Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

*Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, UK
The FASEB Journal (Impact Factor: 5.04). 08/2012; 26(12). DOI: 10.1096/fj.12-213314
Source: PubMed


When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC(50) values for inhibition of cAMP, 5.83 ± 0.11; Ca(2+) mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.-Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs.

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    • "However, the clinical use of these drugs was deemed to be limited due to low solubility and poor pharmacokinetics (90). Orthosteric FFA2 agonists and antagonists have also now been reported (82, 91) and used to demonstrate a role for this receptor in improved glucose uptake, decreased colon motility and contractility, increased GLP-1 secretion, and inhibiting leukocyte activation (81, 82, 89, 92). FFA3 agonists are even less developed although Arena Pharmaceuticals has reported a series of FFA3-selective compounds (89) (Table 1). "
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    ABSTRACT: Dietary free fatty acids (FFAs), such as ω-3 fatty acids, regulate metabolic and anti-inflammatory processes, with many of these effects attributed to FFAs interacting with a family of G protein-coupled receptors. Selective synthetic ligands for free fatty acid receptors (FFA1-4) have consequently been developed as potential treatments for type 2 diabetes (T2D). In particular, clinical studies show that Fasiglifam, an agonist of the long-chain FFA receptor, FFA1, improved glycemic control and reduced HbA1c levels in T2D patients, with a reduced risk of hypoglycemia. However, this ligand was removed from clinical trials due to potential liver toxicity and determining if this is a target or a ligand-specific feature is now of major importance. Pre-clinical studies also show that FFA4 agonism increases insulin sensitivity, induces weight loss, and reduces inflammation and the metabolic and anti-inflammatory effects of short chain fatty acids (SCFAs) are linked with FFA2 and FFA3 activation. In this review, we therefore show that FFA receptor agonism is a potential clinical target for T2D treatment and discuss ongoing drug development programs within industry and academia aimed at improving the safety and effectiveness of these potential treatments.
    Frontiers in Endocrinology 08/2014; 5:137. DOI:10.3389/fendo.2014.00137
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    • "Mutation of Cys1414.57 in hFFA2 (Figure 2) to the Gly corresponding to the bovine receptor transferred a similar ligand selectivity to the human receptor. By introducing a second H242Q mutation in hFFA2, the receptor lost its response to SCFAs, which was taken advantage of in the construction of a receptor activated solely by a synthetic ligand (RASSL) of FFA2 (Hudson et al., 2012). "
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    ABSTRACT: The deorphanization of the free fatty acid (FFA) receptors FFA1 (GPR40), FFA2 (GPR43), FFA3 (GPR41), GPR84, and GPR120 has made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber produces high concentrations of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane (7TM) receptors free fatty acid receptor 2 (FFA2) and FFA3 are activated by these SCFAs, and several lines of evidence indicate that FFA2 and FFA3 mediate beneficial effects associated with a fiber-rich diet, and that they may be of interest as targets for treatment of inflammatory and metabolic diseases. FFA2 is highly expressed on immune cells, in particular neutrophils, and several studies suggest that the receptor plays a role in diseases involving a dysfunctional neutrophil response, such as inflammatory bowel disease (IBD). Both FFA2 and FFA3 have been implicated in metabolic diseases such as type 2 diabetes and in regulation of appetite. More research is however required to clarify the potential of the receptors as drug targets and establish if activation or inhibition would be the preferred mode of action. The availability of potent and selective receptor modulators is a prerequisite for these studies. The few modulators of FFA2 or FFA3 that have been published hitherto in the peer-reviewed literature in general have properties that make them less than ideal as such tools, but published patent applications indicate that better tool compounds might soon become available which should enable studies critical to validate the receptors as new drug targets.
    Frontiers in Endocrinology 10/2012; 3:111. DOI:10.3389/fendo.2012.00111
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    ABSTRACT: Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency, such that acetate (C2) has been described as FFA2 selective while propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations marked variation in ligand-independent, constitutive activity was identified. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity while the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the 2nd extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity, and in most cases also yielded corresponding changes in SCFA potency.
    Journal of Biological Chemistry 10/2012; 287(49). DOI:10.1074/jbc.M112.396259 · 4.57 Impact Factor
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