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Comprehensive notes on the Complex Pharmacology of Free Fatty Acid Receptors (FFA1–FFA4) and related FFAR biology

Introduction

The GI tract of the document outlines how nonesterified or “free” fatty acids (FFAs) regulate cellular function via G protein-coupled receptors (GPCRs), expanding beyond classic orthosteric ligand concepts to include allosteric agonism, allosteric modulation, signaling bias, constitutive activity, and inverse agonism. The FFAs activate a recently defined family of GPCRs, collectively termed the free fatty acid receptors (FFA1–FFA4), which are now pursued as therapeutic targets for metabolic and inflammatory diseases. The review also discusses additional fatty-acid-responsive receptors such as GPR84, Olfr78/OR51E2, and HCA2, and it highlights the pharmacological complexity—orthosteric vs allosteric ligands, ligand bias, receptor constitutive activity, and receptor deorphanization—relevant to drug discovery. The lipid landscape is described in depth: long-chain fatty acids (LCFAs), medium-chain (MCFAs), and short-chain fatty acids (SCFAs), their biosynthesis (dietary uptake, de novo synthesis, gut microbiota fermentation), metabolism, and roles as signaling molecules. The text introduces essential concepts in lipid biology and pharmacology, including lipidomics, PUFA biology, trans fats, and specialized fatty acids like FAHFAs, which may have anti-inflammatory and antidiabetic activities. It emphasizes the need to map molecular targets for uncommon fatty acids (e.g., platinum-induced fatty acids) and to understand how they function amidst a milieu of abundant FFAs. A major theme is how evolving GPCR pharmacology shapes the understanding of FFAR signaling in metabolism and inflammation, guiding therapeutic development. The FFAR family (FFA1–FFA4) is discussed in depth, with attention to receptor expression, ligand interactions, constitutive activity, and synthetic ligand sets, as well as cross-talk among receptors and tissue-specific outcomes. The review notes that while the receptors are promising targets, substantial knowledge gaps remain in physiological contexts and in the availability of pharmacological tools that work across species, particularly for FFA2 and FFA3. The authors disclose potential conflicts of interest, noting cofounding and equity in Caldan Therapeutics (for two authors) and related affiliations.

FFA1 (FFAR1/GPR40)

FFAR1 was the first FFA receptor deorphanized as activated by FFAs, with initial reports in 2003 establishing activation by MCFAs and LCFAs and a predominant Gq/11 signaling profile. FFA1 signals primarily through Gq/11 with occasional Gi and, less commonly, Gs coupling, and there is preliminary evidence for some arrestin recruitment and potential G protein–independent signaling. The receptor is expressed most prominently in pancreatic islets, especially β-cells, with expression also seen in α-cells and various enteroendocrine cells (L cells secreting GLP-1 and PYY; I cells secreting CCK; K cells secreting GIP), plus skeletal muscle, heart, liver, bone, brain, and monocytes. The receptor’s role in metabolism and inflammatory regulation is thus broad and tissue-context dependent.

LCFAs activate FFA1 with modest potency (EC50 values typically > 1 μM across early studies), and SAR analyses show that long-chain polyunsaturated fatty acids (PUFAs) often have higher potency at FFA1. In contrast, trans fatty acids tend to be poor agonists, which is noteworthy given their general health detriments. The receptor’s role in pancreatic β-cells includes acutely enhancing glucose-stimulated insulin secretion (GSIS) via a Gq/11–PLC pathway, leading to PKD phosphorylation and modulation of granule trafficking. FFA1 contributes about half of the LCFA effect on GSIS; the rest is thought to be via additional receptors and pathways. Chronic LCFA exposure can cause lipotoxic effects in β-cells largely independent of FFA1, though some studies suggest context-dependent contributions or protective roles of FFA1 signaling under certain conditions. The LCFA-FFA1 axis also intersects with lipotoxicity debates, with signaling bias proposed as a possible explanation for varied outcomes across different LCFAs.

In the gut, FFA1 is expressed in L, K, and I enteroendocrine cells and promotes LCFA-stimulated secretion of GLP-1, GIP, and CCK, reinforcing the view of FFA1 as a nutrient sensor for dietary fats. However, a notable study found GLP-1 release in rat intestines occurred when LCFAs were delivered via vasculature but not luminally, raising questions about the site of action and absorption dynamics in vivo. The receptor also participates in broader metabolic signaling in other tissues, though to a lesser extent relative to the canonical β-cell and enteroendocrine roles.

