Biology subjects

Forsman, H.

Publications and source records attributed to Forsman, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Free fatty acid 2 receptor regulates the NADPH oxidase activity induced by formyl peptide receptor specific agonists

The neutrophil NADPH oxidase is activated by signals generated by formyl peptide receptor (FPR) agonists recognized by FPR1 (fMLF) and FPR2 (WKYMVM), respectively. Also, the antagonists cyclosporin H (specific for FPR1) and PBP10 (specific for FPR2) inhibit the NADPH oxidase activity when induced by the two peptide agonists. When bound to its receptor, the non-activating positive allosteric modulator Cmp58, specific for the free fatty acid 2 receptor (FFA2R), affects not only the response induced by agonists specific for FFA2R, but also the activating potency but not the efficacy of the two FPR activating peptides. Even though Cmp58 is without effect on the efficacy of the response induced by the FPR agonists, the inhibitory effect of the respective FPR specific antagonists is reduced. This sensitivity shift was reversed by an FFA2R specific antagonist suggesting that two different signals generated by the FPRs activate the NADPH oxidase. According to a receptor trans-regulation model, the activated FPRs generate signals that directly activate the NADPH oxidase and signals that activate the allosterically modulated FFA2Rs to elicit activation of the NADPH.

pharmacology and toxicology↗

bioRxiv2025

Free Fatty Acid Receptor 2 Allosterism is Defined By Cellular Context

BackgroundAllosteric modulators offer a way to fine-tune GPCR signaling in the presence of endogenous ligands. The short-chain fatty acid receptor FFA2R (GPR43) recognizes propionate and allosteric ligands such as Cmp58 and AZ1729. We characterized FFA2R signaling and allosteric modulation using multiple cell models including HEK293, HL60 cells and primary human neutrophils. MethodsFFA2R activation was assessed using complementary assays in HEK293 and HL60 cells as well as primary human neutrophils. G protein activation and {beta}-arrestin recruitment were profiled using ebBRET biosensors. Ca2+ mobilization was measured with Fura-2, and reactive oxygen species (ROS) generation was quantified by isoluminol chemiluminescence. Pharmacological tools included the FFA2R antagonist CATPB, the Gq inhibitor YM-254890, and pertussis toxin (PTX). ResultsPropionate activated all tested G proteins except G12 in HEK293 cells and recruited both {beta}-arrestin1 and {beta}-arrestin2. The allosteric ligands Cmp58 and AZ1729 behaved as pathway-selective ago-PAMs. Alone they engaged a limited subset of G proteins with minimal {beta}-arrestin recruitment, whereas in the presence of propionate they selectively potentiated Gi1 while attenuating Gq/11 and Gi2/3. Fura-2 measurements coupled to YM-254890 treatment established that FFA2R couples to Gq-dependent Ca2+ mobilization in HEK293 cells; Cmp58, but not AZ1729, enhanced Ca2+ responses at submaximal propionate concentrations. In primary neutrophils, propionate elicited Ca2+ transients but did not trigger NADPH oxidase-dependent ROS on its own. Either Cmp58 or AZ1729 enabled propionate-driven ROS, and their combination produced robust ROS. Transient FFA2R expression in HL60 cells reconstituted this neutrophil-like functional profile, including allosteric activation of ROS. ConclusionsAcross systems, Ca2+ mobilization emerged as a conserved, receptor-proximal output of FFA2R, while allosteric modulation by Cmp58 and AZ1729 promoted Gi/o-biased signaling that enabled ROS generation. These data define pathway-selective allosterism at FFA2R and highlight Ca2+ mobilization and ROS as informative readouts for therapeutic strategies that exploit allosteric control.

cell biology↗

bioRxiv2025

The free fatty acid 2 receptor regulates the activation potency of C5a and the sensitivity to the antagonist avacopan

