Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands.
Bottom Line: 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.
Affiliation: From the Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and.Show MeSH
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Mentions: As we were surprised by the relatively minor effect of removing the positively charged arginine residues (at least individually) in hFFA2 on binding of the two antagonists, we next addressed whether this reflected limited importance of the carboxylate functional group for binding of these antagonists. We considered this primarily because in previous models of FFA2 agonist binding, the carboxylate functionality has generally been predicted to form an ionic interaction with the key arginine residues of the receptor (9, 10). Therefore, we synthesized a variant of CATPB in which the carboxylic acid group was replaced with a methyl ester (MeCATPB) (Fig. 1). At wild type hFFA2 MeCATPB also functioned as an antagonist, able to block the ability of C3 to promote recruitment of β-arrestin 2 in a concentration-dependent manner (Fig. 7A). However, the potency of MeCATPB to do so was significantly less than CATPB (Fig. 7A). MeCATPB was also able to compete with [3H]GLPG0974 for binding, but it did so with a 13-fold reduction in affinity (p < 0.01) compared with the carboxylate-containing antagonist CATPB (Fig. 7B and Table 3). This reduction in affinity appeared to result from a loss of ionic interaction with the key arginine residues because, unlike for CATPB, there was no significant decrease in affinity of MeCATPB at either the R180A5.39 or R255A7.35 mutants compared with the wild type receptor (Fig. 7C and Table 3). Indeed, at R255A7.35 MeCATPB displayed a modest trend, that was, however, not statistically significant, toward an increase in affinity compared with the wild type receptor (Fig. 7C and Table 3). This may reflect that the substitution of Arg to Ala opens up the binding pocket to allow more rapid access of this ligand (see below). Next, to extend these analyses and to examine the importance of the carboxylate to antagonists structurally related to GLPG0974, we synthesized a carboxylate (Cmp 71)/methyl ester (MeCmp 71) ligand pair (Fig. 1) based on a representative compound in the GLPG0974 chemical series. These two compounds both contain a p-trifluoromethylbenzyl moiety instead of the m-chlorobenzyl group of GLPG0974 (Fig. 1). MeCmp 71 also acted as an antagonist of C3 (Fig. 7D), and as with the MeCATPB/CATPB pair, MeCmp 71 was significantly less potent than Cmp 71 (Fig. 7D). As anticipated from the above data, MeCmp 71 was also able to compete with [3H]GLPG0974 for binding to hFFA2, but it did so with significantly (p < 0.001) reduced affinity (17-fold) compared with Cmp 71 (Fig. 7E and Table 3). As with MeCATPB, there was no reduction of the binding affinity of MeCmp 71 to either the R180A5.39 or R255A7.35 hFFA2 single mutations compared with wild type hFFA2, and indeed, there was a significant increase in affinity (p < 0.01) at both (Fig. 7F and Table 3). This further supports the hypothesis that it is a loss of an ionic interaction with one or the other of these arginine residues that accounts for the reduced affinity of the methyl ester derivatives. As a further test of the contribution of the carboxylate moiety to binding affinity and function in this chemical series, we synthesized a further carboxylate (Cmp 42)/methyl ester (MeCmp 42) pair from this chemical series (Fig. 1) and performed both functional (Fig. 7G) and binding studies (Fig. 7H). Once again, in both situations the methyl ester was less effective than the carboxylate.
Affiliation: From the Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and.