FFA1 shows ligand-independent constitutive activity in some assay contexts, but apparent constitutive activity can be modulated by fatty acid binding to albumin. Albumin-binding reduces the availability of fatty acids in assay settings, thereby impacting observed constitutive activity. Mechanistic models have implicated an ionic lock between EL2 and the ligand-binding region as a regulator of constitutive activity, and there is evidence of distinct ligand-dependent internalization pathways, with BSA affecting signaling and internalization in different ways.

2.4 Synthetic ligands for FFA1
FFAs attracted substantial pharmacological interest, yielding a broad set of agonists and antagonists. The field has highlighted that agonism at FFA1 is generally therapeutic for diabetes, with the consensus favoring agonists as the preferred modality. In contrast, FFA1 antagonists have been less abundant and often used as pharmacological tools.

  • 2.4.1 FFA1 agonists: Early leads included GW9508 (an N-substituted-3-(4-aminophenyl)-propanoic acid) which activates FFA1 with good potency but also activates FFA4. Thiazolidinedione (TZD) class members (rosiglitazone, pioglitazone, etc.) activate FFA1 and have cross-talk with PPARγ signaling, complicating interpretation since they also modulate FFA1 expression. Subsequent SAR work yielded optimized agonists with improved potency and pharmacokinetic properties, including TUG-469, TAK-875 (fasiglifam), and related analogs (e.g., 10, 11–14). TAK-875, a potent agonist with favorable pharmacokinetics and oral bioavailability, advanced to clinical trials but was terminated due to suspected liver toxicity linked to hepatobiliary transporter inhibition, not a proven target issue. Other series (e.g., 7 and its derivatives) demonstrated in vivo glucose-lowering effects via FFA1 activation in a glucose-dependent fashion, supporting therapeutic potential for T2D, albeit with pharmacokinetic limitations across series.

  • 2.4.2 Mode(s) of agonist interaction: Early homology models placed ligands in the classic GPCR orthosteric pocket with carboxylate–Arg183(5.39)/Arg258(7.35) interactions; however, a high-resolution crystal structure of FFA1 with TAK-875 revealed a surprising pose with TAK-875 extending partly outside the central TM core into the lipid milieu, particularly a gap between TMIII and TMIV. This finding suggested that several ligands may engage multiple binding pockets and that endogenous LCFAs could bind differently from synthetic ligands. Subsequent studies showed AMG-837, AM-8182, AM-1638, and DHA can interact across multiple pockets, with evidence for at least three distinct binding sites. The TAK-875 binding site appears to represent a common site for several ligands, but some ligands (e.g., AM-1638) may bind at alternate allosteric pockets (e.g., Lys622). The structural data imply multiple allosteric sites and potential bitopic-like interactions, complicating orthosteric vs allosteric classification and suggesting ligand-specific signaling outcomes.

  • 2.4.3 Partial agonism and ligand bias: Ligands display varying efficacy; full agonists (e.g., AM-1638) promote broader signaling (Gq/11 and Gs) and robust incretin responses, whereas some ligands (e.g., TAK-875, AMG-837) primarily trigger Gq signaling with limited Gs engagement. The degree of receptor expression and receptor reserve in different cell types can influence apparent potency and efficacy; thus, partial agonists may produce different in vivo outcomes depending on receptor density. Evidence indicates that full agonists and site-dependent ligands can drive signaling bias toward different G proteins or arrestin pathways. Also, some ligands show G protein bias with a shift toward arrestin recruitment, and in arrestin3-deficient islets, TAK-875’s insulinotropic effects are reduced, suggesting arrestin-dependent components of signaling can influence therapeutic outcomes.

  • 2.4.4 FFA1 antagonists: GW1100 was among the first antagonists, but it shows poor cross-species activity, being relatively human-specific. Other antagonists include DC260126 (improves insulin sensitivity in rats but not glucose handling) and a Pfizer-derived 1,2,3,4-tetrahydroisoquinolinone series. These antagonists facilitate mechanistic studies but cross-species considerations are critical. Some antagonists display noncompetitive or allosteric modes, highlighting receptor complexity.

  • 2.4.5 FFA1 fluorescent tracers: Fluorescent ligands and BRET-based approaches enable real-time visualization of ligand–receptor interactions. Early bodipy-labeled fatty acids had limited utility due to lipophilicity and low affinity, but newer tracers derived from TAK-875 or TUG-905 (36–38) offer higher affinity; however, solubility and assay limitations persist. Strategies include antibody-boosted fluorescence and Nanoluciferase-based Bioluminescence Resonance Energy Transfer (BRET) approaches to quantify binding with higher signal-to-noise.

