br Synthetic Antagonists for FFA To date

Synthetic Antagonists for FFA4 To date, only compounds from a single chemical series have been reported as FFA4 ‘antagonists’ (Table 1). ‘Compound 39’ (4-methyl-N-9H-xanthen-9-yl-benzenesulfonamide), now available from commercial vendors as AH-7614, was initially reported as an antagonist at this receptor [56]. This compound, and a closely related molecule, 4-methyl-N-(9H-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 10nM) [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 GDC-0941 mg 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 [Ca2+]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-(9H-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.
Therapeutic Opportunities for FFA4 in Cancer Beyond T2DM, the therapeutic area in which FFA4 has perhaps attracted greatest attention to date is cancer. In part, this reflects appreciated roles for fatty acids and fat-rich diets in either promoting cancer cell growth and motility or the capacity of health-beneficial fatty acids, including omega-3 fatty acids, to reduce the growth of several types of tumour. The significance of a number of the reported studies is hard to define, in that many have focused largely on the effects of fatty acids alone and have frequently suggested potential contributions of both FFA4 and FFA1. Several of these studies have recently been reviewed elsewhere [65]. However, growing appreciation of the developing pharmacology at FFA4 and at FFA1 has provided new insights into the potential for FFA4 ligands. A key example is the contribution of FFA4 to the development of systemic resistance to cisplatin-based chemotherapy. Mesenchymal stem cells produce a pair of polyunsaturated fatty acids [66], 12-S-hydroxy-5,8,10-heptadecatrienoic acid (12-S-HHT), an activator of the leukotriene B4 receptor 2 [67], and hexadeca-4,7,10,13-tetraenoic acid [16:4(n-3)], which, similar to many other fatty acids, can activate both FFA4 and FFA1 with similar potency [36], at least in vitro. Recent studies used a combination of the genetic elimination of FFA4 in mice and a range of both markedly selective and dual-acting FFA4 and FFA1 agonists, in concert with selective antagonists of each receptor, to show that, although both FFA4 and FFA1 are expressed by a key population of splenic macrophages, activation by 16:4(n-3) of FFA4 on these cells specifically induced a signalling cascade that, via activation of cytosolic phospholipase A2, resulted in the release of several species of lysophosphatidic acid into the medium (Figure 3). Such ‘conditioned medium’ was able to induce resistance to cisplatin-induced DNA damage in tumour cells and, subsequently, to limit the effectiveness of cisplatin to inhibit tumour growth when injected into tumour-bearing mice [36]. Moreover, a single lysophosphatidic acid species, C24:1, was able to replicate the effect of 16:4(n-3)-conditioned medium, suggesting C24:1 as a likely end-effector, although the molecular basis for the effect of lysophosphatidic acid C24:1 remains undefined [36]. Addition of either dual-acting FFA4/FFA1 agonists, including TUG-891 [52] and NCG21 [51], but, more importantly, also the highly selective FFA4 agonist TUG-1197 [57], to splenic macrophages isolated from wild-type mice generated conditioned medium that was as effective in producing chemoresistance as treatment with 16:4(n-3) [36]. By contrast, addition of 16:4(n-3) to splenic macrophages isolated from mice lacking expression of FFA4 did not generate conditioned medium that was able to replicate this effect. Moreover, co-addition of the FFA4 antagonist AH-7614 blocked the ‘conditioning’ effect of 16:4(n-3) in cells isolated from wild-type animals [36]. Together, these results suggest the potential of FFA4 antagonists to either limit the development of resistance to platinum-containing chemotherapeutic agents or to spare the dose of such agents required for efficacy. However, the effect of the second platinum-induced fatty acid, 12-S-HHT, was not lost in cells isolated from FFA4 knockout mice [36]. As such, blockade of FFA4 with a synthetic antagonist is unlikely to be fully effective in vivo if used in isolation.