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

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.     

Selective orthosteric free fatty acid receptor 2 (FFA2) agonists: identification of the structural and chemical requirements for selective activation of FFA2 versus FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [(35)S]guanosine 5'-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.     

Defining the Molecular Basis for the First Potent and Selective Orthosteric Agonists of the FFA2 Free Fatty Acid Receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.     

Long-Chain Free Fatty Acid Profiling Analysis by Liquid Chromatography–Mass Spectrometry in Mouse Treated with Peroxisome Proliferator-Activated Receptor α Agonist

A change in the free fatty acid (FFA) profile reflects an alteration in the lipid metabolism of peripheral tissue. A high-throughput quantitative analysis method for individual FFAs therefore needs to be established. We report here an optimized LC-MS assay for a high-throughput and high-sensitivity analysis of the 10 major long-chain FFAs in mouse plasma and liver. This assay enables quantification of individual FFAs by using trace amounts of samples (2 µL of plasma and 10 mg of liver tissue). We apply this method to analyze the FFA profile of plasma and liver samples from an obese mouse model treated with bezafibrate, the peroxisome proliferator-activated receptor α (PPARα) agonist, and show a change in the FFA profile, particularly in the palmitoleic and oleic acid contents. This assay is useful for quantifying individual FFAs and helpful for monitoring the condition of lipid metabolism.     

Extracellular Ionic Locks Determine Variation in Constitutive Activity and Ligand Potency between Species Orthologs of the Free Fatty Acid Receptors FFA2 and FFA3

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [³⁵S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency.     

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.     

Functional Analysis of Free Fatty Acid Receptor GPR120 in Human Eosinophils: Implications in Metabolic Homeostasis

Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.     

The first synthetic agonists of FFA2: Discovery and SAR of phenylacetamides as allosteric modulators

Free fatty acid receptor 2 (FFA2) is a G-protein coupled receptor for which only short-chain fatty acids (SCFAs) have been reported as endogenous ligands. We describe the discovery and optimization of phenylacetamides as allosteric agonists of FFA2. These novel ligands can suppress adipocyte lipolysis in vitro and reduce plasma FFA levels in vivo, suggesting that these allosteric modulators can serve as pharmacological tools for exploring the potential function of FFA2 in various disease conditions.     

Chemotype-selective Modes of Action of κ-Opioid Receptor Agonists

The crystal structures of opioid receptors provide a novel platform for inquiry into opioid receptor function. The molecular determinants for activation of the κ-opioid receptor (KOR) were studied using a combination of agonist docking, functional assays, and site-directed mutagenesis. Eighteen positions in the putative agonist binding site of KOR were selected and evaluated for their effects on receptor binding and activation by ligands representing four distinct chemotypes: the peptide dynorphin A(1–17), the arylacetamide U-69593, and the non-charged ligands salvinorin A and the octahydroisoquinolinone carboxamide 1xx. Minimally biased docking of the tested ligands into the antagonist-bound KOR structure generated distinct binding modes, which were then evaluated biochemically and pharmacologically. Our analysis identified two types of mutations: those that affect receptor function primarily via ligand binding and those that primarily affect function. The shared and differential mechanisms of agonist binding and activation in KOR are further discussed. Usually, mutations affecting function more than binding were located at the periphery of the binding site and did not interact strongly with the various ligands. Analysis of the crystal structure along with the present results provide fundamental insights into the activation mechanism of the KOR and suggest that “functional” residues, along with water molecules detected in the crystal structure, may be directly involved in transduction of the agonist binding event into structural changes at the conserved rotamer switches, thus leading to receptor activation.     

Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120)

The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds 8 and 39 is presented herein.     

Novel free fatty acid receptor 1 (GPR40) agonists based on 1,3,4-thiadiazole-2-carboxamide scaffold

Free fatty acid receptor 1 (FFA1), previously known as GPR40 is a G protein-coupled receptor and a new target for treatment of type 2 diabetes. Two series of FFA1 agonists utilizing a 1,3,4-thiadiazole-2-caboxamide scaffold were synthetized. Both series offered significant improvement of the potency compared to the previously described 1,3,4-thiadiazole-based FFA1 agonists and high selectivity for FFA1. Molecular docking predicts new aromatic interactions with the receptor that improve agonist potency. The most potent compounds from both series were profiled for in vitro ADME properties (plasma and metabolic stability, Log D, plasma protein binding, hERG binding and CYP inhibition). One series suffered very rapid degradation in plasma and in presence of mouse liver microsomes. However, the other series delivered a lead compound that displayed a reasonable ADME profile together with the improved FFA1 potency.     

Mutational analysis of G-protein coupled receptor – FFA2

FFA2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate, and propionate. FFA2 is predominantly expressed in islets, a subset of immune cells, adipocytes, and the gastrointestinal tract which suggest a possible role in inflammatory and metabolic conditions. We have previously described the identification and characterization of novel phenylacetamides as allosteric agonists of FFA2. In the current study, we have investigated the molecular determinants contributing to receptor activation with the endogenous and synthetic ligands as well as allosteric interactions between these two sites. The mutational analysis revealed previously unidentified sites that may allosterically regulate orthosteric ligand’s function as well as residues potentially important for the interactions between orthosteric and allosteric binding sites.     