FFA2 and FFA3 (GPR43 and GPR41)

FFAs for SCFAs activate FFA2 (GPR43) and FFA3 (GPR41). FFA2 couples to Gq/11 and Gi/o, and recruits arrestin-3, suggesting multiple signaling outputs and potential anti-inflammatory signaling through arrestin pathways. FFA3 primarily couples to Gi/o, inhibiting adenylyl cyclase and reducing cAMP; evidence for Gq/11 signaling at FFA3 is weaker. In some contexts, FFA2 and FFA3 can recruit arrestins, though FFA3 arrestin recruitment is less consistently reported than for FFA2. Expression patterns vary: immune cells (especially monocytes and neutrophils) show robust FFA2/FFA3 expression; enteroendocrine cells in the gut express both; pancreatic β-cells express FFA2/FFA3; adipose tissue expresses FFA2 and potentially FFA3; neurons in sympathetic ganglia and enteric nervous system express FFA3; and elevated FFA2/FFA3 expression has been reported in several cancers, with potential tumor-suppressive or tumor-promoting roles depending on context.

3.1 Expression of FFA2 and FFA3
Early studies identified both receptors in immune cells and in the gut, with enteroendocrine cells (L, K, I cells) expressing FFA2/FFA3. Pancreatic β-cells express FFA2/FFA3, and adipose tissue expresses FFA2 (with FFA3 expression variably reported). FFA3 is particularly enriched in sympathetic ganglia and enteric neurons. Expression has also been observed in breast, colon, and liver cancers, with potential tumor-suppressive actions attributed to FFA2 in some settings.

3.2 GPR42 (GPR42 and FFA3 relation)
In primates, a paralogous receptor GPR42 sits near FFA2 and FFA3 on chromosome 19q13.1. GPR42 is highly similar to FFA3 (only six amino-ac acid differences) and shows substantial polymorphism. Some GPR42 variants respond to SCFAs, indicating functional potential. Copy-number variation exists, with a notable proportion of alleles carrying a GPR42 deletion. RT-PCR studies often fail to distinguish FFA3 from GPR42 due to sequence similarity, complicating interpretation. Whether GPR42 activity represents gene dosage effects or a distinct pharmacology relative to FFA3 remains an area of active investigation.

3.3 SCFAs at FFA2 and FFA3
SCFAs (C1–C6) activate FFA2 and FFA3, with clear SAR differences between the receptors. For human receptors, potency rankings for SCFAs differ: FFA2 favors C2 ≈ C3 ≈ C4 > C5 > C6 ≈ C1; FFA3 favors C3 ≈ C4 ≈ C5 > C6 > C2 > C1. C2 is notably more potent at FFA2 than FFA3 in humans, though species differences exist (mouse orthologs show different potency profiles). The residues implicated in SCFA recognition (Arg at positions 5.39 and 7.35; Asn/His at 6.55) mirror those identified in FFA1, indicating conserved carboxylate coordination. A detailed homology-based map places the SCFA carboxylate in contact with these conserved residues, and structural models suggest an essential role for Arg at 7.35 and Arg at 5.39 in SCFA recognition.

3.3.1 SCFAs, FFA2, FFA3 in the GI tract
In the gut, C2 and C3 promote GLP-1 release via FFA2–Gq/11 signaling in primary murine colonic cultures; this effect is absent in FFA2−/− mice and not blocked by pertussis toxin, indicating a Gq/11 pathway rather than Gi/o. In vivo, intracolonic C3 induces GLP-1 and PYY secretion, dependent on FFA2. FFA2 and FFA3 are also expressed in gastric ghrelin-secreting cells; SCFAs can regulate ghrelin, generally inhibiting its secretion via Gi/o signaling with FFA2 as the primary receptor in gastric tissue.

