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. 2017 Jun;91(6):630-641.
doi: 10.1124/mol.116.107821. Epub 2017 Apr 6.

Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4

Kenneth R Watterson  1 , Steffen V F Hansen  1 , Brian D Hudson  1 , Elisa Alvarez-Curto  1 , Sheikh Zahir Raihan  1 , Carlos M G Azevedo  1 , Gabriel Martin  1 , Julia Dunlop  1 , Stephen J Yarwood  1 , Trond Ulven  1 , Graeme Milligan  2
Affiliations

Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4

Kenneth R Watterson et al. Mol Pharmacol. 2017 Jun.

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, 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.

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Figures

Fig. 1.
Fig. 1.
The chemical structures of the FFA4 antagonist AH-7614 and a range of FFA4 agonists (TUG-891, Cpd A, GSK137647A, and TUG-1197).
Fig. 2.
Fig. 2.
AH-7614 is a potent and selective inhibitor of both human and murine FFA4 function. Flp-In T-REx 293 cells induced to express hFFA4-eYFP were pretreated with either vehicle [1% (v/v) DMSO] or increasing concentrations of AH-7614 for 15 minutes, after which time they were treated with either 50 µM 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-Renilla luciferase were preincubated 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. However, AH-7614 had no effect on TUG-770–induced calcium release in 1321N1 cells stably transfected with hFFA1 receptor (G), indicating that AH-7614 is not an antagonist at this receptor. Data represent experiments performed in triplicate three times or more.
Fig. 3.
Fig. 3.
AH-7614 blocks agonist-induced receptor internalization of the human FFA4 receptor. Flp-In T-REx 293 cells induced to express hFFA4-mVenus were pretreated with 10 µM AH-7614 for 15 minutes, after which time cells were further treated with either vehicle [0.1% (v/v) DMSO] or TUG-891 (3 µM) for 30 minutes (A) and subsequently fixed. AH-7614 prevented TUG-891 induced receptor internalization (pIC50 = 7.70 ± 0.10). Cells were pretreated with varying concentrations of AH-7614 and exposed to increasing concentrations of TUG-891 (B) for 30 minutes, after which they were fixed. Cells were imaged using a Cellomics ArrayScan II, and the extent of internalization of the receptor construct was quantified.
Fig. 4.
Fig. 4.
TUG-1387 and TUG-1506 are chemical derivatives of AH-7614 with varying activity at FFA4. (A) Chemical structures of AH-7614, TUG-1387, and TUG-1506. HEK293T cells transiently coexpressing hFFA4-eYFP and β-arrestin-2–Renilla luciferase were preincubated with vehicle (0.1% DMSO) or increasing concentrations of AH-7614, TUG-1387, or TUG-1506. β-Arrestin-2 recruitment to the receptor was then determined in the presence 500 nM TUG-891 (B). All data represent experiments carried out in triplicate at least three times.
Fig. 5.
Fig. 5.
AH-7614 and TUG-1506 are probe-dependent negative allosteric modulators of FFA4. The nature of AH-7614 (A–D) and TUG-1506 (E–H) allosteric modulation of FFA4 function was probed by their ability to limit recruitment of β-arrestin-2 to hFFA4-eYFP induced by varying concentrations of four chemically distinct FFA4 agonists [TUG-891 (A and E), TUG-1197 (B and F), GSK137647A (C and G), and Merck compound A (Cpd A) (D and H)] using HEK293T cells transiently coexpressing hFFA4-eYFP and β-arrestin-2–Renilla luciferase. Data are fitted to an operational model of allosterism (Hudson et al., 2014). Estimated parameters for logα and logβ are shown for both AH-7614 (I and J) and TUG-1506 (K and L) when assessed at each of the four agonists. Data shown are from representative experiments (N numbers for each agonist-NAM combination are reported in Table 1) fit to an operational model of allosterism (Hudson et al., 2014).
Fig. 6.
Fig. 6.
TUG-1387 does not antagonize the effects of distinct FFA4 agonist chemotypes. HEK293T cells transiently expressing mFFA4-eYFP and β-arrestin-2–Renilla luciferase constructs were pretreated for 30 minutes with vehicle, 10 µM AH-7614, or 10 µM TUG-1387. They were then treated with TUG-891 (A), Cpd A (B), GSK137647A (C), or TUG-1197 (D) to assess receptor-mediated arrestin recruitment. AH-7614 was able to block β-arrestin-2 recruitment in all cases, whereas TUG-1387 did not have any effect except to produce a small but significant (**P < 0.001) reduction of the efficacy of GSK137647A (C). Data represent experiments performed in triplicate three/four times. mBRET, units are equal to the 535/475 nm emission ratio multiplied by 1000.
Fig. 7.
Fig. 7.
AH-7614, but not TUG-1387, blocks agonist-induced elevation of intracellular inositol monophosphates and phosphorylation of FFA4. (A) FFA4-mediated inositol monophosphate (IP1) production was measured in cells expressing hFFA4-mVenus. TUG-891 (closed circles) promoted accumulation of IP1. Pretreatment with either 10 µM AH-7614 (inverted triangles) or increasing concentrations of this molecule (open squares) blocked IP1 accumulation. By contrast, pretreatment with TUG-1387 was without effect (open diamonds). (B) mFFA4-eYFP cells were pretreated with vehicle or various concentrations of AH-7614 or TUG-1387 for 30 minutes followed by treatment with 10 µM TUG-891 for 5 minutes. Cell lysates were prepared and resolved by SDS-PAGE and immunoblotted with an mFFA4-specific phospho-antiserum that recognizes phosphorylation of residues Thr347 and Ser350 (Prihandoko et al., 2016). Quantification of a series of such immunoblots is shown in the right-hand panel. ns, P > 0.05.
Fig. 8.
Fig. 8.
mRNA levels of FFA4 and PPARγ are upregulated in C3H10T1/2 cells by differentiation toward the adipocyte phenotype. (A) C3H10T1/2 cells were differentiated toward an adipocyte phenotype by treatment with IID medium. Confirmation of effective differentiation after 5 days of treatment was obtained by staining for triglyceride droplets within the cells using Oil Red O. RT-qPCR was performed to assess changes in expression levels of PPARγ (B), Runx2 (C), and mFFA4 (D) under conditions of subconfluence, confluence, 5 days postconfluence, and 5 days postconfluence in the presence of IID. Data are presented as the group mean ± S.E.M. of three independent experiments (*different at P < 0.05, **P < 0.01, and ***P < 0.001). Scale bar in (A) = 400 μm.
Fig. 9.
Fig. 9.
Adipocyte differentiation of C3H10T1/2 cells requires FFA4 signaling. (A) C3H10T1/2 cells were differentiated (IID) in the presence of vehicle (0.1% DMSO), 10 μM AH-7614, or 10 μM TUG-1387 compared with a nondifferentiated control and then stained with Oil Red O. (B) Oil Red O staining was subsequently dissolved in isopropanol and then quantified by measuring absorbance at 405 nm (n = 10, data presented as mean ± S.E.M.). RT-qPCR analysis of PPARγ (n = 5) (C) and Runx2 (n = 4) (D) levels was then performed on differentiated (IID) and nondifferentiated (vehicle) C3H10T1/2 cells in the presence or absence of 10 µM AH-7614. Data are presented as the mean ± S.E. (**P < 0.01, and ***P < 0.001). Scale bar in (A) = 400 μm.
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