
receptors are activated by the same group of SCFAs (Brown
Cani et al., 2013). In recent times, these receptors, free fatty
et al., 2003; Stoddart et al., 2008a,b), that the expression
acid 2 receptor (FFA2) (previously designated GPR43) and
patterns of the two receptors can overlap (Nøhr er al., 2013),
free fatty acid 3 receptor (FFA3) (previously GPR41), have
and that synthetic ligands capable of selectively activating or
attracted considerable attention, not least because under-
inhibiting FFA2 and FFA3 have been limited in availability
standing of the role of the microbiota in the regulation of
and detailed characterization (Hudson et al., 2011). In recent
health has developed and deepened (Tan et al., 2014). Indeed,
times, this situation has improved somewhat for FFA2, with
broad appreciation of the role of the microbiota in areas
the description and use of both orthosteric agonists and
including metabolic health and the regulation of inflamma-
antagonists (Schmidt et al., 2011; Hudson et al., 2012a,
tory processes has encouraged detailed analysis of the SCFA
2013a) as well as a group of phenylacetamide-based ago-
receptors and resulted in suggestions that they might be novel
allosteric modulators (Lee et al., 2008; Wang et al., 2010;
and effective therapeutic targets (Ulven, 2012; Hara et al.,
Smith et al., 2011). These compounds have subsequently been
2013; Yonezawa et al., 2013; Tan et al., 2014). To date,
used to define the contribution of FFA2 in SCFA-mediated
understanding of the specific roles of FFA2 and FFA3 has
inhibition of lipolysis (Lee et al., 2008; Hudson et al., 2013a),
release of the incretin GLP-1 from enteroendocrine cells
This work was supported in part by grants from the Danish Council for
(Hudson et al., 2013a), and in neutrophil chemotaxis (Vinolo
et al., 2011). In contrast, only two reports to date have
examined the action of a FFA3-selective agonist, AR420626
[N-(2,5-dichlorophenyl)-4-(furan-2-yl)-2-methyl-5-oxo-1,4,5,6,7,
Strategic Research (to T.U. and G.M.) [11-116196] and a Canadian Institutes of
Health Research fellowship (to B.D.H.).
dx.doi.org/10.1124/mol.114.093294.
s
This article has supplemental material available at molpharm.aspetjournals.
org.
ABBREVIATIONS: eYFP, enhanced yellow fluorescent protein; FFA2, free fatty acid 2 receptor; FFA3, free fatty acid 3 receptor; hFFA, human FFA;
mFFA, mouse FFA; NAM, negative allosteric modulator; PAM, positive allosteric modulator; pERK, phosphorylation of extracellular signal-regulated
kinase; SAR, structure-activity relationship; SCFA, short chain fatty acid.
200

Complex Pharmacology of FFA3 Receptor Ligands
201
doxycycline (100 ng/ml21) to induce receptor expression. Cells were
then incubated in serum-free Dulbecco’s modified Eagle’s medium for
5–6 hours prior to the assay. Test compounds were added to the cells
and incubated for 5 minutes at 37°C before cells were lysed and
assayed for pERK1/2 using an Alphascreen-based detection kit
according to the manufacturer’s protocol (Perkin Elmer, Beaconsfield,
Buckinghamshire, UK).
cAMP Assay. cAMP experiments were performed using a homoge-
nous time-resolved fluorescence resonance energy transfer-based
detection kit (CisBio Bioassays, Codolet, France), according to the
manufacturer’s protocol. Cells already induced to express the desired
receptor were plated in low-volume 384-well plates, and the inhibition
of 5 mM forskolin-stimulated cAMP production was assessed following
a 30-minute coincubation with test compounds.
Data Analysis and Curve Fitting. All data presented represent
mean 6 S.E. of at least three independent experiments. Data analysis
and curve fitting were carried out using the GraphPad Prism soft-
ware package v5.0b. Concentration-response data were fit to three-
parameter sigmoidal concentration-response curves. Global curve
fitting of allosterism data was carried out using the following op-
erational model equation described previously (Keov et al., 2011;
Smith et al., 2011):
8-hexahydro-quinoline-3-carboxamide], indicating this compound
produces a modest but significant stimulation of GLP-1 release
from murine colonic crypt cultures (Nøhr et al., 2013) and
inhibits ghrelin secretion from murine gastric ghrelin cells
(Engelstoft et al., 2013). However, recent suggestions of a key
role for FFA3 in mediating the effects of the SCFA propionate
(C3) on allergic inflammation (Trompette et al., 2014) have
further heightened the need to identify and characterize FFA3-
selective compounds.
In this study, we describe the characterization of ligands
reported in the patent literature to have activity at FFA3
(Leonard et al., 2006), and of some analogs of these. A number
of moderately potent ligands were identified that either
activate this receptor as allosteric agonists, act as positive
allosteric modulators (PAMs) of the effect of SCFAs at the
receptor without detectable direct agonism, or inhibit the
effects of SCFAs in a noncompetitive manner as negative
allosteric modulators (NAMs). Although the detailed phar-
macology of this group of ligands is shown to be complex,
careful selection may provide useful tool compounds to further
define the role of FFA3 in both human and rodent cells and
tissues.
n
EmðtA½AꢀðKB 1ab½BꢀÞ1tB½BꢀKAÞ
E5
n
n
ð½AꢀKB 1KAKB 1½BꢀKA 1a½Aꢀ½BꢀÞ 1ðtA½AꢀðKB 1ab½BꢀÞ1tB½BꢀKAÞ
where E is the measured response, and A and B represent the
orthosteric and allosteric ligands, respectively. In this equation, Em is
the maximal system response, a is a measure of the allosteric
Materials and Methods
Materials and Compounds. Tissue culture reagents were from
Life Technologies (Paisley, UK). Compounds 1–8 were synthesized
as described in the supplemental information (Supplemental
Methods). The radiochemical [35S]GTPgS was from PerkinElmer
Life and Analytical Sciences (Beaconsfield, Buckinghamshire, UK).
All other experimental reagents were from Sigma-Aldrich (Poole,
UK).