3.3.2 SCFAs, FFA2, FFA3, and adipose tissue
SCFAs inhibit lipolysis in adipocytes via FFA2–Gi/o signaling. SCFAs promote adipogenesis in 3T3-L1 cells, with FFA2 expression increasing during differentiation. In vivo, FFA2−/− mice on a high-fat diet show altered adiposity, but results are strain-dependent; some FFA2-overexpressing models are lean, others show obesity, suggesting complex in vivo regulation and potential species/strain differences. Leptin regulation by SCFAs shows inconsistency across studies, with some data suggesting FFA2 or FFA3 involvement and others arguing against a major role for FFA2 in adipocyte leptin dynamics. SCFAs modulate glucose uptake in adipocytes, with evidence that FFA3 contributes to certain uptake responses; however, results vary by tissue (adipose vs muscle vs liver) and by species.

3.3.3 SCFAs, FFA2, FFA3, and insulin secretion
In rodent islets, SCFAs can enhance GSIS via FFA2–Gq/11 signaling, but conflicting studies report SCFAs inhibiting GSIS via FFA2 and FFA3 Gi/o signaling. The net effect in mouse islets appears to favor Gq/11 signaling-enhanced GSIS; in human islets, SCFAs do not significantly alter GSIS, suggesting species differences and potential context-dependency in signaling pathway engagement.

3.3.4 SCFAs, FFA2, FFA3, and inflammation
FFAs influence immune cell function; FFA2 promotes neutrophil chemotaxis via Gi/o signaling, and FFA2 activation can modulate cytokine production in mononuclear cells. In intestinal epithelial cells, SCFAs can induce anti-inflammatory signaling via both FFA2 and FFA3, indicating a coordinated role for these receptors in mucosal immunity.

3.4 FFA2, FFA3, and the Gut Microbiota
Disentangling microbiota-driven effects from host receptor signaling is challenging due to high SCFA concentrations and species-specific receptor pharmacology. Germ-free and antibiotic-treated mouse models show that the microbiota regulates inflammatory and metabolic phenotypes via FFA2 and FFA3, with SCFAs restoring certain functions in FFA2−/− or FFA3−/− animals. Several studies link gut microbial ecology to systemic metabolic outcomes through SCFAs and FFA2/FFA3, including effects on regulatory T cell populations in the gut and systemic leukocyte trafficking and inflammation. The intricate interactions between diet, microbiota, SCFAs, and FFARs remain an active area of research, with potential implications for obesity, diabetes, inflammatory diseases, and tissue-specific metabolic regulation.

3.5 Synthetic Ligands for FFA2
Developing selective ligands for FFA2 has been challenging due to cross-reactivity with FFA3 and species differences between human and rodent FFARs. Orthosteric and allosteric ligands, including agonists and antagonists, have been described, with relative selectivity for FFA2 over FFA3 improving over time but not yet universal.

3.5.1 Orthosteric FFA2 agonists
Early orthosteric ligands for FFA2 include short-chain fatty acids (SCFAs) and short-chain carboxylates (SCAs). Among orthosteric selective ligands, trans-2-methylcrotonic acid and cyclopent-1-enecarboxylic acid show FFA2 selectivity over FFA3, though their potencies are not superior to SCFAs. Propiolic acid and 2-butynoic acid represented orthosteric activators with some selective activity; they can potentiate GSIS in wild-type but not FFA2−/− mice. Additional mutational analyses identified residues around the orthosteric pocket and expanded insights into the FFA2 binding landscape. Purportedly orthosteric FFA2 ligands based on patent literature include 39–45, with 39 and 40 (orthosteric agonists) showing clear FFA2 activity, while 46 (CATPB) and 47 (GLPG0974) are notable for human-specific antagonism at orthosteric sites. 46 blocks acetate-stimulated responses and acts as an inverse agonist; 48 represents a class lacking a carboxylate but retaining orthosteric-like activity in some contexts, complicating cross-species pharmacology.

3.5.2 Orthosteric antagonists
CATPB (46) and GLPG0974 (47) are among the best-characterized orthosteric antagonists for human FFA2, blocking acetate- and 39/40-driven responses in a surmountable fashion and reducing receptor activation. However, these antagonists show poor cross-species affinity, with reduced or no efficacy in rodent FFAR2. A newer antagonist class (48) lacks a carboxylate and is relatively low potency but still orthosteric, with some reports of GLP-1 or ghrelin modulation at high concentrations. The lack of broad cross-species antagonists remains a challenge for in vivo translation.

