Identification and Pharmacological Characterization of Multiple Allosteric Binding Sites on the Free Fatty Acid 1 Receptor

Abstract

Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium-and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits.

Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer

“…5 In fact, studies using radiolabeled ligands have indicated that FFA1 possesses at least three distinct binding sites that recognize structurally closely related ligands and that each site is able to allosterically modulate ligand function at the others. 6 The recently published crystal structure of FFA1 in complex with 1 lent further support to the concept of multiple FFA1 binding sites, revealing several potential binding sites in addition to the site of 1. 7 Most importantly from a drug development standpoint, the specific binding site an FFA1 agonist interacts with may influence the ligand's downstream signaling outcomes, 2c and thus may have clear implications for the ultimate therapeutic efficacy of any developed FFA1 agonists.…”

“…It is therefore possible that at least some aspects of the discrepancy between potency and affinity observed for certain chemical series of FFA1 agonists could be related to binding kinetics. A third factor that may contribute to the discrepancy is the apparent presence of the multiple binding sites in FFA1, [5][6] implying the possibility of partial or lack of overlap with 4. In line with these observations, lauric acid was found to behave as an allosteric compound incompetent to fully displace 4, in contrast to the synthetic agonists that all behaved as fully competitive with 4, indicating at least partially overlapping binding sites.…”

“…finding is consistent with previous reports that the fatty acids bind to a different site than many synthetic FFA1 agonists. [5][6] Global curve fitting to an allosteric ternary complex model of the lauric acid data allowed for an estimation of its pK B (4.46 ± 0.15) and allosteric cooperativity factor (logα = -0.29 ± 0.09) values.…”

Les récepteurs membranaires des acides gras de la cellule β

“…Récem-ment, une autre série d'agonistes de GPR40, incluant AMG-837 et ses dérivés (Amgen), a été proposée [18,19]. Certaines de ces molécules agissent comme des modulateurs allostériques et potentialisent les effets insulinotropes des acides gras [52]. Des agonistes de GPR119 sont également en cours de développement clinique pour le traitement du DT2 [53].…”

The Therapeutic Potential of Allosteric Ligands for Free Fatty Acid Sensitive GPCRs

“…Such has been the case in developing selective allosteric agonists for individual subtypes of both muscarinic [37] and metabotropic glutamate receptors [38]. Finally, since a GPCR presumably has only one orthosteric binding site, yet may have multiple allosteric sites as evidenced by detailed studies of for example muscarinic acetylcholine [37] and FFA1 receptors [39], developing allosteric ligands also represents a viable strategy in situations where the orthosteric site is simply not amenable to drug development.…”