Cell Culture and Transfection. All cells used in these experi-
ments were derived from Flp-In T-REx 293 cells designed to express
the desired receptor on demand following induction with the
antibiotic doxycycline. All cells used in these studies were previously
described and designed to express either human, mouse, or rat FFA3
(Hudson et al., 2012a); human (h)FFA2 (Stoddart et al., 2008a); or
mutated forms of hFFA3 (Stoddart et al., 2008a). In all cases, the
receptor construct expressed was fused in frame to enhanced yellow
cooperativity on ligand-binding affinity, and
b is an empirical
measure of the allosteric effect on efficacy. KA and KB are measures
of the binding affinities of the orthosteric and allosteric ligands,
respectively. The value n represents the slope factor of the trans-
duction function, whereas the abilities of the orthosteric and allosteric
ligands to directly activate the receptor are incorporated through the
values tA and tB. To fit experimental data to this equation, in all cases
the system maximum (Em) and slope (n) functions were constrained,
allowing for estimations of a, b, tA, tB, KA, and KB. To fit the data
presented in Figs. 7B, 8B, 9B, and 9C, the KA value for C3 was
constrained to the average value obtained in the experiments
presented in Fig. 4 (summarized in Table 1). Finally, it was also
required that the value for tB be constrained to a value of effectively
0 to fit data for allosteric modulators that did not produce any direct
agonism on their own (compounds 4 and 6).
fluorescent protein (eYFP) at its
C terminal. The cells were
maintained in Dulbecco’s modified Eagle’s medium without sodium
pyruvate, supplemented with 10% (v/v) fetal bovine serum and
penicillin/streptomycin mixture (Sigma-Aldrich) in
a humidified
Results
atmosphere containing 5% CO2. To induce receptor expression, cells
were incubated overnight with 100 ng/ml21 doxycycline.
Based on a patent disclosing synthetic ligands as regulators
of the FFA3 receptor, we initially synthesized the represen-
tative compound 1 (4-(furan-2-yl)-2-methyl-5-oxo-N-(o-tolyl)-
1,4,5,6,7,8-hexahydroquinoline-3-carboxamide) (Fig. 1). FFA3
is known to couple to the Gi family of heterotrimeric G
proteins (Brown et al., 2003; Le Poul et al., 2003; Stoddart
et al., 2008a,b). In [35S]GTPgS-binding assays performed on
membranes of Flp-In T-Rex 293 cells that had been induced to
express a C-terminally eYFP-tagged form of hFFA3, com-
pound 1 increased incorporation of this radionucleotide in
a concentration-dependent fashion with pEC50 of 5.65 6 0.07
(Fig. 2A). In this assay, 1 produced a maximal response
similar to the endogenous SCFA propionate (C3) but was
approximately 100-fold more potent (pEC50 for C3 5 3.47 6
0.09). These effects of both 1 and C3 reflected interactions
with hFFA3, as no significant response to either ligand was
observed using membranes derived from the cells that had not
been pretreated with doxycycline to induce hFFA3-eYFP
[
35S]GTPgS Incorporation Assay. Cell membrane preparations
were first generated as described previously (Stoddart et al., 2008a).
[
35S]GTPgS-binding experiments were then performed using a de-
scribed method (Smith et al., 2011). Briefly, cell membrane prepara-
tions were added to assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 100 mM NaCl, 1 mM EDTA, 1 mM GDP, and 0.1% fatty acid-
free bovine serum albumin) containing the appropriate concentra-
tions of ligand and allowed to reach equilibrium by preincubating for
15 minutes at 25°C. [35S]GTPgS was then added to initiate the assay,
before incubation for 1 hour at 25°C. The reaction was terminated by
filtration through GF/C glass-fiber filters, and unbound [35S]GTPgS
was washed from the filters with ice-cold wash buffer (50 mM Tris-
HCl, pH 7.4, and 10 mM MgCl2) before the remaining [35S]GTPgS was
quantified by liquid scintillation spectrometry.
Extracellular Signal-Regulated Kinase 1/2 Phosphorylation
Assay. Phosphorylation of extracellular signal-regulated kinase
(pERK)1/2 was assessed using a previously described protocol
(Hudson et al., 2012b). Briefly, 80,000 cells were seeded per well in
a 96-well plate, allowed to attached, before incubating overnight with

202
Hudson et al.
Fig. 1. Chemical structure of compounds used in this study.
expression (Fig. 2A). Furthermore, 1 was highly selective for from that occupied by the endogenous SCFA agonists. Such
hFFA3 over the closely related hFFA2 receptor because this allosteric agonists are often also found to act as allosteric
compound did not increase [35S]GTPgS incorporation in modulators, exhibiting binding cooperativity or altering
membranes of Flp-In TREx 293 cells induced to express the maximal response of orthosteric agonists (Smith et al.,
hFFA2-eYFP, although C3 did generate the expected re- 2011; Hudson et al., 2013b), and such compounds are of-
sponse (pEC50 5 4.18 6 0.16) (Fig. 2B). In addition to ten described as PAM-agonists. As measured in functional
stimulating [35S]GTPgS incorporation, compound 1 also assays, these allosteric effects will manifest in alterations
produced similar signaling responses to those of C3 in other in the measured potency or maximal signaling response to
endpoint measures of hFFA3 function in cells induced to the orthosteric ligand when the allosteric modulator is
express hFFA3-eYFP. These included inhibition of forskolin- present. Indeed, coaddition of concentrations of 1 ranging
stimulated cAMP production (Fig. 2C), with pEC50 values of from 100 nM to 10 mM resulted in observed increases in both
5.77 6 0.05 and 4.58 6 0.09 for 1 and C3, respectively, and the potency and maximal signaling response for C3 in the
pERK1/2 (Fig. 2D): pEC50 values of 5.27 6 0.16 for compound
[
35S]GTPgS-binding assay (Fig. 4A), suggesting that 1 is
1 and 3.68 6 0.13 for C3. As in the [35S]GTPgS incorporation a PAM-agonist of hFFA3. Reciprocal studies showed that
assay, in both cAMP and pERK1/2 assays 1 produced similar increasing concentrations of C3 (30 mM to 3 mM) also
maximal responses to that of C3.
