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The G protein-coupled receptor FFA2 is a key mediator of short chain fatty acid signalling, which are produced in the gut via fermentation of poorly digested carbohydrates by the gut microbiota. Therefore, FFA2 has attracted interest as a potential therapeutic target for metabolic and inflammatory diseases. However, several limitations have hindered validation of FFA2 as a drug target, including the limited understanding of the molecular determinants of ligand binding and species-specific differences in pharmacology. Herein, novel tool compounds and assay systems were developed for FFA2 and utilised to address some of these limitations. Following the characterisation of functional assays for detection of FFA2 signalling, these were employed to examine the structure-activity relationship and pharmacology of FFA2 agonists versus antagonists. To assess how the pharmacology of FFA2 ligands is defined by their mode of binding, a radioligand binding assay was developed using a tritiated form of FFA2 antagonist GLPG0974 that was utilised in combination with site-directed mutagenesis and homology modelling to explore FFA2 ligand binding sites. These studies showed that FFA2 agonist binding was defined by an essential interaction between the ligand carboxylate and an orthosteric Arg-His-Arg triad. In contrast, FFA2 antagonists only required one orthosteric arginine for high-affinity binding and could tolerate modifications of the carboxylate moiety. This knowledge was applied to develop an antagonist-based fluorescent tracer for FFA2 that was utilised in BRET binding assays but displayed complex pharmacological behaviour that was shown to be based on the bitopic nature of FFA2 antagonists. The secondary binding site of FFA2 antagonists was also related to their lack of action at rodent orthologues of FFA2, whose molecular basis was explored using homology models of human and murine FFA2. This facilitated the identification of a single lysine to arginine variation at position 2.60 that might provide a basis for antagonist selectivity. Extending these studies to agonist function demonstrated that removal of the positive charge at this position produced a signalling-biased form of FFA2, in which only coupling to Gi G proteins was fully maintained. In summary, these findings contribute to understanding the complex pharmacology of FFA2 ligands and the underlying mechanisms that define their function, and conclusions drawn from these studies may help advance future efforts to validate the therapeutic potential of targeting FFA2. |