The Action and Mode of Binding of Thiazolidinedione Ligands at Free Fatty Acid Receptor 1

The endogenous ligands for free fatty acid receptor 1 (FFA1) are medium and longer chain free fatty acids. However, a range of selective, small molecule ligands have recently been developed as tool compounds to explore the therapeutic potential of this receptor, whereas clinically employed thiazolidinedione “glitazone” drugs are also agonists at FFA1. Each of these classes of agonist was able to promote phosphorylation of the ERK1/2 mitogen-activated protein (MAP) kinases in cells able to express human FFA1 on demand. However, although both lauric acid and the synthetic agonist GW9508X produced rapid and transient ERK1/2 MAP kinase phosphorylation, the thiazolidinedione rosiglitazone produced responses that were sustained for a substantially longer period. Despite this difference, the effects of each ligand required FFA1 and were transduced in each case predominantly via G proteins of the Gα_q/Gα₁₁ family. Different glitazone drugs also displayed markedly different efficacy and kinetics of sustainability of ERK1/2 MAP kinase phosphorylation. A number of orthosteric binding site mutants of FFA1 were generated, and despite variations in the changes of potency and efficacy of the three ligand classes in different functional end point assays, these were consistent with rosiglitazone also binding at the orthosteric site. Four distinct polymorphic variants of human FFA1 have been described. Despite previous indications that these display differences in function and pharmacology, they all responded in entirely equivalent ways to lauric acid, rosiglitazone, and GW9508X in measures of ERK1/2 MAP kinase phosphorylation, enhancement of binding of [³⁵S]GTPγS (guanosine 5′-O-(3-[³⁵S]thio)triphosphate) to Gα_q, and elevation of intracellular [Ca²⁺], suggesting that individuals expressing each variant are likely to respond equivalently to orthosteric agonists of FFA1.     

Homology modeling and explicit membrane molecular dynamics simulation to delineate the mode of binding of thiazolidinediones into FFAR1 and the mechanism of receptor activation

Free fatty acid receptor 1 (FFAR1) is a member of a previously characterized cluster of orphan G protein-coupled receptors (GPCRs). Later, this orphan receptor was identified as a target of medium- to long-chain free fatty acids in β-cells of the pancreas. Administration of FFAR1 agonists has been proved to potentiate glucose-stimulated insulin secretion from pancreatic β-cells. It was reported that some thiazolidinediones (TZDs), the best studied PPARγ agonists, are also able to stimulate FFAR1 in a dose-dependent manner. In the present study, a homology model of the human FFAR1 was constructed and inserted into a pre-equilibrated DPPC/TIP3P membrane system. This system was then simulated for 20 ns in complex with the FFAR1 agonist GW9085, as well as rosiglitazone and pioglitazone. We noticed that the salt bridge between Glu172 and Arg258 and the H bond between Glu145 and His153 could be responsible for the stabilization of the receptor in the inactive state. Moreover, we described for the first time the binding mode of TZDs in the binding site of FFAR1. The thiazolidinedione head forms a hydrogen bonding network with the critical polar residues in the binding site, Arg258 and Asn244, while the rest of the molecule is embedded into the receptor hydrophobic pocket. Based on this modeling study, we arrived at a proposal of the pharmacophore required for binding to both PPARγ and FFAR1. Insights gained from this investigation should provide future directions for the design of novel dual acting antidiabetic agents.     

Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the Free Fatty Acid Receptor FFA4

In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr³⁴⁷, Thr³⁴⁹, Ser³⁵⁰, Ser³⁵⁷, and Ser³⁶⁰) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu³⁴¹, Asp³⁴⁸, and Asp³⁵⁵ located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from G_q/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling.     

Free fatty acid receptor 1 (GPR40) agonists containing spirocyclic periphery inspired by LY2881835

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly’s drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC₅₀ = 55 nM) was reached. Four lead compounds (EC₅₀ range 55–410 nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability (F = 10.3%) and plasma levels achieved on oral administration.     

Discovery of HWL-088: A highly potent FFA1/GPR40 agonist bearing a phenoxyacetic acid scaffold

Based on a previously reported phenoxyacetic acid scaffold, compound 7 (HWL-088) has been identified as a superior free fatty acid receptor 1 (FFA1) agonist by comprehensive structure-activity relationship study. Our results indicated that the introduction of ortho-fluoro greatly increased the activity of phenoxyacetic acid series, and the unique structure-activity relationship in biphenyl moiety is different from previously reported FFA1 agonists. Moreover, the modeling study was also performed to better understand the binding mode of present series. Compound 7 significantly improved glucose tolerance both in normal and diabetic models, and even exerted greater potential on glucose control than that of TAK-875. These findings provided a novel candidate HWL-088, which is currently in preclinical study to evaluate its potential for the treatment of diabetes.