enhanced the measured potency and maximal signaling
It has previously been established that the carboxylic acid response of 1 (Fig. 4B). Global analyses of the data were
functional group of C3 and other SCFAs are integral to their performed using an operational model of allosteric modula-
function at FFA3, forming key ionic interactions with arginine tion, as described previously (Keov et al., 2011; Smith et al.,
residues at positions 5.39 and 7.35 [numbering system of 2011). This led to estimations of allosteric effects on binding
Ballesteros and Weinstein (1995)] (Stoddart et al., 2008a). cooperativity (loga) and modulation of the maximal signal-
However, compound 1 does not contain a carboxylic acid ing response (logb) between C3 and 1; estimates of the ability
functional group, nor a negatively charged carboxylic acid of C3 and 1 to directly activate hFFA3 (logtA and logtB); as
bioisostere. We assessed, therefore, whether 1 functioned as well as estimates of the affinities of these two ligands for the
an orthosteric ligand for hFFA3. It did not: although mutation receptor (pKA and pKB) (Table 1). These analyses demon-
to Ala of either Arg5.39 or Arg7.35 completely eliminated strated reciprocal allosteric modulation between C3 and 1
response to C3 in the [35S]GTPgS-binding assay (Fig. 3A), at that is primarily attributed to a positive binding coopera-
both of these mutants the function of 1 was essentially tivity (a values of 20 and 16 for the reciprocal experiments),
unaltered (Fig. 3B). It is also important to note that both the although some positive modulation of the maximal response
R5.39A and R7.35A mutants of hFFA3-eYFP were expressed was also indicated (b values of 1.7 and 2.8 for the reciprocal
effectively from the inducible locus of stable Flp-In TREx 293 experiments). It is important to note that similar values
lines, yielding 137 6 5 and 179 6 4% of wild-type hFFA3- were obtained for a in each data set regardless of whether C3
eYFP levels, respectively, as measured by eYFP fluorescence or 1 was treated as modulator, as this value is expected to be
(Fig. 3C).
conserved due to the reciprocal nature of allosterism (Keov
The above results suggested that 1 most likely functioned et al., 2011). In contrast, the values for b need not be
as an allosteric agonist of hFFA3, binding to a site distinct conserved, in large part because this value is expected to

Complex Pharmacology of FFA3 Receptor Ligands
203
Fig. 2. Compound 1 is a selective activator of hFFA3. The concentration
response of 1 to stimulate [35S]GTPgS incorporation into membranes
from cells either induced, or not (2Dox) to express hFFA3-eYFP is
shown in (A). The effects of C3 and 1 in the [35S]GTPgS assay on
membranes from cells expressing hFFA2-eYFP are in (B). Concentration
responses for C3 and 1 showing inhibition of forskolin-stimulated cAMP
production (C) and pERK1/2 (D) were generated from cells induced to
express hFFA3-eYFP.
depend on the intrinsic efficacy of the modulator ligand, and
therefore is predicted to track with changes in the t value of
the modulator (Keov et al., 2011). However, in this case,
given that the tA and tB values were similar (average tA
5
2.3 and tB 5 1.8), it is not surprising that similar values were
obtained for b in the reciprocal experiments.
To extend these studies, comparable reciprocal allosterism
experiments were also performed in the cAMP (Fig. 4, C and
D) and pERK1/2 (Fig. 4, E and F) assays. In these experi-
ments, similar response patterns were observed, and global
fitting of these data indicated that again C3 and 1 exhibited
positive binding cooperativity and, to a lesser degree, en-
hancement of each other’s maximal responses (Table 1). In
Fig. 3. Compound 1 is an allosteric agonist of hFFA3. The capacity of C3
(A) and compound 1 (B) to promote binding of [35S]GTPgS to each of wild-
type, R5.39A, and R7.35A hFFA3-eYFP is shown. (C) As defined by levels
of eYFP fluorescence, both R5.39A and R7.35A hFFA3-eYFP were
expressed more highly than the wild-type receptor.
both cases, there were no statistical differences between the Combined ab values are often reported as a means to
a values in reciprocal experiments, as would be predicted. measure the overall cooperativity between orthosteric and
Interestingly, although the largest b value was observed for allosteric ligands, taking into account both the allosteric
the modulation of 1 on C3 function in the cAMP assay (b 5 effects on binding cooperativity and maximal response
5.6), this does appear to track with the observed t values as (Smith et al., 2011). Whereas analysis of these values with
the largest average t value was indeed observed for 1 in the C3 and 1 did show some differences between assays (Table 1),
cAMP assay (average tB 5 2.8). Similarly, the smallest again, given that statistically similar values were obtained for
average t value was obtained for C3 in the pERK1/2 assay a, and b values were found to track with modulator t value, the
(average t 5 1.6) and, as would be predicted from this, C3 assay differences observed in ab cannot be attributed to bias
modulation of 1 in this assay also yielded the lowest value for modulation.
b (1.4). A more complete analysis suggested that the average
In addition to estimates of the degree of allosterism and
t value obtained within an assay for the compound used as agonist activity of C3 and 1, global fitting of the allosterism
a modulator did correlate extremely well with the correspond- experiments also provided estimates of the affinities of
ing estimated b values in the same assay (Supplemental Fig. these two ligands (Table 1). As would be expected, these val-
1). Together, these results suggest that, although some ues were generally very similar across each assay and agonist/
differences are observed between ligands and across assays modulator combination, yielding an average pKA for C3 of
in the values for b, these differences can be attributed to 3.27 6 0.15 and an average pKB for 1 of 5.01 6 0.16. Given the
differences in the intrinsic efficacy of the ligands and do not difficulties in developing binding assays for the FFA class of
appear to represent bias in the allosteric modulation of FFA3. receptors (Hudson et al., 2011), such estimations may be

204
Hudson et al.
Fig. 4. Compound 1 is a PAM-agonist of hFFA3. Various fixed concentrations of 1 were added to concentration-response assays for C3 in each of
[
35S]GTPgS binding (A), cAMP inhibition (C), and pERK1/2 activation (E) studies. Reciprocal experiments in which various fixed concentration of C3 was
added to concentration-response curves for compound 1 are shown for [35S]GTPgS binding (B), cAMP inhibition (D), and pERK1/2 (F). See text and tables
for details of the curve fit analyses.
particularly useful in predicting the affinity of ligands for the the presence of increasing concentrations of C3 (Fig. 5D).
FFA3 receptor.