The complement derived neutrophil chemoattractant C5a, is a potent activator of the neutrophil superoxide anion generating NADPH oxidase. An allosteric modulator specific for the free fatty acid 2 receptor increases the activating potency but not the efficacy of C5a. The allosteric modulator also decreases the inhibitory effect of the C5a receptor antagonist avacopan, suggesting that the NADPH oxidase is activated by two different signaling pathways downstream of the receptor for C5a. While the allosteric modulator affected the C5a-mediated activation of the NADPH oxidase, the C5a-induced rise in the intracellular concentration of free calcium ions was unaffected. The C5a receptor and the free fatty acid receptor belong to the family of G protein-coupled receptors family. Our results show that the activated C5a receptors generate signals that directly activate the NADPH oxidase and allosterically modulated free fatty acid receptors which secondarily generate signals that elicit NADPH oxidase activity. This is in line with an earlier described receptor transactivation model, by which the fatty acid receptor is activated by receptor downstream signals generated by several different neutrophil receptors to which we now add the receptor for C5a. In addition, the fatty acid receptor was higher ranked than the receptor for C5a, in the neutrophil receptor hierarchy. The dual receptor trans-regulatory effects, by which the receptor for C5a activates the fatty acid receptor and by which this receptor reduces the C5a response, represent new regulatory mechanisms of importance for the NADPH oxidase activity in neutrophils.

cell biology↗

bioRxiv2025

THE ACTIN CYTOSKELETON CONTROLS NADPH OXIDASE ACTIVATION AND G PROTEIN RECRUITMENT MEDIATED BY NEUTROPHIL G-alpha-q-COUPLED RECEPTORS

Signaling by formyl peptide receptor 1 (FPR1), the prototype G protein-coupled receptor (GPCR) expressed in neutrophil leukocytes, is initiated by an activation of a G protein containing a Gi subunit. FPR1 activation results in an increase in the cytosolic concentration of free calcium ions ([Ca2+]i), and an activation of the superoxide anion producing NADPH oxidase. Receptor downstream signals generated by the danger molecule ATP recognized by the purinergic receptor P2Y2 are transduced by a G protein containing a Gq subunit. The neutrophil response induced by ATP also includes a transient rise in [Ca2+]i, but the downstream signals do not activate the NADPH oxidase. ATP can, however, activate this enzyme system through a receptor transactivation mechanism dependent not only on the ATP receptor but also on the free fatty acid receptor FFA2R, provided that this receptor is allosterically modulated. This occurs through a novel mechanism whereby FFA2R is activated from the cytosolic side of the plasma membrane by Gq transduced signals generated by the ATP receptor. Furthermore, in neutrophils with a disrupted actin cytoskeleton, ATP (as well as platelet activating factor; recognized by the Gq-coupled PAFR) becomes a potent NADPH oxidase activating agonist. At high concentrations of the actin cytoskeleton disrupting drug latrunculin A the activation was only partly reduced by Gq inhibition. More importantly, this response was also partly inhibited by pertussis toxin. The effects on the ATP-induced NADPH oxidase activity, of the Gq inhibitor and pertussis toxin were more and less pronounced, respectively, when the concentration of latrunculin A was reduced. Taken together, we show that in primary human neutrophils the actin cytoskeleton is part of the regulatory machinery that determines the activation of NADPH oxidase activation and the G protein recruitment profile downstream of activated of Gq-coupled GPCRs. HighlightsO_LIATP is a biased signaling agonist unable to activate the neutrophil NADPH oxidase C_LIO_LIATP activates the NADPH oxidase through P2Y2R mediated transactivation of FFA2R C_LIO_LIActin cytoskeleton disruption enables ATP to activate the NADPH oxidase C_LIO_LICytoskeleton regulated NADPH oxidase activation depends on Gi and Gq signaling C_LIO_LIThe actin cytoskeleton regulates the G protein recruitment profile of P2Y2R C_LI

immunology↗

bioRxiv2024

Activation and signaling characteristics of the hydroxy-carboxylic acid 3 receptor identified in human neutrophils through a microfluidic flow cell technique

