The first described synthetic FFA active agonist GW phenoxyp
The first described synthetic FFA1 active agonist, GW9508 (4- [(3-phenoxyphenyl)methyl]amino benzenepropanoic acid), was immediately shown to also activate FFA4, although with some 100-fold lower potency . Therefore, in the initial absence of FFA4-selective synthetic agonist ligands, GW9508 was used as a FFA4 agonist in several studies using cells and tissues that lacked detectable levels of co-expressed FFA1 (e.g., ). Probably because this ligand is available commercially, this approach has continued (reviewed in ). Early efforts to produce FFA4-selective ligands reported only modest success. For example, Suzuki et al. modified PPARγ-active molecules to generate a ligand, 4- 4-[2-(phenyl-pyridin-2-yl-amino)-ethoxy]-phenyl -butyric acid, later named NCG21 (Table 1), which displayed modest selectivity for FFA4 over FFA1, but also only modest potency . With appropriate recognition that it probably acts as a combined FFA4 and FFA1 activator, this compound has been used recently alongside other more-selective ligands to help unravel the contribution of each long-chain fatty PR-957 receptor to the ability of the omega-3 fatty acid, hexadeca-4,7,10,13-tetraenoic acid [16:4(n-3)], to generate systemic resistance to the DNA-damaging chemotherapeutic cisplatin  (discussed below). In the first significant advance in developing FFA4-selective agonist ligands, Shimpukade et al. reported the ortho-biphenyl ligand 4- [4-fluoro-4′-methyl(1,1′-biphenyl)-2-yl]methoxy -benzenepropanoic acid (TUG-891) (Table 1). This molecule showed good potency at both human and mouse FFA4 and 1000-fold selectivity over human FFA1 in assays based on the induced interactions between the receptor and β-arrestin 2. These studies also reported loss of agonist function of TUG-891 at an Arg99 to Gln mutant of human FFA4 and, alongside the parallel mutagenesis studies of Watson et al., provided the first direct evidence of the key role of this arginine residue in coordinating the carboxylate of fatty acids and fatty acid-like synthetic ligands. GPCRs responsive to fatty acids have been shown to display substantial variation in pharmacology between species orthologues . More extensive studies with TUG-891 illustrated that selectivity reported between human FFA4 and FFA1 was significantly less pronounced at mouse orthologues  and also varied when measuring G protein-mediated Ca2+ elevation versus receptor interactions with an arrestin . Although there is no comprehensive analysis of these features for many ligands, which are rarely reported beyond humans versus mice, the major reason for the lower selectivity between the mouse orthologues is their higher potency at mouse FFA1 rather than reduced potency at mouse FFA4. In practise, although a potent and selective FFA4 agonist in human cells and tissues, TUG-891 is best described as a dual FFA4 and FFA1 agonist in equivalent tissues from rodents . More recently reported compounds have provided greater levels of selectivity. For example, Adams et al. reported a chromane propionic acid-based agonist series where specific members are at least 300-fold more selective for both human and mouse FFA4 compared with FFA1. Similarly, Sparks et al. described a phenylpropanoic acid series with an exemplar compound showing between 40- and 130-fold selectivity over FFA1 across human, mouse, and rat orthologues. Similar to free fatty acids, all of the compounds described above contain a carboxylate that has been shown directly (or at least modelled) to interact with Arg99 of FFA4. However, a pair of recent reports has also described sulfonamide-containing FFA4 agonists 56, 57. GSK137647A [4-methoxy-N-(2,4,6-trimethylphenyl)benzenesulfonamide] (Table 1) is reported to have greater than 50-fold selectivity for FFA4 over FFA1 and that this is preserved across species . Similarly, a potent nonacidic sulfonamide FFA4 agonist, TUG-1197 2-[3-(pyridin-2-yloxy)phenyl]-2,3-dihydrobenzo[d]isothiazole 1,1-dioxide (Table 1) is described as having no detectable activity at FFA1 . Despite the nonacidic nature of the compound, both mutational and modelling studies indicated that it likely binds within the same orthosteric binding pocket as the carboxylate-containing agonists that resemble synthetic fatty acids . Clearly, the more selective nature of several recently disclosed ligands offers potential for more defined analysis of FFA4-mediated functions, and several such ligands (e.g., GSK137647A) are now available from commercial sources. However, not all of the more recently described ligands are suitable for in vivo studies due to poor pharmacokinetic and pharmacodynamic properties . By contrast, although not currently available from commercial suppliers, phenylpropanoic acid ‘compound 29’ , nonacidic sulfonamide ‘compound 34’ , and chromane propionic acid ‘compound 18’  have each been used for rodent in vivo studies to explore glucose handing and aspects of regulation of insulin sensitivity. In each case, these have provided clear support for an important role of FFA4 in the regulation of glucose homeostasis. The emergence of chemically distinct series of FFA4 agonists allows the possibility of using pairs of compounds from different series to provide greater support for specific roles of FFA4 . Even if the full off-target profile of each ligand is not currently available, it is reasonable to assume that compounds derived from different chemotypes will produce varying non-FFA4-mediated effects. Although no FFA4 agonist has yet entered clinical studies, there is considerable expectation that such ligands may offer novel combinations of benefits in T2DM 53, 59.