Global curve fit analysis of these data was performed, and the
We have previously noted marked differences in effective- resulting estimated parameters are shown in Table 2. Indeed,
ness of a number of synthetic FFA2 ligands between species through these reciprocal experiments, positive binding coop-
orthologs (Hudson et al., 2012a,b; Hudson et al., 2013a) and erativity was observed and with nearly identical values for a
next, therefore, assessed the activity of 1 at both rat and (Table 2). However, the values for a obtained were lower than
mouse (m) orthologs of FFA3 (mFFA3). In the [35S]GTPgS- those observed for hFFA3, suggesting the allosteric coopera-
binding assay, as anticipated from our previous work (Hudson tivity is not as strong at mFFA3 as it is at hFFA3. However, as
et al., 2012a), C3 was significantly more potent at both rat was the case with hFFA3, some positive allosteric modulation
FFA3 (pEC50 5 4.82 6 0.13) and mFFA3 (pEC50 5 5.25 6 of the maximum signaling response was also observed with
0.10) than at hFFA3 (3.47 6 0.09) (Fig. 5A). In contrast, 1 mFFA3, and again the magnitude of this correlated with the
displayed modestly reduced potency at the rodent orthologs observed t value for the modulator. Specifically, 1 displayed
(pEC50 5.27 6 0.08 for rat, and 5.20 6 0.07 for mouse) a lower average t value (average tB 5 1.3) than C3 (average
compared with its potency at the human receptor (5.65 6 0.07) tA 5 1.9) in the cAMP assay at mFFA3 and, as would be
(Fig. 5B). Given that 1 was found to be a PAM-agonist of predicted from this, also yielded a lower b value when used as
hFFA3, we next assessed whether this allosteric modulation the modulator (b 5 2.1) than did C3 (b 5 4.2). It was also
of C3 by 1 was also observed at mFFA3. For this we generated interesting to note that the average affinities between these
concentration-response curves to C3 in the presence of reciprocal experiments at mFFA3 suggest a pKA of 4.41 for C3
increasing concentrations of 1 (Fig. 5C), as well as reciprocal and pKB of 4.74 for 1. Comparison of these values with those
experiments generating concentration-response curves to 1 in obtained for hFFA3 (Fig. 4; Table 1) demonstrated that,
TABLE 1
Operational model analysis of C3 and compound 1 at hFFA3
[
35S]GTPgS
cAMP
pERK1/2
Agonista
C3
1
1
C3
C3
1
1
C3
C3
1
1
C3
Modulatorb
loga
logb
1.30 6 0.16
0.23 6 0.07
0.30 6 0.05
0.06 6 0.02
3.16 6 0.11
5.86 6 0.07
34
1.21 6 0.13
0.44 6 0.04
0.42 6 0.05
0.40 6 0.05
3.18 6 0.09
4.92 6 0.08
44
1.11 6 0.31
0.75 6 0.12
0.05 6 0.05
0.45 6 0.16
3.96 6 0.16
5.15 6 0.17
72
1.70 6 0.27
0.28 6 0.09
0.34 6 0.09
0.44 6 0.10
3.41 6 0.18
4.85 6 0.18
95
1.34 6 0.16
0.31 6 0.06
0.31 6 0.04
0.10 6 0.05
2.96 6 0.08
4.92 6 0.10
44
1.40 6 0.19
0.16 6 0.07
0.03 6 0.07
0.33 6 0.05
2.97 6 0.09
4.84 6 0.13
50
c
logtAd
logtB
c
pKAd
pKB
ab
aAgonist is the compound used to generate concentration-response information.
bModulator is the compound used in fixed concentrations.
ctA and pKA are values estimated for C3.
dtB and pKB are values estimated for 1.

Complex Pharmacology of FFA3 Receptor Ligands
205
Fig. 5. Compound 1 displays similar function at human and rodent orthologs of FFA3. The orthosteric agonist C3 is more potent at rat (r) and mouse (m)
orthologs of FFA3 than at the human receptor (A). By contrast, compound 1 displays similar potency at these species orthologs (B). The effects of adding
various fixed concentrations of 1 to the concentration response of C3 were assessed in the cAMP assay (C). Reciprocal experiments using fixed
concentrations of C3 are also shown (D).
although 1 does show slightly reduced affinity for mFFA3 ring and is reported to have pEC50 values of 6.57 and 6.92 in
compared with hFFA3, C3 appears to have significantly inositol phosphate accumulation and cAMP assays, respectively
higher affinity for the mouse ortholog than it has for the (Engelstoft et al., 2013; Nøhr et al., 2013). We found 8 to be a full
human. Both of these assessments are in good agreement with agonist with potency resembling 1 (pEC50 5 5.74 6 0.11) in the
the relative potency of these compounds in functional assays [35S]GTPgS-binding assay (Fig. 6C).
at mFFA3 compared with hFFA3 (Fig. 5, A and B).
Analysis of the analogs tested failed to identify any
The Arena Pharmaceuticals patent (Leonard et al., 2006) compounds with significantly improved potency compared
lists 14 ligands designated as either agonists or antagonists of with 1. Indeed, across the series there were only relatively
FFA3. To explore the structure-activity relationship (SAR) modest differences in potency among compounds that dis-
and extend the pharmacology of compound 1, we next played agonism (Table 3). There were, however, compounds
explored the functions of an additional reported agonist 2 producing varying maximal responses, including two that
and an antagonist 3 (see Fig. 1 for compound structures) at appeared to be superagonists (2, 7), and another that was
hFFA3-eYFP in the [35S]GTPgS-binding assay (Fig. 6A; a partial agonist of hFFA3 (5). Considering this partial
Table 3). The 3-furyl analog 2 showed increased maximal agonist, we next assessed whether 5 also allosterically
signaling response but reduced potency, whereas 3, carrying modulated C3 function at hFFA3, as this might suggest the
a 4-phenoxyphenyl in the place of the furyl, did not exhibit compound series could also be used to identify pure allosteric
significant activity on its own, in agreement with its modulators of FFA3 that do not possess intrinsic efficacy. For
purported antagonist activity. To further examine the SAR, this, we first confirmed the function of 5 in the pERK1/2
2-bromophenyl (4), 3-biphenyl (5), 3-phenoxyphenyl (6), and assay, in which, as in the [35S]GTPgS assay, it acted as a weak
4-biphenyl (7) analogs were assessed (Fig. 6B; Table 3). Among partial agonist (pEC50 5 5.08 6 0.40; EMax 5 24 6 8%) (Fig.