Human neutrophils express numerous G protein-coupled receptors (GPCRs) of importance for immune regulation. Several of the functionally known neutrophil GPCRs, are not part of the human neutrophil proteome. To identify GPCRs not earlier shown to be expressed in human neutrophils, we utilized a microfluidic flow cell technique in combination with subcellular granule fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach added the hydroxy-carboxylic acid 3 receptor (HCA3R) to the human neutrophil proteome. The {beta}-oxidation intermediate 3-hydroxy-octanoic acid (3-OH-C8) is the primary endogenous agonist for this receptor which is highly expressed in adipocytes where it has anti-lipolytic effects. Literature describing the role and function of HCA3R in human neutrophils is scarce. We now show that 3-OH-C8 as well the synthetic HCA3R agonist IBC 293 activate human neutrophils determined as an increase in the intracellular concentration of free calcium ions ([Ca2+]i) and activation of the NADPH oxidase. However, in contrast to the rise in [Ca2+]i which could be triggered in naive neutrophils, pre-treatment of neutrophils was required for HCA3Rs to activate the NADPH oxidase. That is, the HCA3R-mediated NADPH oxidase activation occurred only in neutrophils pre-treated with either an actin cytoskeleton disrupter or an allosteric modulator targeting the GPCR termed free fatty acid receptor 2 (FFA2R). Our findings demonstrate that HCA3R is not only a new member of the human neutrophil proteome, but also exhibits functional activity with complex signaling pathways when stimulated with endogenous and synthetic HCA3R agonists.

immunology↗

bioRxiv2024

LTB4 is converted into a potent human neutrophil NADPH oxidase activator via a receptor transactivation mechanism in which the BLT1 receptor activates the free fatty acid receptor 2

The endogenous neutrophil chemoattractant leukotriene B4 (LTB4) is a biased signalling agonist that potently increases the intracellular concentration of free calcium ions ([Ca2+]i), but alone is a weak activator of the neutrophil superoxide anion (O2-)-generating NADPH oxidase. However, in this study we show that an allosteric modulator of the free fatty acid 2 receptor (FFA2R) converts LTB4 into a potent NADPH oxidase activating agonist. While an allosteric modulation of FFA2R was required for LTB4 receptor 1 (BLT1R)-mediated activation of the NADPH oxidase, the LTB4-induced increase in [Ca2+]i was not affected by the modulator. Thus, the biased BLT1R signalling pattern was altered in the presence of the allosteric FFA2R modulator, being biased with a preference towards the signals that activate the NADPH oxidase relative to an increase in [Ca2+]i. Both BLT1R and FFA2R belong to the family of G protein-coupled receptors (GPCRs), and our results show that a communication between the activated BLT1R and the allosterically modulated FFA2Rs generates signals that induce NADPH oxidase activity. This is consistent with a previously described receptor transactivation (crosstalk) model whereby the function of one neutrophil GPCR can be regulated by receptor downstream signals generated by another GPCR. Furthermore, the finding that an allosteric FFA2R modulator sensitises not only the response induced by orthosteric FFA2R agonists but also the response induced by LTB4, violates the receptor restriction properties that normally define the selectivity of allosteric GPCR modulators.

immunology↗

bioRxiv2023

Larixol is not an inhibitor of Gαi containing G proteins and lacks effect on signaling mediated by human neutrophil expressed formyl peptide receptors

Neutrophils express several G protein-coupled receptors (GPCRs) connected to intracellular Gi or Gq containing G proteins for down-stream signaling. To dampen GPCR mediated inflammatory processes, several inhibitors targeting the receptors and/or their down-stream signals, have been developed. Potent and selective inhibitors for Gq containing G proteins are available, but potent and specific inhibitors of Gi containing G proteins are lacking. Recently, Larixol, a compound extracted from the root of Euphorbia formosana, was shown to abolish human neutrophil functions induced by N-formyl-methionyl-leucyl-phenylalanine (fMLF), an agonist recognized by formyl peptide receptor 1 (FPR1) which couple to Gi containing G proteins. The inhibitory effect was suggested to be due to interference with/inhibition of signals transmitted by {beta}{gamma} complexes of the Gi containing G proteins coupled to FPR1. In this study, we applied Larixol, obtained from two different commercial sources, to determine the receptor- and G protein-selectivity of this compound in human neutrophils. However, our data show that Larixol not only lacks inhibitory effect on neutrophil responses mediated through FPR1, but also on responses mediated through FPR2, a Gi coupled GPCR closely related to FPR1. Furthermore, Larixol did not display any features as a selective inhibitor of neutrophil responses mediated through the Gq coupled GPCRs for platelet activating factor and ATP. Hence, our results imply that the inhibitory effects described for the root extract of Euphorbia formosana are not mediated by Larixol and that the search for a selective inhibitor of G protein dependent signals generated by Gi coupled neutrophil GPCRs must continue.

