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

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.

A Potent Antagonist of Protease-Activated Receptor 2 That Inhibits Multiple Signaling Functions in Human Cancer Cells

“…A characteristic of allosterism is cooperativity between orthosteric and allosteric ligands, known as "probe dependence" (Valant et al, 2012). Allosteric modulators can use different mechanisms of inhibition against different agonists of GPCRs (Watterson et al, 2017). However, for PARs it is difficult to characterize allosteric mechanisms because there is no exogenous orthosteric ligand.…”

“…Using an operational model of allosterism (Gregory et al, 2012;Kenakin, 2013;Watterson et al, 2017), we calculated the cooperativity for both affinity (a) and efficacy (b) of the "probe" 2f-LIGRL-NH 2 in the calcium response. The finding that a 5 0.85 , 1.0 and b 5 0.04 , 1.0 suggests that I-191 may be a negative allosteric modulator of 2f-LIGRL-NH 2 -induced PAR2 activation in Ca 21 mobilization.…”

FFA4/GPR120: Pharmacology and Therapeutic Opportunities

“…Although neither competitive nor displaying more than moderate affinity (approximately 10 nM) [60] for the receptor, AH-7614 has recently been used in a range of studies; for example, to confirm a specific role for FFA4 as the long-chain fatty acid receptor on splenic macrophages responsible for release of a lysophosphatidic acid species that is able to produce systemic resistance to platinum-containing chemotherapeutics [36] ; to identify a role of the receptor in activation of brown fat [61] ; to explore whether various effects of arachidonic acid are produced via FFA4 [62] ; and the contribution of FFA4 to docosahexaenoic acid [20:6(n-3)]-mediated effects in GnRH-producing neurones [63] . However, as noted by Watterson et al [60] , AH-7614 is a simple xanthene-containing chemical and, although this molecule does not block agonist effects at FFA1 [60] , other potential sites of action have not been explored. Therefore, it is noteworthy that, in studies in rat round spermatids, AH-7614 itself induced an increase in [Ca 2+ ] i in the absence of FFA4 receptor-activating ligands [64] , akin to those produced by the omega-6 fatty acid arachidonic acid [20:4(n–6)].…”

“…This compound, and a closely related molecule, 4-methyl- N -(9 H -thioxanthen-9-yl)benzenesulfonamide (TUG-1506) [60] ( Table 1 ), act as noncompetitive, negative allosteric modulators of the action of a range of FFA4 agonist chemotypes [60] . Although neither competitive nor displaying more than moderate affinity (approximately 10 nM) [60] for the receptor, AH-7614 has recently been used in a range of studies; for example, to confirm a specific role for FFA4 as the long-chain fatty acid receptor on splenic macrophages responsible for release of a lysophosphatidic acid species that is able to produce systemic resistance to platinum-containing chemotherapeutics [36] ; to identify a role of the receptor in activation of brown fat [61] ; to explore whether various effects of arachidonic acid are produced via FFA4 [62] ; and the contribution of FFA4 to docosahexaenoic acid [20:6(n-3)]-mediated effects in GnRH-producing neurones [63] . However, as noted by Watterson et al [60] , AH-7614 is a simple xanthene-containing chemical and, although this molecule does not block agonist effects at FFA1 [60] , other potential sites of action have not been explored.…”

“…Therefore, it is noteworthy that, in studies in rat round spermatids, AH-7614 itself induced an increase in [Ca 2+ ] i in the absence of FFA4 receptor-activating ligands [64] , akin to those produced by the omega-6 fatty acid arachidonic acid [20:4(n–6)]. Given such concerns, Watterson et al [60] suggested as a control the parallel use of 4-methyl- N -(9 H -xanthen-9-yl)benzamide (TUG-1387) ( Table 1 ), an analogue of both AH-7614 and TUG-1506 that has no activity at FFA4. Whereas AH-7614 blocked autocrine differentiation of mouse preadipocytes towards an adipocyte phenotype, TUG-1387 did not [60] , providing stronger evidence for a direct role of FFA4 in this process.…”