this series, whereas the 4-biphenyl substituted 7 retained full 7A). Interestingly, 5 was also a strong PAM of C3 function in
agonist activity, although with lower potency, the 3-biphenyl this assay. However, unlike compound 1 that primarily
compound 5 showed reduced maximal signaling response, and increased the potency of C3 (Fig. 4E), 5 significantly increased
the two phenoxyphenyl compounds (3, 6) and the 2-bromophenyl the maximal response of C3 with little apparent effect on
4 were essentially inactive. We also synthesized AR420626 (8), potency (Fig. 7B). Global curve fitting of these data confirmed
which is identical to 1 apart from a 2,5-dichlorophenyl terminal this observation, indicating a loga value of only 0.11 6 0.29,
TABLE 2
Operational model analysis of C3 and compound 1 at mFFA3
c
d
c
d
Agoa
Modb
loga
logb
tA
tB
pKA
pKB
C3
1
1
C3
0.64 6 0.36 0.32 6 0.14 0.31 6 0.05
0.29 6 0.16 4.40 6 0.11 4.96 6 024
0.65 6 0.27 0.62 6 0.14 0.24 6 0.06 20.18 6 0.11 4.42 6 0.21 4.51 6 0.10
aAgo is the compound used to generate concentration-response information.
bMod is the compound used in fixed concentrations.
ctA and pKA are values estimated for C3.
dtB and pKB are values estimated for 1.

206
Hudson et al.
TABLE 3
Potency and efficacy of chemical variants of 4-(furan-2-yl)-5-oxo-N-
(o-tolyl)-5,6,7,8-tetrahydroquinoline-3-carboxamide (compound 1)
hFFA3
a
Compound
hFFA3 pEC50
Efficacyb
C3
3.47 6 0.09
5.65 6 0.07
5.24 6 0.08
,4
100%
101 6 4%
135 6 8%
1
2
3
4
NR
5
5.70 6 0.43
,4
4.74 6 0.20
5.74 6 0.11
42 6 8%
6
7
132 6 4%
120 6 4%
8 (AR420626)
NR, no response.
apEC50 values using hFFA3-eYFP in a [35S]GTPgS-binding assay.
bPercentage of C3 response using hFFA3-eYFP in a [35S]GTPgS-binding assay.
compounds were most likely NAMs of hFFA3. By contrast, 4
enhanced the potency of the C3 response without altering
maximal response and, therefore, appeared to act as a PAM of
C3. To explore this in further detail, increasing concentra-
tions of 4 were used in combination with C3 concentration-
response curves (Fig. 8B). Global fitting of these data to the
operational model of allosterism indicated that compound 4
has a pKB of 5.43 6 0.17. Furthermore, these analyses
confirmed
a modest but positive binding cooperativity
between C3 and 4 (loga 5 0.79 6 0.10), with effectively no
modulation of the maximal C3 signaling response (logb 5 0.08 6
0.05). Although this represents only modest cooperativity
between compound 4 and C3, it does suggest that this com-
pound may well be a promising lead to develop further and
more effective PAMs of hFFA3.
Next, we explored further details of the two compounds that
in the initial studies appeared to be FFA3 NAMs, 3 and 6. We
generated C3 concentration-response curves in the presence
of increasing fixed concentrations of either 3 (Fig. 9A) or 6
(Fig. 9B) in the [35S]GTPgS-binding assay. In each case, there
was a clear concentration-dependent decrease in the C3
maximal response (but not potency), suggesting that com-
pounds 3 and 6 are indeed NAMs of C3. Global curve fitting of
the data for compound 6 yielded a pKB for this compound of
5.46 6 0.38. Interestingly, the a and b values generated from
these analyses suggested that, whereas a negative effect on
the signaling response was observed (logb 5 21.92 6 0.20),
a positive binding cooperativity (loga 5 1.20 6 0.39) was also
observed between C3 and compound 6. As this was a some-
what surprising result, we also examined the allosteric effects
of 6 on C3 in the pERK1/2 assay, which yielded a similar
pattern of responses to C3 in the presence of compound 6 (Fig.
9C). Once more, whereas the most striking observation was
a clear inhibition of C3 maximal response by 6, the global
curve fit analyses demonstrated that this resulted from
a combination of a negative allosteric b (logb 5 21.88 6
0.25) and a positive allosteric a (loga 5 0.98 6 0.07). As
a means to depict the apparently divergent effects on C3
maximal signaling response and potency produced by com-
pound 6 in the pERK1/2 assay, measures of these two
parameters (EMax and pEC50) obtained from individual
three-parameter concentration-response curve fits to C3 in
the presence of increasing concentrations of 6 were plotted
(Fig. 9D). This clearly demonstrated opposing effects on C3
maximal response and potency by this compound, and that
both effects were produced with near-identical pEC50 values
Fig. 6. Analogs of compound 1 display diverse activity and maximal
signaling response. The concentration-response curves as direct agonists
at hFFA3 in [35S]GTPgS-binding studies of various ligands are shown in
(A–C). Response curves to C3 (A and B) or 1 (C) are shown for comparison
as dashed lines.
but a logb of 1.33 6 0.21 for the allosteric effect of 5 on C3,
suggesting that 5 has little positive binding cooperativity with
C3, but does positively enhance the C3 signaling response in
this assay. These analyses also were used to estimate the
affinity of 5, indicating a pKB value of 3.95 6 0.18.
The strong allosteric modulation of C3 by 5, despite this
compound displaying limited agonist activity, suggested that
compounds appearing to be inactive when tested as agonists
might, in fact, be allosteric modulators if they still bind to the
allosteric site of hFFA3. To explore this, we first examined the
effect of a single high concentration (100 mM) of the reported
antagonist 3 as well as the two additional inactive com-
pounds, 4 and 6, on the concentration response to C3 in the
[
35S]GTPgS assay (Fig. 8A). In these experiments, 3 largely
eliminated response to C3, as did 6, suggesting that the

Complex Pharmacology of FFA3 Receptor Ligands
207
Fig. 8. Analogs of compound 1 display diverse pharmacology, with
compound 4 acting as a hFFA3 PAM. In (A), the effects of single
concentrations of compounds 4, 3, and 6 (100 mM) as allosteric modulators
of C3 function at hFFA3-eYFP in the [35S]GTPgS assay are shown.
Testing the effects of additional concentrations of 4 in this assay (B)
indicates that, although lacking direct agonist activity, 4 is a positive
allosteric modulator of the potency of C3.
Fig. 7. The weak partial agonist compound 5 acts as a PAM of C3
activity. The effect of compound 5 as a direct agonist in the pERK1/2
assay was compared with C3 (A). Although a weak partial agonist, 5
acted as a positive allosteric modulator of the efficacy and potency of
C3 (B).