molecular biology↗

bioRxiv2022

The ketone body acetoacetate activates human neutrophils through FFA2R

Neutrophils express many surface receptors that sense environmental changes. One such sensor is FFA2R (free fatty acid receptor 2), a receptor that detects gut microbiota-derived short chain fatty acids. As such, FFA2R has been regarded as a molecular link between metabolism and inflammation. Our recent studies on FFA2R, using its endogenous agonist propionate in combination with allosteric modulators, have identified several novel aspects of FFA2R regulation. A recent study has also identified the ketone body acetoacetate as an endogenous ligand for mouse FFA2R. Whether human FFA2R also recognizes acetoacetate and how this recognition modulates human neutrophil functions has not been earlier investigated. In this study, we found that acetoacetate can induce a decrease of cAMP and translocation of {beta}-arrestin in cells overexpressing FFAR2. In addition, we show that similar to propionate, FFA2R specific allosteric modulators enhance acetoacetate-induced transient rise in cytosolic calcium, production of reactive oxygen species and cell migration in human neutrophils. In summary, we demonstrate that human neutrophils recognize the ketone body acetoacetate through FFA2R. Thus, our data further highlight the key role of FFA2R in inflammation and metabolism.

cell biology↗

bioRxiv2022

THE ALLOSTERICALLY MODULATED FREE FATTY ACID RECEPTOR 2 IS TRANSACTIVATED BY AN INCREASE IN THE CYTOSOLIC CONCENTRATION OF CALCIUM IONS

Allosterically modulated free fatty acid receptor 2 (FFA2R/GPR43) can be activated without the involvement of any orthosteric FFA2R agonist, by signals generated for example by P2Y2R, the G protein coupled receptor for ATP. An FFA2R specific positive allosteric modulator (PAM; Cmp58) was used to disclose the molecular mechanism by which signals generated by ATP/P2Y2R transactivates FFA2R. The P2Y2R induced signal that transactivates the allosterically modulated FFA2R was generated downstream of the Gq containing G protein that couple to P2Y2R. A receptor induced rise in the cytosolic concentration of ionized calcium ([Ca2+]i) was hypothesized to be the receptor transactivation signal. The Gq dependent transient rise in [Ca2+]i induced by the ATP activated P2Y2Rs was not affected by Cmp58. The hypothesis gained, however, support from the finding that the modulator transferred FFA2R to a Ca2+ sensitive state. The rise in [Ca2+]i induced by the Ca2+ specific ionophore ionomycin, activated the allosterically modulated FFA2R. The response induced by ionomycin was rapidly terminated and the FFA2Rs could then no longer be activated by the orthosteric FFA2R agonist propionate or be transactivated by the signal generated by the activated ATP receptor. The desensitized/non-responding state of FFA2R was, however, revoked by an earlier described cross-sensitizing/activating allosteric FFA2R modulator. The receptor transactivation of the allosterically modulated FFA2Rs, represent a unique regulatory receptor cross-talk mechanism by which the activity of a G protein coupled receptor is controlled by a signaling system operating from the cytosolic side of the plasma membrane. One-sentence summaryA P2Y2 receptor signal generated downstream of a Gq containing G protein transactivates the allosterically modulated FFA2 receptor

cell biology↗

bioRxiv2022

Signals Generated by Neutrophil Receptors for Danger Molecules Transactivate Allosterically Modulated FFA2R: Distinct response patterns are mediated by modulators recognized by different allosteric receptor sites

Positive allosteric modulators for free fatty acid receptor 2 (FFA2R/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFA2R was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFA2R from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFA2R in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFA2Rs, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFA2R activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFA2R activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFA2R activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFA2R modulator. O_LIThe allosterically modulated FFA2R is transactivated by signals generated by other GPCRs. C_LIO_LIThe PAF and ATP receptors transactivate FFA2R from the cytosolic side of the membrane. C_LIO_LIThe mechanisms that regulates activation of FFA2R from outside and inside differ. C_LI

cell biology↗

bioRxiv2021

Structural determinants in the Staphylococcus aureus derived phenol-soluble modulin-α2 peptide required for neutrophil formyl peptide receptor activation