3.5.3 Allosteric agonists (and modulators)
49 (4-CMTB, AMG7703) is a prominent FFAR2 allosteric agonist and PAM, activating FFAR2 and enhancing the potency of SCFAs. 49 also binds to orthosteric receptor variants, classifying it as an allosteric agonist with potential probe-dependence (its allosteric effect varies with the orthosteric agonist used). Allosteric communication with the SCFAs appears to involve extracellular loop 2 (EL2) of FFAR2, with Leu173 in EL2 contributing to communication between orthosteric and allosteric interactions. The series has limited potency improvements, but 49 remains a widely used FFAR2 allosteric tool; analogs 50–51 have been explored in vivo and in scFAs signaling studies. The allosteric approach provides a path to receptor subtype selectivity and saturable effects dependent on endogenous ligand presence, which can be advantageous for safety and therapeutic control.

FFA3 ligands and selectivity

3.6 Synthetic Ligands for FFA3
3.6.1 Orthosteric ligands
Despite SCFAs triggering FFA3, potent and selective orthosteric ligands for FFA3 are rare. Some SCAs demonstrate partial FFA3 selectivity, but no orthosteric ligands with robust potency and cross-species activity are widely reported. Beta-hydroxybutyrate has also been described as a potential FFA3 ligand, but its role as an agonist vs antagonist remains unclear. The lack of robust orthosteric FFAR3 agonists presents a challenge for dissecting FFAR3 biology.

3.6.2 Allosteric ligands
There are allosteric FFA3 ligands based on hexahydroquinolone-3-carboxamides (ARENA-derived). AR420626 is reported to be an FFAR3-selective allosteric agonist with modest potency, promoting GLP-1 release in murine colonic crypts, and providing some mechanistic separation of FFAR3 and FFAR2 functions. Other hexahydroquinolone derivatives, including 52–54, exhibit a range of allosteric effects, including PAM, NAM, and NAM–PAM combinations, highlighting the potential for allosteric ligands to generate diverse signaling outputs (including bias) at FFAR3. However, the in vivo utility of these FFAR3 allosteric ligands is limited by modest potency and by the challenge of predicting metabolic outcomes given the complexity of FFAR2/FFAR3 cross-talk and tissue context.

FFA4 (GPR120)

FFA4 (GPR120) is activated by LCFA and MCFA with signaling dominated by Gq/11, leading to calcium signaling. There is also evidence for Gi/o signaling in certain contexts, including inhibition of ghrelin release from ghrelin-expressing cells, which implicates Gi/o in gastric tissue. Receptor internalization upon agonist binding is rapid, and arrestin recruitment (Arrestin-2/3) is robust, particularly for GPR120, which is used as a readout of agonist activity. The receptor interacts with arrestins as well as G proteins, implying both canonical and noncanonical signaling outputs. The signaling architecture is important for understanding how GPR120 mediates anti-inflammatory and metabolic responses.

4.1 Expression of FFA4
FFA4 is widely expressed including in the lower intestine, lung, spleen, adipose tissue, taste buds, and pancreatic islets, with notable presence in enteroendocrine cells. FFA4 expression in the intestine is modulated by diet and obesity models, and FFA4 expression correlates with obesity in certain human and rodent contexts. Expression in taste buds is linked to fat taste sensing, but FFA4 is not the sole mediator of fat taste; CD36 also contributes. FFA4 has been reported in various cancers, with evidence for tumor-promoting roles in colorectal cancer and potential protective effects in some contexts in other tissues.

4.2 FFA4 splice variation
Humans express both long and short FFA4 isoforms, the long form differing by a 16 amino-acid insertion in intracellular loop 3. The long form shows impaired ability to elevate intracellular Ca2+, suggesting reduced Gq/11 signaling relative to the short form, though some studies report Ca2+ signaling via the long isoform in response to certain fatty acids. In rodents, only the short isoform is present, complicating cross-species interpretation and in vivo translation.

4.3 FFA4 genetic polymorphisms
The R254H polymorphism in the short FFAR4 isoform (R270H in long isoform) is associated with obesity in some European cohorts. Functional studies suggest R254H reduces Gq/11 and Gi/o signaling, but arrestin signaling persists and may preserve anti-inflammatory outcomes. Population studies yield mixed results on fasting glucose and diabetes risk; allele frequency varies by population. The functional relevance of FFAR4 polymorphisms, especially with respect to metabolic and inflammatory phenotypes, remains under investigation.

