Extracellular Loop 2 of the Free Fatty Acid Receptor 2 Mediates Allosterism of a Phenylacetamide Ago-Allosteric Modulator

Abstract

Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-␣-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu 173 or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.

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

“…Despite this, some subsequent studies have indicated very limited ability of this ligand to produce elevation of Ca 21 levels in transfected cells (discussed later). Moreover, further analyses of the pharmacological properties of this ligand revealed an ability to exert positive cooperativity with both C3 and C2, indicating that 4-CMTB behaves as an allosteric agonist at FFA2 (Lee et al, 2008;Wang et al, 2010;Smith et al, 2011). Mutagenic studies have revealed that extracellular loop 2 plays an important role in the allosteric effect of 4-CMTB.…”

“…The main endogenous SCFAs (C2, C3, and C4) activate FFA2 and FFA3 with varying potency (denoted by the thickness of the arrow), and the rank order of activity is not maintained between human and mouse species orthologs (Hudson et al, 2012b). Synthetic allosteric agonists at FFA2 are represented by phenylacetamides, where 4-CMTB is the most potent ligand (Lee et al, 2008;Wang et al, 2010;Smith et al, 2011) and it maintains its activity across human and mouse FFA2 (Hudson et al, 2012b). Allosteric modulators at FFA3 are represented by a class of synthetic ligands that comprise allosteric agonists, such as AR420626, and allosteric antagonists, such as 2-methyl-5-oxo-4-(3-phenoxyphenyl)-N-(o-tolyl)-1, 4,5,6,7,8-hexahydroquinoline-3-carboxamide (compound 6) .…”

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

“…Somewhat surprisingly, although significant species variation has been observed among the orthosteric SCFAs at FFA2, 14 appears to show little variation in potency and activity between human, rodent [83] and bovine (unpublished observation) orthologues of FFA2. There is, however, also some indication that 14 may produce different signaling responses at FFA2 than the SCFAs, specifically 14 appears to be a full agonist in some pathways but only a partial agonist in others [89], perhaps suggesting some degree of functional selectivity. This, however, needs to be more clearly defined in future studies.…”

“…In particular, (S)-4-chloro--(1-methylethyl)-N-2-thiazolylbenzeneacetamide (14) was identified in a high throughput screen and found to be a selective ago-allosteric modulator of FFA2 [87]. As is often the case with allosteric ligands, subsequent attempts to explore the SAR of this series were unable to improve on its modest potency despite modification of all parts of the structure [88,89]. Defining the allosteric site where 14 binds has also presented a substantial challenge, with several modeling and mutagenesis studies unable to determine a precise mode of binding [88][89][90][91].…”

“…As is often the case with allosteric ligands, subsequent attempts to explore the SAR of this series were unable to improve on its modest potency despite modification of all parts of the structure [88,89]. Defining the allosteric site where 14 binds has also presented a substantial challenge, with several modeling and mutagenesis studies unable to determine a precise mode of binding [88][89][90][91]. Somewhat surprisingly, although significant species variation has been observed among the orthosteric SCFAs at FFA2, 14 appears to show little variation in potency and activity between human, rodent [83] and bovine (unpublished observation) orthologues of FFA2.…”