Highly pathogenic Staphylococcus aureus strains produce phenol-soluble modulins (PSMs), peptides which are formylated N-terminally. Nanomolar concentrations of PSM2 are recognized by formyl peptide receptor 2 (FPR2), but unlike the prototypic FPR2 agonist WKYMVM, PSM2 is a biased signaling agonist. A shortened N-terminal PSM2 variant, consisting of the five N- terminal residues, is selectively recognized by the closely related FPR1, showing that the C- terminal part of PSM2 confers FPR2 selectivity, while the N-terminal part may interact with the FPR1 binding site. In the present study, a combined pharmacological and genetic approach, involving primary neutrophils and engineered FPR "knock-in" and "knock-out" cells, was used to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides and the receptor downstream signaling induced by these peptides. In comparison to the full-length PSM2, we show that the peptide in which the N-terminal part of PSM2 was replaced by fMIFL (an FPR1- selective peptide agonist) potently activates both FPRs for production of superoxide anions and {beta}- arrestin recruitment. A shortened analogue of PSM2 (PSM21-12), lacking the nine C-terminal residues activated both FPR1 and FPR2 to produce ROS, whereas {beta}-arrestin recruitment was only mediated through FPR1. However, a single amino acid replacement (Gly-2 to Ile-2) in PSM21-12 was sufficient to alter FPR2 signaling to include {beta}-arrestin recruitment, highlighting a key role of Gly-2 in conferring FPR2 biased signaling. In conclusion, we provide novel structural insights into FPR1 and FPR2 recognition as well as the signaling induced by interaction with formyl peptides derived from PSM2, originating from Staphylococcus aureus bacteria.

immunology↗

bioRxiv2021

Allosteric Receptor Modulation uncovers an FFAR2 antagonist as a positive orthosteric modulator/agonist in disguise

Two earlier described Free Fatty Acid Receptor 2 (FFAR2)-specific antagonists (CATPB and GLPG0974) have different receptor-interaction characteristics at the molecular/functional level. The inhibitory effect of the two antagonists, on the novel receptor-cross-talk activation signals generated by the ATP-receptor, show that both antagonists inhibit the effect of the positive allosteric FFAR2 modulators (PAMs) AZ1729 and Cmp58. No neutrophil activation was induced by AZ1729 or Cmp58 alone, but together they were co-agonistic PAMs and activated the superoxide generating NADPH-oxidase in neutrophils. This response was inhibited by CATPB but not by GLPG0974; in contrast, GLPG0974 acted as a positive modulator that increased the potency but not the efficacy of the response. At the signaling level, GLPG0974 changed the biased signaling induced by the co-agonistic PAMs, to include a rise in the cytosolic concentration of free calcium ions (Ca2+). This effect was reciprocal, i.e., GLPG0974 triggers a rise in intracellular Ca2+, demonstrating that GLPG0974 may act as an FFAR2 agonist. In summary, by studying the effects of the FFAR2 ligand GLPG0974 on neutrophils activation induced by the co-agonists AZ1729 and Cmp58, we reveal that GLPG0974 in addition to be an antagonist, displays also agonistic and positive FFAR2 modulating functions that affects the NADPH-oxidase activity and the receptor down-stream signaling induced by the two co-agonistic PAMs.

pharmacology and toxicology↗

bioRxiv2020

The two formyl peptide receptors differently regulate GPR84-mediated neutrophil NADPH-oxidase activity