(5.77 6 0.10 and 5.75 6 0.24, respectively). As would be
predicted, these are similar to the pKB value obtained from
the global curve fit analysis of the [35S]GTPgS data with this
compound (Fig. 9B). Together, these findings indicate that,
although 6 may have initially appeared to be a hFFA3 NAM,
this compound may be better described as a PAM-antagonist,
showing positive binding cooperativity with the orthosteric
ligand, while also negatively modulating orthosteric ligand-
signaling responses.
Finally, as these represent the first compounds described
that could be used as functional antagonists of FFA3, we also
wished to establish whether the negative allosteric effect on
C3 signaling of compound 6 was also observed at the mouse
ortholog of FFA3. To do so, concentration-response curves
were generated to C3 in the pERK1/2 assay in the absence or
presence of a 100 mM concentration of 6 (Fig. 9E). The
presence of 6 at this concentration substantially reduced C3
efficacy to 23 6 7% of the control, suggesting that this
compound may be useful as a functional FFA3 antagonist in
murine systems as well as human.
demonstrated in this respect in relation to both metabolism
(Kimura et al., 2013) and inflammation (Trompette et al.,
2014), and this has stimulated interest in the receptors as
novel therapeutic targets (Ulven, 2012; Dranse et al., 2013;
Yonezawa et al., 2013). However, clearly defining the specific
roles of FFA2 versus FFA3, and importantly, which would
ultimately be a more effective therapeutic target in distinct
pathologic conditions, has been challenging in the absence of
selective pharmacological tool compounds. This has been
particularly problematic for FFA3, for which, until recently,
only a single publically available patent describes a series
of FFA3-selective, but poorly characterized, compounds (Leonard
et al., 2006). Recently, a representative compound from this
series, designated AR420626, was used to demonstrate a con-
tribution of FFA3 to SCFA-mediated GLP-1 (Nøhr er al., 2013)
and ghrelin (Engelstoft et al., 2013) secretion. However, the
detailed pharmacology of this compound series had remained
largely unknown. In the present study, we have begun to ex-
plore this by demonstrating that compounds from within this
series act as selective ligands for FFA3 but with varied
pharmacological properties.
Initially, we synthesized and studied 4-(furan-2-yl)-2-
methyl-5-oxo-N-(o-tolyl)-1,4,5,6,7,8-hexahydroquinoline-3-
Discussion
The SCFA receptors FFA2 and FFA3 have generated carboxamide (compound 1) as an exemplar. Although able to
increasing interest in recent years, particularly for their roles selectively regulate a series of signaling endpoints via hFFA3
linking the microfloral composition of the gut to health (Tan with similar maximal responses to the endogenous SCFA C3,
et al., 2014). Involvement of these receptors has now been the structure of 1 suggested it was unlikely to be an orthosteric

208
Hudson et al.
Fig. 9. Compounds 3 and 6 are PAM-antagonists of the
function of C3 at FFA3. Fixed concentrations of compounds
3 (A) and 6 (B, C) were added to concentration-response
curves of propionate (C3) in either [35S]GTPgS-binding (A
and B) or pERK1/2 (C) assays. In (D), effects of 6 on both the
potency (right y-axis) and measured maximal response (left
y-axis) of C3 in pERK1/2 assays are plotted separately to
highlight the separate and distinct allosteric effects of 6 on
the potency and maximal response of C3. Concentration-
response curves to C3 at mouse FFA3-eYFP in the absence
and presence of 100 mM 6 are shown in (E).
agonist. Indeed, this was confirmed, as 1 still acted as an the metabotropic glutamate receptors, in which these chem-
effective agonist at forms of hFFA3 in which either of the two ical modifications have been described as molecular switches
arginine residues known to be key in coordination of the capable of altering the function of the allosteric ligands (Wood
carboxylate function of SCFAs (Stoddart et al., 2008b) was et al., 2011). The SAR of the FFA3 ligand series suggests that
mutated. Although 1 was entirely selective for FFA3 over such switches may also be present among these compounds.
FFA2, this compound displayed only modest potency for FFA3, In particular, the presence of either a 2-bromophenyl (4) or
particularly when compared with what had been described a 3- or 4-phenoxyphenyl (3, 6) substituent on the hexahy-
recently for the similar compound AR420626 (Engelstoft et al., droquinolone scaffold appears to eliminate intrinsic agonism
2013; Nøhr er al., 2013). To establish whether this was related of the ligand. In the case of the phenoxyphenyl variants, this
to intrinsic differences in potency between these two com- also appears to result in the gain of a negative modulatory
pounds, or instead due to the use of distinct assay systems, we effect on endogenous agonist maximal signaling response.
synthesized AR420626 (compound 8) and found it to have Although such molecular switches result in a series of
similar potency to 1 in the [35S]GTPgS assay. This suggests compounds with diverse and interesting pharmacology, they
that, despite lower measured potency in this assay, 1 is likely to clearly also complicate ligand optimization and, potentially,
be an equally useful selective FFA3 ligand as AR420626.
present a significant problem in selecting compounds that
By extending the SAR of compound 1, we also identified might be used in vivo (Wood et al., 2011).
a number of compounds with activity at FFA3 but with
Another consideration that may complicate the use of these
diverse pharmacological properties. Interestingly, relatively compounds is that the two compounds identified with
minor chemical modifications had substantial impact on antagonistic properties, 3 and 6, displayed further complexity
ligand function, switching compounds from PAM-agonists, in their pharmacology. Specifically, whereas they did nega-
to PAMs lacking intrinsic agonism, or to PAM-antagonists tively modulate C3 maximal signaling response, at the
having divergent effects on orthosteric ligand potency and same time they exhibited positive binding cooperativity with
maximal signaling response. Similar observations have been C3. Similar pharmacology has been observed for allosteric
reported previously, particularly for allosteric modulators of modulators of other G protein–coupled receptors, with certain

Complex Pharmacology of FFA3 Receptor Ligands
209
receptor allosteric modulators display both agonist and signaling pathway speci-
ficity. Mol Pharmacol 83:322–338.
Ballesteros JA, and Weinstein H (1995) Integrated methods for modeling G-protein
coupled receptors. Methods Neurosci 25:366–428.