4.4 LCFAs at FFA4
FFAR4 is activated by a broad range of FFAs (MCFA and LCFA), with potencies typically in the 1–20 μM range; SFAs are generally partial agonists relative to PUFAs. The receptor recognizes the carboxylate moiety; esterification of the carboxylate abrogates activity. FFA4 has been proposed as a receptor for n−3 PUFAs (e.g., DHA, EPA), but comprehensive SAR suggests a broader LCFA/pufa signaling landscape. Arg992.64 is a key interaction residue for carboxylate coordination in FFA4, distinct from FFAR1/FFAR2/FFAR3 residues. FAHFAs and other unusual fatty acids have been proposed as FFAR4 ligands, though replication and potency remain uncertain. While FFAR4 generally responds to FFAs, differential efficacy across fatty acids suggests that SFAs and PUFAs may produce divergent signaling and biological outcomes through FFAR4.

4.4.1 LCFAs, FFA4, and gut hormone secretion
Early work linked FFAR4 activation to GLP-1 secretion from enteroendocrine cells; this effect has been variably reproduced. In vivo data indicate that oral LCFA administration enhances GLP-1 secretion in some contexts but not others, and some findings point to FFAR1 rather than FFAR4 as the primary driver for GLP-1. FFAR4 appears to contribute to GIP secretion in vivo. FFAR4 has been implicated in ghrelin regulation, with LCFAs inhibiting ghrelin secretion in gastric cells via Gi/o, whereas GLP-1 and other hormone secretions appear to be driven by Gq/11 signaling in enteroendocrine contexts. In taste buds, fatty acids can influence taste-related signaling via FFAR4, though other receptors also contribute to fat taste perception.

4.4.2 LCFAs, FFA4, and adipocyte function
FFAR4 is expressed in adipocytes and upregulated during adipocyte differentiation. It has been implicated in promoting GLUT4 translocation in adipocytes and, thus, acute LCFA-stimulated glucose uptake via FFAR4–Gq/11 signaling. FFA4 signaling may also regulate gene expression related to glucose metabolism (e.g., GLUT4) with longer-term knockdown studies suggesting broader metabolic control. Anti-inflammatory effects of n−3 PUFAs (e.g., EPA, DHA) involve FFAR4 signaling and link to improved insulin sensitivity in certain models.

4.4.3 LCFAs, FFA4, and inflammation
N−3 PUFAs exert anti-inflammatory actions via FFAR4; DHA can inhibit NF-κB and downregulate COX-2 and PGE2 in macrophages in an arrestin-3–dependent manner, though some studies indicate Gq/11 involvement as well, creating a complex picture of signaling cross-talk. FFAR4 appears to contribute to anti-inflammatory effects and to insulin sensitivity improvements in vivo, with some knockout studies supporting FFAR4 as a mediator of dietary n−3 PUFA benefits, while other studies show FFAR4 independence in certain metabolic endpoints. The role of FFAR4 in inflammation is thus nuanced and context-dependent, requiring more detailed dissection of signaling bias and cell-type specificity.

4.5 Synthetic Ligands for FFA4
FFAR4 drug discovery has focused on agonists due to the therapeutic rationale of benefiting metabolism and inflammation. Selectivity over FFAR1 is challenging, but several ligands show FFAR4 preference.

4.5.1 Orthosteric agonists
GW9508 is a well-known FFAR1 agonist that also activates FFAR4 but with lower potency. NCG21 and grifolic acid represent earlier FFAR4-selective scaffolds with partial agonism. The most significant FFAR4 agonist is TUG-891, a potent full agonist with high selectivity for FFAR4 over FFAR1 in arrestin-3 recruitment readouts, though in some Ca2+-based assays FFAR4 potency is closer to FFAR1. TUG-891 exhibits cross-species activity (mouse FFAR4 is more sensitive than mouse FFAR1 in some assays). The ligand set includes diarylsulfonamides that show FFAR4 selectivity over FFAR1 but suffer from solubility issues, limiting in vivo use. Other FFAR4-selective agonists with good potency and selectivity include 60 and 61, with in vivo data showing improvements in glucose handling in mice on normal diets and FFAR4-dependent metabolic improvements in high-fat diet models, including effects on obesity, insulin sensitivity, and inflammation.

4.5.2 Mode of ligand interaction with FFA4
A FFAR4 homology model (based on a β2-adrenergic receptor active-state template) has been used to examine ligand interactions. The orthosteric pocket appears to lie among TMII–TMVII, and residues important for binding include Arg992.64, W104, F115, F211, W277, and F304, among others. Mutations to these residues often abolish responses in FFAR4-arrestin-3 or FFAR4-Gq signaling assays, indicating a robust orthosteric binding pocket that coordinates carboxylate ligands through Arg992.64. Binding pose comparisons between known FFAR4 agonists (57 and 60) show similar poses, suggesting a conserved orthosteric interaction, although GW9508 occupies a slightly different pocket. Some ligands (61) have been proposed to be biased toward arrestin signaling, though this remains to be validated. Overall, the model predicts that the orthosteric pocket is the major site for FFAR4 ligand recognition, with some evidence for potential allosteric interactions.