Neutrophils express many G protein-coupled receptors (GPCRs) including the two formyl peptide receptors (FPR1 and FPR2) and the medium chain fatty acid receptor GPR84. The FPRs are known to define a hierarchy among neutrophil GPCRs, i.e., the GPCR-mediated response can be either suppressed or amplified by signals generated by FPRs. In this study, we investigated the position of GPR84 in the FPR-defined hierarchy regarding the activation of neutrophil NADPH-oxidase, an enzyme system designed to generate reactive oxygen species (ROS). When naive neutrophils are activated by GPR84 agonists a modest ROS release was induced. However, vast amounts of ROS production was induced by these GPR84 agonists in FPR2-desensitized neutrophils, and the response is inhibited not only by a GPR84 antagonist but also by an FPR2 specific antagonist. This suggests that the amplified GPR84 agonist response is achieved through a reactivation of the desensitized FPR2. In addition, the GPR84-mediated FPR2 reactivation was independent of {beta}-arrestin recruitment and sensitive to a protein phosphatase inhibitor. In contrast, the modest ROS production induced by GPR84 agonists was primarily suppressed in FPR1-desensitized neutrophils through hierarchical desensitization of GPR84 by FPR1 generated signals. In summary, our data show that FPRs control the NADPH-oxidase activity mediated through GPR84 in human neutrophils. While an amplified ROS generation is achieved by GPR84 agonists through reactivation of desensitized FPR2, FPR1 heterologously desensitizes GPR84 and by that suppresses the release of ROS induced by GPR84 agonists.

immunology↗

bioRxiv2020

Multiple ligand recognition sites in free fatty acid receptor 2 (FFAR2) direct distinct neutrophil activation patterns

Non-activating positive allosteric modulators specific for free fatty acid receptor 2 (FFAR2) increased the activity induced by orthosteric agonists to trigger a rise in intracellular Ca2+ ([Ca2+]i) and activate the O2- producing neutrophil NADPH-oxidase. In addition, two allosteric modulators (Cmp58 and AZ1729) recognized by different receptor domains on FFAR2, cooperatively triggered activation without any rise in [Ca2+]i. To gain insights into FFAR2 modulation and signaling, we set out to identify structurally diverse allosteric FFAR2 modulators. Initially, we identified two molecules that directly activate neutrophils and these were classified as an allosteric FFAR2 agonists and an orthosteric agonist, respectively. Based on the sensitizing effect on the neutrophil response to propionate, ten non-direct-activating molecules were classified as allosteric FFAR2 modulators. One of these synergistically activated neutrophils when combined with AZ1729, but not when combined with Cmp58. The remaining nine compounds synergistically induced the same type of biased neutrophil signaling but only when combined with Cmp58. The activation signals down-stream of FFAR2 when stimulated by two allosteric modulators with different binding sites were in most cases biased in that two complementary modulators together triggered an activation of the NADPH-oxidase, but no increase in [Ca2+]i. The neutrophil activation pattern achieved when two functionally "AZ1729- or "Cmp58-like" allosteric FFAR2 modulators were combined, supporting a model for activation in which FFAR2 has two different sites that selectively bind allosteric modulators. The novel neutrophil activation patterns and receptor down-stream signaling mediated by two cross-sensitizing allosteric modulators represent a new regulatory mechanism that controls FFAR2 receptor function.

cell biology↗

bioRxiv2020

Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2-complex

Two formyl peptide receptors (FPR1 and FPR2), abundantly expressed by neutrophils, regulate both pro-inflammatory tissue recruitment of neutrophils and resolution of inflammatory reactions. This dual functionality of the FPRs, opens for a possibility to develop receptor selective therapeutics as mechanism for novel anti-inflammatory treatments. In line with this, high throughput screening studies have identified numerous FPR ligands belonging to different structural classes, but a potent FPR1 agonist with defined biased signaling and functional selectivity has not yet been reported. In this study, we used an FPR1 selective small compound agonist (RE) that represents a chemical entity developed from NOX2 activators identified from our earlier screening studies (WO2012127214). This FPR1 agonist potently activates neutrophils to produce reactive oxygen species (ROS, EC50 ~1 nM), whereas it is a weaker chemoattractant than the prototype FPR1 agonist fMLF. At the signaling level, RE has a strong bias towards the PLC-PIP2-Ca2+ pathway and ERK1/2 activation but away from {beta}-arrestin recruitment and the ability to recruit neutrophils chemotactically. In addition, FPR1 when activated by RE could cross-regulate other receptor-mediated neutrophil functions. In comparison to the peptide agonist fMLF, RE is more resistant to oxidization-induced inactivation by the MPO-H2O2-halide system. In summary, this study describes as a novel FPR1 agonist displaying a biased signaling and functional selectivity when activating FPR1 in human blood neutrophils. RE could possibly be a useful tool compound not only for further mechanistic studies of the regulatory role of FPR1 in inflammation in vitro and in vivo, but also for developing FPR1specific drug therapeutics.

