Search

The formyl peptide receptors FPR1 and FPR2 are abundantly expressed by neutrophils, in which they regulate proinflammatory tissue recruitment of inflammatory cells, the production of reactive oxygen species (ROS), and resolution of inflammatory reactions. The unique dual functionality of the FPRs makes them attractive targets to develop FPR‐based therapeutics as novel anti‐inflammatory treatments. The small compound RE‐04‐001 has earlier been identified as an inducer of ROS in differentiated HL60 cells but the precise target and the mechanism of action of the compound was has until now not been elucidated. In this study, we reveal that RE‐04‐001 specifically targets and activates FPR1, and the concentrations needed to activate the neutrophil NADPH‐oxidase was very low (EC50 ∼1 nM). RE‐04‐001 was also found to be a neutrophil chemoattractant, but when compared to the prototype FPR1 agonist N‐formyl‐Met‐Leu‐Phe (fMLF), the concentrations required were comparably high, suggesting that signaling downstream of the RE‐04‐001‐activated‐FPR1 is functionally selective. In addition, the RE‐04‐001‐induced response was strongly biased toward the PLC‐PIP2‐Ca2+ pathway and ERK1/2 activation but away from β‐arrestin recruitment. Compared to the peptide agonist fMLF, RE‐04‐001 is more resistant to inactivation by the MPO‐H2O2‐halide system. In summary, this study describes RE‐04‐001 as a novel small molecule agonist specific for FPR1, which displays a biased signaling profile that leads to a functional selective activating of human neutrophils. RE‐04‐001 is, therefore, a useful tool, not only for further mechanistic studies of the regulatory role of FPR1 in inflammation in vitro and in vivo, but also for developing FPR1‐specific drug therapeutics. [ABSTRACT FROM AUTHOR]

[Display omitted] Formyl peptide receptor 1 (FPR1), a G protein-coupled receptor expressed in phagocytes, recognizes short N-formylated peptides originating from proteins synthesized by bacteria and mitochondria. Such FPR1 agonists are important regulators of neutrophil functions and by that, determinants of inflammatory reactions. As FPR1 is implicated in promoting both pro-inflammatory and pro-resolving responses associated with inflammatory diseases, characterization of ligands that potently and selectively modulate FPR1 induced functions might be of high relevance. Accordingly, a number of FPR1 specific antagonists have been identified and shown to inhibit agonist binding or receptor down-stream signaling as well as neutrophil functions such as granule secretion and NADPH oxidase activity. The inhibitory effect on neutrophil chemotaxis induced by FPR1 agonists has generally not been part of basic antagonist characterization. In this study we show that the inhibitory effects on neutrophil chemotaxis of established FPR1 antagonists (i.e., cyclosporin H, BOC1 and BOC2) are limited. Our data demonstrate that the recently described small molecule AZ2158 is a potent and selective FPR1 antagonist in human neutrophils. In contrast to the already established FPR1 antagonists, AZ2158 also potently inhibits chemotaxis. Whereas the cyclosporin H inhibition was agonist selective, AZ2158 inhibited the FPR1 response induced by both a balanced and a biased FPR1 agonist equally well. In accordance with the species specificity described for many FPR1 ligands, AZ2158 was not recognized by the mouse orthologue of FPR1. Our data demonstrate that AZ2158 may serve as an excellent tool compound for further mechanistic studies of human FPR1 mediated activities. [ABSTRACT FROM AUTHOR]

Pepducins constitute a unique class of G-protein coupled receptor (GPCR) modulating lipopeptides. Pepducins with inhibitory effects on neutrophils could potentially be developed into anti-inflammatory pharmaceuticals. A pepducin with a peptide sequence identical to the third intracellular loop of FPR1 was found to inhibit neutrophil functions including granule mobilization and superoxide production. This FPR1-derived pepducin selectively inhibited signaling and cellular responses through FPR2, but not FPR1 as expected. Binding to the neutrophil surface of a conventional FPR2 agonist is also inhibited. The fatty acid is essential for inhibition and pepducins with shorter peptides lose in potency. In summary, a pepducin designed to target FPR1 was found to hijack FPR2 and potently inhibit neutrophil functions. [ABSTRACT FROM AUTHOR]

Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPRdes) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPRdes back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR) and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPRdes as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPRdes also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPRdes initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species. [ABSTRACT FROM AUTHOR]