Bellahcene M, O’Dowd JF, Wargent ET, Zaibi MS, Hislop DC, Ngala RA, Smith DM,
Cawthorne MA, Stocker CJ, and Arch JR (2013) Male mice that lack the G-protein-
coupled receptor GPR41 have low energy expenditure and increased body fat
content. Br J Nutr 109:1755–1764.
modulators of the CB1 cannabinoid receptor being par-
ticularly notable examples (Price et al., 2005; Baillie et al.,
2013). Indeed, this class of compound might be termed
PAM-antagonists and possess the particularly interesting
property that they should increase the potency of their
antagonism with increasing amounts of orthosteric agonist
present. Whereas this complex pharmacology may complicate
the use of these compounds as tools, as long as they are used
in sufficient concentrations, and under carefully planned
experimental conditions, they should still act effectively
as functional antagonists that will help define further the role
of FFA3.
Bergman EN (1990) Energy contributions of volatile fatty acids from the gastroin-
testinal tract in various species. Physiol Rev 70:567–590.
Bjursell M, Admyre T, Göransson M, Marley AE, Smith DM, Oscarsson J,
and Bohlooly-Y M (2011) Improved glucose control and reduced body fat mass in
free fatty acid receptor 2-deficient mice fed a high-fat diet. Am J Physiol Endocrinol
Metab 300:E211–E220.
Brown AJ, Goldsworthy SM, Barnes AA, Eilert MM, Tcheang L, Daniels D, Muir AI,
Wigglesworth MJ, Kinghorn I, and Fraser NJ et al. (2003) The Orphan G protein-
coupled receptors GPR41 and GPR43 are activated by propionate and other short
chain carboxylic acids. J Biol Chem 278:11312–11319.
A further key set of observations within these studies is
that compounds including 1 and 6 are effective regulators of
the mouse ortholog of FFA3 as well as the human receptor.
This is important given that allosteric ligand binding sites
have been predicted to be under less evolutionary pressure
than orthosteric sites and, therefore, less likely to be con-
served across species (Hudson et al., 2013b). Interestingly, the
opposite may be true among the SCFA receptors, in which
significant species differences have been described for both
FFA2 and FFA3 orthosteric ligands (Hudson et al., 2012a,b,
2013a), whereas the function of the allosteric ligands de-
scribed in this study appears more similar across species. This
may result from the nature of the SCFA receptors as
nutritional sensors (Dranse et al., 2013; Milligan et al., 2014)
that respond to ligands derived from fiber fermented by the
gut microbiota. Specifically, as different species rely on
markedly different amounts of fiber in their diet, they are
exposed to significantly different concentrations of SCFAs
(Bergman, 1990). It might then be predicted that the affinity
of these receptors for the endogenous SCFA ligands will also
differ significantly between species (Hudson et al., 2012a).
Indeed, as noted in this study, C3 is substantially more potent
at mouse and rat FFA3 than at the human ortholog, and we
have previously reported on significant variation in SCFA
potency at human, rodent, and ruminant orthologs of FFA2
(Hudson et al., 2012a,b).
Cani PD, Everard A, and Duparc T (2013) Gut microbiota, enteroendocrine functions
and metabolism. Curr Opin Pharmacol 13:935–940.
Dranse HJ, Kelly ME, and Hudson BD (2013) Drugs or diet?–Developing novel
therapeutic strategies targeting the free fatty acid family of GPCRs. Br J Phar-
macol 170:696–711.
Engelstoft MS, Park WM, Sakata I, Kristensen LV, Husted AS, Osborne-Lawrence S,
Piper PK, Walker AK, Pedersen MH, and Nøhr MK et al. (2013) Seven trans-
membrane G protein-coupled receptor repertoire of gastric ghrelin cells. Mol Metab
2:376–392.
Hara T, Kimura I, Inoue D, Ichimura A, and Hirasawa A (2013) Free fatty acid
receptors and their role in regulation of energy metabolism. Rev Physiol Biochem
Pharmacol 164:77–116.
Hudson BD, Smith NJ, and Milligan G (2011) Experimental challenges to targeting
poorly characterized GPCRs: uncovering the therapeutic potential for free fatty
acid receptors. Adv Pharmacol 62:175–218.
Hudson BD, Christiansen E, Tikhonova IG, Grundmann M, Kostenis E, Adams DR,
Ulven T, and Milligan G (2012a) Chemically engineering ligand selectivity at the
free fatty acid receptor 2 based on pharmacological variation between species
orthologs. FASEB J 26:4951–4965.
Hudson BD, Tikhonova IG, Pandey SK, Ulven T, and Milligan G (2012b) Extracel-
lular 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 287:41195–41209.
Hudson BD, Due-Hansen ME, Christiansen E, Hansen AM, Mackenzie AE, Murdoch
H, Pandey SK, Ward RJ, Marquez R, and Tikhonova IG et al. (2013a) Defining the
molecular basis for the first potent and selective orthosteric agonists of the FFA2
free fatty acid receptor. J Biol Chem 288:17296–17312.
Hudson BD, Ulven T, and Milligan G (2013b) The therapeutic potential of allosteric
ligands for free fatty acid sensitive GPCRs. Curr Top Med Chem 13:14–25.
Keov P, Sexton PM, and Christopoulos A (2011) Allosteric modulation of G protein-
coupled receptors: a pharmacological perspective. Neuropharmacology 60:24–35.
Kim MH, Kang SG, Park JH, Yanagisawa M, and Kim CH (2013) Short-chain fatty
acids activate GPR41 and GPR43 on intestinal epithelial cells to promote in-
flammatory responses in mice. Gastroenterology 145:396–406.
Kimura I, Ozawa K, Inoue D, Imamura T, Kimura K, Maeda T, Terasawa K,
Kashihara D, Hirano K, and Tani T et al. (2013) The gut microbiota suppresses
insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43.
Nat Commun 4:1829.
Le Poul E, Loison C, Struyf S, Springael JY, Lannoy V, Decobecq ME, Brezillon S,
Dupriez V, Vassart G, and Van Damme J et al. (2003) Functional characterization
of human receptors for short chain fatty acids and their role in polymorphonuclear
cell activation. J Biol Chem 278:25481–25489.
Lee T, Schwandner R, Swaminath G, Weiszmann J, Cardozo M, Greenberg J, Jaeckel
P, Ge H, Wang Y, and Jiao X et al. (2008) Identification and functional charac-
terization of allosteric agonists for the G protein-coupled receptor FFA2. Mol
Pharmacol 74:1599–1609.
Leonard JN, Chu ZL, Bruce MA, and Boatman PD (2006) GPR41 and modulators
thereof for the treatment of insulin-related disorders. PCT Int Appl WO
2006052566.
Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, Schilter HC, Rolph MS,
Mackay F, and Artis D et al. (2009) Regulation of inflammatory responses by gut
microbiota and chemoattractant receptor GPR43. Nature 461:1282–1286.
Milligan G, Ulven T, Murdoch H, and Hudson BD (2014) G-protein-coupled receptors
for free fatty acids: nutritional and therapeutic targets. Br J Nutr 111 (Suppl 1):
S3–S7.
Despite some potential challenges, as described above,
members of this series of FFA3 allosteric ligands represent
the best currently available options to selectively target this
receptor pharmacologically. The diverse and complex phar-
macology observed, even within the relatively small number
of analogs explored in detail to date, suggests that further
development of this series may yet provide allosteric
agonists, PAM-agonists, PAMs, NAMs, or PAM-antagonists
for the FFA3 receptor with even more useful pharmacolog-
ical properties.
Authorship Contributions
Nøhr MK, Pedersen MH, Gille A, Egerod KL, Engelstoft MS, Husted AS, Sichlau RM,
Grunddal KV, Seier Poulsen S, and Han S et al. (2013) GPR41/FFAR3 and GPR43/
FFAR2 as cosensors for short-chain fatty acids in enteroendocrine cells vs
FFAR3 in enteric neurons and FFAR2 in enteric leukocytes. Endocrinology 154:
3552–3564.
Price MR, Baillie GL, Thomas A, Stevenson LA, Easson M, Goodwin R, McLean A,
McIntosh L, Goodwin G, and Walker G et al. (2005) Allosteric modulation of the
cannabinoid CB1 receptor. Mol Pharmacol 68:1484–1495.
Schmidt J, Smith NJ, Christiansen E, Tikhonova IG, Grundmann M, Hudson BD,
Ward RJ, Drewke C, Milligan G, and Kostenis E et al. (2011) Selective orthosteric
free fatty acid receptor 2 (FFA2) agonists: identification of the structural and
chemical requirements for selective activation of FFA2 versus FFA3. J Biol Chem
286:10628–10640.
Participated in research design: Hudson, Murdoch, Ulven,
Milligan.
Conducted experiments: Hudson, Murdoch, Jenkins.
Contributed new reagents or analytic tools: Madsen, Hansen,
Christiansen, Ulven.
Performed data analysis: Hudson, Murdoch, Jenkins, Milligan,
Ulven.
Wrote or contributed to the writing of the manuscript: Hudson,
Milligan, Ulven.
Sina C, Gavrilova O, Förster M, Till A, Derer S, Hildebrand F, Raabe B, Chalaris A,
Scheller J, and Rehmann A et al. (2009) G protein-coupled receptor 43 is essential
for neutrophil recruitment during intestinal inflammation. J Immunol 183:
7514–7522.
References
Baillie GL, Horswill JG, Anavi-Goffer S, Reggio PH, Bolognini D, Abood ME,
McAllister S, Strange PG, Stephens GJ, and Pertwee RG et al. (2013) CB(1)

210
Hudson et al.
Smith NJ, Ward RJ, Stoddart LA, Hudson BD, Kostenis E, Ulven T, Morris JC,
Tränkle C, Tikhonova IG, and Adams DR et al. (2011) Extracellular loop 2 of the
free fatty acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric
modulator. Mol Pharmacol 80:163–173.
Stoddart LA, Smith NJ, Jenkins L, Brown AJ, and Milligan G (2008a) Conserved
polar residues in transmembrane domains V, VI, and VII of free fatty acid receptor
2 and free fatty acid receptor 3 are required for the binding and function of short
chain fatty acids. J Biol Chem 283:32913–32924.
Stoddart LA, Smith NJ, and Milligan G (2008b) International Union of Pharmacol-
ogy. LXXI. Free fatty acid receptors FFA1, -2, and -3: pharmacology and patho-
physiological functions. Pharmacol Rev 60:405–417.
Tan J, McKenzie C, Potamitis M, Thorburn AN, Mackay CR, and Macia L (2014) The
role of short-chain fatty acids in health and disease. Adv Immunol 121:91–119.
Tolhurst G, Heffron H, Lam YS, Parker HE, Habib AM, Diakogiannaki E, Cameron J,
Grosse J, Reimann F, and Gribble FM (2012) Short-chain fatty acids stimulate
glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2. Di-
abetes 61:364–371.
Vinolo MA, Ferguson GJ, Kulkarni S, Damoulakis G, Anderson K, Bohlooly-Y M,
Stephens L, Hawkins PT, and Curi R (2011) SCFAs induce mouse neutrophil
chemotaxis through the GPR43 receptor. PLoS One 6:e21205.
Wang Y, Jiao X, Kayser F, Liu J, Wang Z, Wanska M, Greenberg J, Weiszmann J, Ge
H, and Tian H et al. (2010) The first synthetic agonists of FFA2: discovery and SAR
of phenylacetamides as allosteric modulators. Bioorg Med Chem Lett 20:493–498.
Wood MR, Hopkins CR, Brogan JT, Conn PJ, and Lindsley CW (2011) “Molecular
switches” on mGluR allosteric ligands that modulate modes of pharmacology.
Biochemistry 50:2403–2410.
Yonezawa T, Kurata R, Yoshida K, Murayama MA, Cui X, and Hasegawa A (2013)
Free fatty acids-sensing G protein-coupled receptors in drug targeting and thera-
peutics. Curr Med Chem 20:3855–3871.
Zaibi MS, Stocker CJ, O’Dowd J, Davies A, Bellahcene M, Cawthorne MA, Brown AJ,
Smith DM, and Arch JR (2010) Roles of GPR41 and GPR43 in leptin secretory
responses of murine adipocytes to short chain fatty acids. FEBS Lett 584:
2381–2386.
Trompette A, Gollwitzer ES, Yadava K, Sichelstiel AK, Sprenger N, Ngom-Bru C, Blanchard
C, Junt T, Nicod LP, and Harris NL et al. (2014) Gut microbiota metabolism of dietary
fiber influences allergic airway disease and hematopoiesis. Nat Med 20:159–166.
Ulven T (2012) Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41
as new potential therapeutic targets. Front Endocrinol 3:111.
Address correspondence to: Dr. Graeme Milligan, Wolfson Link Building
253, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
E-mail: Graeme.Milligan@glasgow.ac.uk