cell biology↗

bioRxiv2019

Interdependent allosteric FFA2R modulators synergistically induce functional selective activation and desensitization in neutrophils

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFA2R), transfers the orthosteric FFA2R agonists propionate and the P2Y2R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFA2R modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFA2R when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free Ca2+. In addition, following AZ1729/Cmp58 activation, signaling by the desensitized FFA2Rs was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca2+. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFA2R modulators represents a new regulatory mechanism that controls receptor signaling. Significance StatementA novel activation mechanism of a G-protein coupled free fatty acid receptor (FFA2R), is synergistically triggered by two otherwise non-activating allosteric modulators in the absence of orthosteric agonists. The receptor down-stream signaling proved to be functionally selective (biased); a superoxide generating enzyme is assembled and activated without involvement of the Ca2+ signaling pathway. The novel activation mechanism and the receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFA2R modulators, represents a new regulatory mechanism for control of GPCR-signaling.

pharmacology and toxicology↗

bioRxiv2019

The putative Gαq-inhibiting pepducin P4Pal10 distinctly modulates function of the Gαi-coupled receptors FPR2 and FFA2R in neutrophils

A novel mechanism of action was described, when a protease-activated receptor 4 (P4Pal10) derived lipopeptide (pepducin) was shown to inhibit signaling downstream of several unrelated Gq-coupled receptors. We show that this putative Gq-inhibiting pepducin lacks inhibitory effects on signaling downstream of the Gq-coupled receptors for ATP (P2Y2R) and PAF (PAFR) expressed in human neutrophils. P4Pal10 inhibited however, signaling in neutrophils activated with agonists for the Gi-coupled formyl peptide receptor 2 (FPR2) but not the closely related FPR1. In addition, the P4Pal10 pepducin was turned into an activating agonist in the presence of an allosteric modulator selective for free fatty acid receptor 2 (FFA2R). The results presented thus reveal Gq-independent effects of P4Pal10 in modulating FPR2- and FFA2R-mediated neutrophil activation.

immunology↗

bioRxiv2019

Functional and signaling characterization of the neutrophil FPR2 selective agonist Act-389949

Despite the steadily increased numbers of formyl peptide receptor (FPR) ligands identified over the years, few have been characterized in studies using animal disease models and even less have entered clinical trials in human subjects. A small-molecule compound, Act-389949, was however recently tested in a phase I clinical trial and found to be safe and well tolerated in healthy human subjects. The desired anti-inflammatory property of Act-389949 was proposed to be mediated through FPR2, one of the FPRs expressed in neutrophils, but no basic characterization was included in the study. To gain more insights into FPR2 recognition of this first-in-class compound for future utility of the agonist, we have in this study determined the receptor preference and down-stream signaling characteristics induced by Act-389949 in human blood neutrophils isolated from healthy donors. Our data demonstrate that Act-389949 is an agonist for FPR2 that triggers functional/signaling repertoires comparable to what has been earlier described for other FPR2 agonists, including neutrophil chemotaxis, granule mobilization and activation of the NADPH-oxidase. In fact, Act-389949 was found to be as potent as the prototype FPR2 peptide agonist WKYMVM and had the advantage of being resistant to oxidation by the MPO-H2O2-halide derived oxidants, as compared to the sensitive WKYMVM. The down-stream signals generated by Act-389949 include an FPR2-dependent and Gq-independent transient rise in intracellular Ca2+ and recruitment of {beta}-arrestin. In summary, our data show that Act-389949 serves as an excellent tool-compound for further dissection of FPR2-regulated activities in vitro and in vivo. Potent and stable FPR ligands such as Act-389949 may therefore be used to develop the next generation of FPR signaling regulating anti-inflammatory therapeutics.

pharmacology and toxicology↗