4.5.3 FFAR4 antagonists
AH 7614 is the most widely cited FFAR4 antagonist, potentially acting at an allosteric site rather than orthosteric, given its noncompetitive profile in some assays. The pharmacology of FFAR4 antagonists remains underdeveloped relative to FFAR1/FFAR2, with few well-characterized cross-species-acting antagonists and limited in vivo use.

Other potential fatty acid receptors

Beyond the canonical FFA1–FFA4 family, several receptors have been proposed as fatty acid targets.

5.1 GPR84
GPR84 is considered an orphan receptor activated by medium-chain fatty acids (MCFAs; C9–C14) with the highest potency for C10–C11, and some hydroxy-MCFAs (2- or 3-hydroxy) also activate GPR84. GPR84 is linked to pro-inflammatory responses, and knockout studies suggest its involvement in inflammatory signaling and leukocyte chemotaxis. Species differences and the presence of loss-of-function mutations in some mouse strains complicate in vivo interpretation. A limited set of synthetic ligands exists, including early Takeda-derived compounds (63, 64) with poor potency, and more potent surrogate agonist 65; embelin (66) has been reported, though off-target effects limit its utility. More recently, additional GPR84 agonists (67–68) and antagonists (69) have been reported, with some studied in colitis models and inflammatory diseases. The GPR84 literature underscores the need for high-affinity, selective tools and careful strain selection in in vivo studies.

5.2 Olfr78/OR51E2
The olfactory receptor Olfr78 (OR51E2 in humans) is expressed in kidney and other tissues and responds to SCFAs (acetate, propionate) to regulate blood pressure. It is also expressed in PYY-secreting enteroendocrine cells. A study found Olfr78 activated by lactate as well as SCFAs in carotid body glomus cells, suggesting lactate as a physiologic endogenous ligand in certain contexts. The receptor’s role outside the olfactory system is an area of active investigation.

5.3 HCA2 (GPR109A)
HCA2 is typically considered the receptor for β-hydroxybutyrate, with butyrate activating HCA2 at high concentrations. HCA2 is expressed in adipocytes (lipolysis regulation), certain immune cells, and colon tissue where anti-inflammatory effects are observed. Nicotinic acid (niacin) and several synthetic HCA2 ligands exist (including allosteric activators). Arg111 in HCA2 appears to anchor the carboxylate moiety similarly to other FFAR residues. The physiological relevance of butyrate acting via HCA2 remains a topic of investigation, given the receptor’s higher endogenous agonist affinity and the potential for HCA2 to mediate inflammation-related signaling in colon and adipose tissue.

Conclusions and Future Perspectives

The landscape of GPCR pharmacology has expanded from simple orthosteric antagonism/agonism to include allosteric modulation, signaling bias, constitutive activity, and inverse agonism. This expanded framework has been crucial for understanding FFAR pharmacology, given the diverse endogenous ligands (SCFAs, MCFAs, LCFAs) and the tissue-specific signaling outputs that influence metabolism and inflammation. The FFAR family offers substantial therapeutic promise for metabolic diseases (e.g., obesity, type 2 diabetes) and inflammatory conditions, but several challenges remain: cross-species differences in receptor pharmacology (especially for FFA2/FFA3), limited cross-species active antagonists for in vivo use, and a need for a broader array of potent, selective orthosteric and allosteric ligands, including for FFAR3. A major goal is to develop ligands with expanded chemical diversity that can reveal biased signaling properties and enable precise therapeutic interventions. The discovery of multiple binding sites on FFAR1 and FFAR2, and the evidence for FFAR4 bias signaling, highlight the possibility that endogenously circulating fatty acids engage FFARs in complex ways that could be leveraged therapeutically while minimizing adverse effects. The review suggests that future efforts should focus on: (i) developing cross-species antagonists to enable robust in vivo testing and translation; (ii) discovering orthosteric FFAR3 agonists with high potency and cross-species activity; (iii) expanding chemical scaffolds to identify biased ligands with tailored signaling profiles; (iv) understanding tissue- and cell-type–specific receptor signaling to determine when agonism or antagonism might be most beneficial; and (v) leveraging allosteric modulators to achieve saturable effects and improved safety profiles. The authors advocate continued exploration of synthetic FFAR ligands, better pharmacological tools, and careful consideration of receptor context and signaling bias to unlock therapeutic potential across metabolic and inflammatory diseases. Overall, FFAR pharmacology remains a rich field with significant therapeutic promise, meriting ongoing development of cross-species tools and refined mechanistic understanding of receptor signaling in health and disease.

