Abstract
Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.
Highlights
There is a degree of selectivity in rank-order of potency for the SCFAs between free fatty acid 2 (FFA2) and free fatty acid 3 (FFA3) with FFA2 preferentially activated by the shorter SCFAs [4, 5] and, in general, by short carboxylic acids with sp2 or sp-hybridized ␣-carbon atoms [11]
GLPG0974 (Fig. 1) has recently been described as a selective FFA2 receptor antagonist that is able to block C2-mediated chemotaxis of human neutrophils [19]. We synthesized this compound and demonstrated that it was able to antagonize, in a concentration-dependent manner, both C3 and Cmp 1-mediated activation of a form of hFFA2 that had been C-terminally tagged with enhanced yellow fluorescent protein
GLPG0974 was highly selective for hFFA2, showing no inhibitory effect on C3-mediated activation of the closely related SCFA receptor hFFA3 (Fig. 2D)
Summary
Materials and Compounds—FFA2 ligands Cmp 1 and CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)phenyl) butanoic acid) were synthesized as described previously [10]. To assess the inhibitory ability of prospective antagonist ligands, test compounds were added to the cells followed by incubation for 5 min at 37 °C. [35S]GTP␥S Incorporation Assay—Cell membranes were generated as described previously [9] from Flp-InTM T-RExTM cells either uninduced or treated with doxycycline (100 ng/ml unless otherwise indicated) to induce expression of the receptor construct of interest. [35S]GTP␥S binding assays [26, 27] were performed in reactions with 5 g of cell membrane protein pre-incubated for 15 min at 25 °C in assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 100 mM NaCl, 1 mM EDTA, 1 M GDP, and 0.1% fatty acid-free bovine serum albumin) containing the indicated concentrations of ligands. Statistical analysis of curve fit parameters was carried out by independently fitting
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