The neutrophil formyl peptide receptors, FPRl and FPR2, play critical roles for inflammatory reactions, and receptor-specific antagonists/inhibitors can possibly be used to facilitate the resolution of pathological inflammatory reactions. A 10-aa-long rhodamine-linked and membrane-permeable peptide inhibitor (PBP10) has such a potential. This FPR2 selective inhibitor adopts a phosphatidylinositol 4,5-bisphosphate-binding sequence in the cytoskeletal protein gelsolin. A core peptide, RhB-QRLFQV, is identified that displays inhibitory effects as potent as the full-length molecule. The phosphatidylinositol 4,5-bisphosphate-binding capacity of PBP10 was not in its own sufficient for inhibition. A receptor in which the presumed cytoplasmic signaling C-terminal tail of FPR2 was replaced with that of FPR1 retained the PBP10 sensitivity, suggesting that the tail of FPR2 was not on its own critical for inhibition. This gains support from the fact that the effect of cell-penetrating lipopeptide (a pepducin), suggested to act primarily through the third intracellular loop of FPR2, was significantly inhibited by PBP10. The third intracellular loops of FPR1 and FPR2 differ in only two amino acids, but an FPR2 mutant in which these two amino acids were replaced by those present in FPR1 retained the PBP10 sensitivity. In summary, we conclude that the inhibitory activity on neutrophil function of PBP10 is preserved in the core sequence RhB-QRLFQV and that neither the third intracellular loop of FPR2 nor the cytoplasmic tail of the receptor alone is responsible for the specific inhibition. [ABSTRACT FROM AUTHOR]

Abstract: The neutrophil formyl peptide receptors (FPR1 and FPR2) are G-protein coupled receptors that can induce pro-inflammatory as well as anti-inflammatory activities when activated. Accordingly, these receptors may become therapeutic targets for the development of novel drugs to be used for reducing the inflammation induced injuries in asthma, rheumatoid arthritis, Alzheimer''s disease, cardiovascular diseases and traumatic shock. We screened a library of more then 50K small compounds for an ability of the compounds to induce a transient rise in intracellular Ca2+ in cells transfected to express FPR2 (earlier called FPRL1 or the lipoxin A4 receptor). Ten agonist hits were selected for further analysis representing different chemical series and five new together with five earlier described molecules were further profiled. Compounds 1–10 gave rise to a calcium response in the FPR2 transfectants with EC50 values ranging from 4×10−9 M to 2×10−7 M. All 10 compounds activated human neutrophils to release superoxide, and based on the potency of their activity, the three most potent activators of the neutrophil NADPH-oxidase were further characterized. These three agonists were largely resistant to inactivation by neutrophil produced reactive oxygen species and shown to trigger the same functional repertoire in neutrophils as earlier described peptide agonists. Accordingly they induced chemotaxis, granule mobilization and secretion of superoxide. Interestingly, the oxidase activity was largely inhibited by cyclosporine H, an FPR1 selective antagonist, but not by PBP10, an FPR2 selective inhibitor, suggesting that FPR1 is the preferred receptor in neutrophils for all three agonists. [Copyright &y& Elsevier]

Abstract: Human neutrophils express formyl peptide receptor 1 and 2 (FPR1 and FPR2), two highly homologous G-protein-coupled cell surface receptors important for the cellular recognition of chemotactic peptides. They share many functional as well as signal transduction features, but some fundamental differences have been described. One such difference was recently presented when the FPR2-specific ligand MMK-1 was shown to trigger a unique signal in neutrophils [S. Partida-Sanchez, P. Iribarren, M.E. Moreno-Garcia, et al., Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose, J. Immunol. 172 (2004) 1896–1906]. This signal bypassed the emptying of the intracellular calcium stores, a route normally used to open the store-operated calcium channels present in the plasma membrane of neutrophils. Instead, the binding of MMK-1 to FPR2 was shown to trigger a direct opening of the plasma membrane channels. In this report, we add MMK-1 to a large number of FPR2 ligands that activate the neutrophil superoxide-generating NADPH-oxidase. In contrast to earlier findings we show that the transient rise in intracellular free calcium induced by MMK-1 involves both a release of calcium from intracellular stores and an opening of channels in the plasma membrane. The same pattern was obtained with another characterized FPR2 ligand, WKYMVM, and it is also obvious that the two formyl peptide receptor family members trigger the same type of calcium response in human neutrophils. [Copyright &y& Elsevier]

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 MPO-H 2 O 2 -halide derived oxidants, as compared to the sensitive WKYMVM. The down-stream signals generated by Act-389949 include an FPR2-dependent and Gαq-independent transient rise in intracellular Ca2+ and recruitment of β-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. [ABSTRACT FROM AUTHOR]