Table 1 (Summary of Free Fatty Acid Receptors)

  • FFA1 (GPR40)

    • Ligands: MCFAs, LCFAs; orthosteric and allosteric agonists; occasional antagonists; key pharmacology involves Arg183^{5.39}, Arg258^{7.35}, Asn244^{6.55}; signaling via Gq/11 (primarily), occasional Gi; tissue: pancreatic β-cells, α-cells, enteroendocrine cells; functions: acute GSIS enhancement; potential role in lipotoxicity; synthetic ligands include GW9508, TAK-875 (fasiglifam) and related compounds; allosteric interactions with TAK-875 and AMG-837; bias between G proteins and arrestins reported; some ligands show multiple binding pockets on FFAR1.

  • FFA2 (GPR43)

    • Ligands: SCFAs (C2–C6) and SCAs; orthosteric ligands include SCFAs, with allosteric modulators such as 49 (4-CMTB/AMG-7703) and 50–51; antagonists include CATPB (46) and GLPG0974 (47) with species-specific activity; signaling via Gq/11 and Gi/o; arrestin-3 recruitment observed; tissue: immune cells, enteroendocrine cells, adipose tissue, pancreatic β-cells; functions: GLP-1 and PYY secretion in GI tract, inhibition of lipolysis in adipocytes, regulation of adipogenesis, insulin secretion, and inflammation; microbiota–SCFA–FFA2 axis linked to Tregs and inflammatory responses; allosteric modulators show probe-dependence.

  • FFA3 (GPR41)

    • Ligands: SCFAs (orthosteric), hexahydroquinolone allosteric ligands (AR420626, 52–54 series); orthosteric ligands limited; signaling primarily Gi/o; arrestin recruitment less pronounced than FFA2; tissue: enteroendocrine cells, pancreatic β-cells, sympathetic ganglia; functions: insulin secretion regulation, adiposity modulation; allosteric ligands act as PAMs with complex signaling switches (e.g., PAM vs NAM effects).

  • FFA4 (GPR120)

    • Ligands: LCFA/MFCA; orthosteric agonists (GW9508 with FFAR4 activity; TUG-891 as a potent FFAR4-selective agonist; NCG21, grifolic acid as FFAR4-active scaffolds); allosteric and biased ligands reported; Arg992.64 coordinates ligation to carboxylate; tissue: adipocytes, enteroendocrine cells, taste buds, immune cells, pancreatic islets; functions: GLP-1, GIP, and ghrelin regulation; anti-inflammatory actions largely arrestin-3–mediated; in adipocytes, FFAR4 promotes glucose uptake; in macrophages, FFAR4 modulates inflammatory signaling.

  • Other receptors and related notes

    • GPR84: MCFA receptor with pro-inflammatory signaling; synthetic ligands with limited potency; knockout studies show altered inflammatory responses.

    • Olfr78/OR51E2: olfactory receptor expressed in kidney and enteroendocrine cells; activated by SCFAs and lactate in some contexts; role in blood pressure regulation.

    • HCA2 (GPR109A): receptor for β-hydroxybutyrate and butyrate at higher concentrations; expressed in adipocytes and immune cells; anti-inflammatory signaling and metabolic regulation; nicotinic acid receptor with various agonists and allosteric ligands.

Key concepts throughout Table 1 and the notes include the existence of multiple ligand-binding pockets per receptor, ligand bias (G protein vs arrestin), constitutive activity/inverse agonism, and the need for cross-species pharmacology tools to translate findings from rodent models to humans. The pharmacology of FFARs remains a dynamic and evolving field with significant implications for metabolic and inflammatory disease therapies. The future direction emphasizes development of cross-species antagonists, orthosteric FFAR3 agonists, diverse allosteric ligands to exploit bias signaling, and deeper mechanistic understanding of receptor signaling in different tissues to guide therapeutic use.