Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands

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.

The Pharmacology and Function of Receptors for Short-Chain Fatty Acids

“…Recently, Sergeev and colleagues (2016) analyzed the binding interaction of [ 3 H]GLPG0974 at hFFA2. From this study it emerged that the orthosteric antagonists GLPG0974 and CATPB do not require interaction with both arginine residues, Arg 5.39 and 7.35, in the orthosteric binding pocket to engage with the receptor (Sergeev et al, 2016). In addition, it was found that these different classes of antagonists displayed preferential interaction with different arginine residues (Sergeev et al, 2016).…”

“…From this study it emerged that the orthosteric antagonists GLPG0974 and CATPB do not require interaction with both arginine residues, Arg 5.39 and 7.35, in the orthosteric binding pocket to engage with the receptor (Sergeev et al, 2016). In addition, it was found that these different classes of antagonists displayed preferential interaction with different arginine residues (Sergeev et al, 2016). The characterization of ligand-receptor interactions is likely to be important for the design of ligands that also display antagonism at rodent orthologs of the receptor.…”

“…Interestingly, this residue differs Pharmacology of Short-Chain Fatty Acid Receptors between human (serine) and rodent (glycine) orthologs. More recently, a further homology model of this receptor, based on the X-ray structure of the related receptor FFA1, has indicated that tyrosine 90 (residue position 3.33) is also in close proximity to this phenyl ring (Sergeev et al, 2016), and mutation of this residue has also previously been shown to markedly reduce the potency of compound 1 (Hudson et al, 2013a).…”

Complex Pharmacology of Free Fatty Acid Receptors

“…While confirming the direct roles of arginine residues 5.39 and 7.35 in coordinating the carboxylate this model suggests that the key contribution of the histidine at position 6.55 is to organize the binding pocket by making a direct interaction with the arginine at 7.35 to effectively position this residue. 257 Assessing the biological functions of the SCFAs at FFA2 and FFA3 has proven particularly challenging. 136 This is in large part due to similar expression profiles, the very low potencies of the SCFAs at these receptors, and the relatively similar pharmacology between them.…”

Application of GPCR Structures for Modelling of Free Fatty Acid Receptors

“…In contrast, docking of CATPB predicts that the carboxylate could form interactions with only the arginines. More importantly, a recent mutagenesis study suggests that CATPB preferably binds to R255 7.35 , whereas GLPG0974, another antagonist binds to R180 5.39 (Sergeev et al 2016). It appears that the carboxylate of the agonists and antagonists differently coordinate the arginine network of interactions.…”