Structural optimization of a peptidomimetic antagonist of formyl peptide receptor 2 (FPR2) was explored by an approach involving combination of elements from the two most potent FPR2 antagonists described: a Rhodamine B-conjugated 10-residue gelsonin-derived peptide (i.e., PBP10, RhB-QRLFQVKGRR-OH) and the palmitoylated α-peptide/β-peptoid hybrid Pam-(Lys-βNspe)6-NH2. This generated an array of hybrid compounds from which a new subclass of receptor-selective antagonists was identified. The most potent representatives displayed activity in the low nanomolar range. The resulting stable and potent FPR2-selective antagonists (i.e., RhB-(Lys-βNphe)n-NH2; n = 4-6) are expected to become valuable tools in further elucidation of the physiological role of FPR2 in health and disease. [ABSTRACT FROM AUTHOR]

The formyl peptide receptor (FPR) gene family has a complex evolutionary history and comprises eight murine members but only three human representatives. To enable translation of results obtained in mouse models of human diseases, more comprehensive knowledge of the pharmacological similarities/differences between the human and murine FPR family members is required. Compared to FPR1 and FPR2 expressed by human neutrophils, very little is known about agonist/antagonist recognition patterns for their murine orthologues, but now we have identified two potent and selective formylated peptide agonists (fMIFL and PSMα2) for Fpr1 and Fpr2, respectively. These peptides were used to determine the inhibition profile of a set of antagonists with known specificities for the two FPRs in relation to the corresponding murine receptors. Some of the most potent and selective antagonists for the human receptors proved to be devoid of effect on their murine orthologues as determined by their inability to inhibit superoxide release from murine neutrophils upon stimulation with receptor-specific agonists. The Boc-FLFLF peptide was found to be a selective antagonist for Fpr1, whereas the lipidated peptidomimetic Lau-(Lys-βNSpe) 6 -NH 2 and the hexapeptide WRW 4 were identified as Fpr2-selective antagonists. [ABSTRACT FROM AUTHOR]

Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists for this receptor display potent activity for the human receptor (FPR2) but low activity for the mouse receptor orthologue (Fpr2), rendering them inapplicable in murine models of human disease. Here we describe a novel FPR2 agonist, the proteolytically stableα-peptide/βNphe)6-NH2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca2+ concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated with lauric acid (C12 fatty acid), which is linked to a peptide/peptoid repeat ((Lys-βNphe)6-NH2). Both the fatty acid moiety and the (S)-Aoc residue were required for FPR2/Fpr2 activation. This type of proteolytically stable FPR2-specific peptidomimetics may serve as valuable tools for future analysis of FPR2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases. [ABSTRACT FROM AUTHOR]

Proper recruitment and activation of neutrophils to/at sites of infection/inflammation relies largely on the surface expression of chemoattractant receptors of which a formyl peptide receptor (FPR1) was the first to be cloned and characterized in more detail. This receptor displays high affinity for bacterial- or mitochondrial-derived peptides that contain a formylated methionine in the N-terminus. The neutrophil chemoattractant receptors belong to the group of 7-transmembrane domain receptors that signal through activation of heterotrimeric G proteins. These receptors have been shown to be important in host defense against microbial intruders and in regulating inflammatory reactions. The two FPRs (FPR1, FPR2) expressed in neutrophils share significant sequence homology and bind many structurally diverse activating (agonistic) and inhibiting (antagonistic) ligands, ranging from peptides to lipopeptides containing peptide sequences derived from intracellular regions of the FPRs. Recent structural and functional studies of the two neutrophil FPRs have generated important information for our understanding of general pharmacological principles, governing regulation of neutrophil function and inflammation and increased knowledge of more general G-protein coupled receptor features, such as ligand recognition, biased signaling, allosteric modulation, and a unique receptor cross-talk phenomenon. This article aims to summarize recent discoveries and pharmacological characterization of neutrophil FPRs and to discuss unmet challenges, including recognition by the receptors of diverse ligands and how biased signals mediate different biological effects. [ABSTRACT FROM AUTHOR]

Several G-protein-coupled receptors (GPCRs) can be activated or inhibited in a specific manner by membrane-permeable pepducins, which are short palmitoylated peptides with amino acid sequences identical to an intracellular domain of the receptor to be targeted. Unlike the endogenous P2Y 2 R agonist ATP, the P2Y 2 Pal IC2 pepducin, which has an amino acid sequence corresponding to the second intracellular loop of the human ATP receptor (P2Y 2 R), activated the superoxide anion-generating NADPH-oxidase in neutrophils. In addition to having a direct effect on neutrophils, the P2Y 2 R pepducin converted naïve neutrophils to a primed state, which secondarily responded to ATP by producing superoxide. A pepducin with a peptide identical to the third intracellular loop of P2Y 2 R (P2Y 2 Pal IC3 ) exhibited the same basic functions as P2Y 2 Pal IC2 , whereas one with a peptide that was identical to the first intracellular loop (P2Y 2 Pal IC1 ) lacked these functions. The responses induced in neutrophils by the P2Y 2 R pepducins were not inhibited by the P2Y 2 R antagonist AR-C118925, and the receptor desensitization profile suggested the involvement of FPR2 rather than P2Y 2 R. Accordingly, antagonists/inhibitors of FPR2 attenuated the activities of the P2Y 2 R pepducins, which also selectively activated FPR2-overexpressing cells. In summary, we show that pepducins supposed to target P2Y 2 R activate human neutrophils through FPR2. We also show that the P2Y 2 Pal IC2 pepducin can convert ATP from a non-activating agent to a potent neutrophil NADPH-oxidase activator. The molecular basis of this phenomenon involves cross-talk between the receptor/ligand pairs of P2Y 2 R/ATP and FPR2/P2Y 2 -pepducin. [ABSTRACT FROM AUTHOR]

Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs. [ABSTRACT FROM AUTHOR]

Abstract: Neutrophils express several G-protein coupled receptors (GPCRs) and they cross regulate each other. We described a novel cross-talk mechanism in neutrophils, by which signals generated by the receptor for ATP (P2Y2) reactivate desensitized formyl peptide receptors (FPRs) so that these ligand-bound inactive FPRs resume signaling. At the signaling level, the cross-talk was unidirectional, i.e., P2Y2 ligation reactivated FPR, but not vice versa and was sensitive to the phosphatase inhibitor calyculinA. Further, we show that the cross talk between P2Y2 and FPR bypassed cytosolic Ca2+ transients and did not rely on the actin cytoskeleton. In summary, our data demonstrate a novel cross-talk mechanism that results in reactivation of desensitized FPRs and, an amplification of the neutrophil response to ATP. [Copyright &y& Elsevier]

Abstract: The neutrophil formyl peptide receptors (FPR1 and FPR2) are members of the G-protein coupled receptor family. The signals generated by occupied FPRs are both pro-inflammatory and anti-inflammatory. Accordingly, these receptors have become a therapeutic target for the development of novel drugs that may be used to reduce injuries in inflammatory diseases including asthma, rheumatoid arthritis, Alzheimer''s disease and cardiovascular diseases. To support the basis for a future pharmacological characterization, we have identified a small molecular non-peptide inhibitor with selectivity for FPR1. We used the FPR1 and FPR2 specific ligands fMLF and WKYMVM, respectively, and an earlier described ratio technique, to determine inhibitory activity combined with selectivity. We show that the compound 3,5-dichloro-N-(2-chloro-5-methyl-phenyl)-2-hydroxy-benzamide (BVT173187) fulfills the criteria for an FPR1 inhibitor selective for FPR1 over FPR2, and it inhibits the same functional repertoire in neutrophils as earlier described peptide antagonists. Accordingly, the new inhibitor reduced neutrophil activation with FPR1 agonists, leading to mobilization of adhesion molecules (CR3) and the generation of superoxide anion from the neutrophil NADPH-oxidase. The effects of a number of structural analogs were determined but these were either without activity or less active/specific than BVT173187. The potency of the new inhibitor for reduction of FPR1 activity was the same as that of the earlier described FPR1 antagonist cyclosporine H, but signaling through the C5aR and CXCR (recognizing IL8) was also affected by BVT173187. [Copyright &y& Elsevier]

Abstract: Neutrophil accumulation at an inflammatory site or an infected tissue is dependent on the recognition of chemotactic peptides that bind to G-protein coupled receptors (GPCRs) exposed on the surface of the inflammatory cells. A GPCR activated by a chemoattractant quickly becomes refractory to further stimulation by ligands using the same receptor. This desensitization phenomenon has been used frequently to characterize new receptor agonists and to determine receptor hierarchies. In this study we show that desensitization patterns differ depending on what read out systems are used to follow neutrophil activity. When monitoring release of superoxide, neutrophils were readily desensitized against repeated stimulations with the prototypical agonist formylmethionyl-leucyl-phenylalanine (fMLF). In contrast, neutrophils were not desensitized for fMLF when cell activity was determined by intracellular calcium ([Ca2+]i). The difference observed was dependent on inactivation of the agonist in one read out system but not in the other, and we suggest several different solutions to the problem. Agonist inactivation occurs through a myeloperoxidase (MPO)/hydrogen peroxide catalyzed reaction, and the problem could be avoided by using a FACS based technique to measure the change in [Ca2+]i, by the use of an agonist insensitive to the MPO/hydrogen peroxide-system or, by adding an MPO inhibitor or a scavenger that removes either superoxide/hydrogen peroxide or the MPO-derived metabolites. [Copyright &y& Elsevier]