Huamei Forsman

Christina Kalderén

Anna Nordin

Erik Nordling

Annika Jernmalm Jensen

Claes Dahlgren
email: claes.dahlgren@microbio.gu.se

Stable Formyl Peptide Receptor Agonists That Activate the Neutrophil NADPH-oxidase Identified Through Screening of a Compound Library

Keywords: chemoattractant receptors, formyl peptide, receptor agonists, NADPH-oxidase, library neutrophils, formyl peptide receptors, receptor antagonists, signal transduction, NADPH-oxidase, chemoattractant receptors

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 50 K small compounds for an ability of the compounds to induce a transient rise in intracellular Ca 2+ in cells transfected to express FPR2 (earlier called FPRL1 or the lipoxin A 4 receptor). Ten agonists 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 EC 50 values ranging from 4 x 10 -9 M to 2 x 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.

Introduction

Professional phagocytes such as neutrophil granulocytes play important roles in host defence against pathogenic microbes, but they are also key regulators in the fine-tuning of inflammatory reactions. The functional repertoires of these cells are triggered and regulated through binding of different inflammatory mediators that are recognized by specific cell surface receptors expressed on resting or primed cells [START_REF] Murphy | The molecular biology of leukocyte chemoattractant receptors[END_REF][START_REF] Miller | Chemotaxis receptors and signaling[END_REF]. Receptors that belong to the "Gprotein coupled, seven transmembrane family of receptors (GPCRs)" are well represented on neutrophils, and the function of one of these, the formyl peptide receptor (FPR1) has been extensively studied. This receptor is a member of the chemoattractant receptor subfamily that recognizes N-formylated peptides [START_REF] Ye | Structure and function of leukocyte chemoattractant receptors[END_REF][START_REF] Fu | Ligand recognition and activation of formyl peptide receptors in neutrophils[END_REF][START_REF] Ye | International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family[END_REF]. As such peptides are derived from bacterial or mitochondrial proteins, it has been proposed that a primary FPR1 function is to promote trafficking of phagocytic myeloid cells to sites of infection and tissue damage, where they exert antibacterial effector functions and clear cell debris.

Accordingly the prototype agonist formylmethionyl-leucyl-phenylalanine (fMLF) is a bacterial produced high-affinity FPR1 agonist that triggers a variety of biologic activities in neutrophils, including chemotaxis, granule secretion, cytokine production, and superoxide release, the latter generated through an activation of an electron transporting NADPH-oxidase [START_REF] Arruda | NADPH oxidase activity: In the crossroad of neutrophil life and death[END_REF]. The reactive oxygen species (ROS) derived from the activated NADPH-oxidase are essential for a successful clearance of bacteria during infection as illustrated by the fact that patients lacking a functional oxidase suffer repeatedly from severe bacterial infections [START_REF] Nauseef | How human neutrophils kill and degrade microbes: an integrated view[END_REF]. In addition to FPR1, neutrophils express also a closely related receptor, FPR2, that belongs to the same receptor family [START_REF] Fu | Ligand recognition and activation of formyl peptide receptors in neutrophils[END_REF][START_REF] Ye | International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family[END_REF]. This receptor was initially orphan, but over the last 15 years, a wide range of agonists have been identified. All peptide A c c e p t e d M a n u s c r i p t 4 agonists described but two, possess the same pro-inflammatory activities as the FPR1 agonists. The two exceptions are i) peptides cleaved off from the N-terminus of the calcium regulated protein annexin I [START_REF] Hayhoe | Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement[END_REF][START_REF] Martin | Annexin-1 modulates repair of gastric mucosal injury[END_REF] and ii) a 21 amino acids long synthetic peptide identified through a library screen [START_REF] Hecht | s c r i p t 29[END_REF]. Activation of FPR2 with these two peptide agonists triggers a unique signalling pathway that inhibits neutrophil function and induces anti-inflammatory activities in different model systems. The same functional link -FPR2 and induction of antiinflammatory activitieshas been established for the eicosanoid LXA 4 [START_REF] Serhan | Anti-inflammatory and proresolving lipid mediators[END_REF], but our earlier published data suggest that this lipid mediator uses a receptor distinct from FPR2 [START_REF] Christophe | Phagocyte activation by Trp-Lys-Tyr-Met-Val-Met, acting through FPRL1/LXA4R, is not affected by lipoxin A4[END_REF][START_REF] Forsman | Lipoxin A(4) metabolites/analogues from two commercial sources have no effects on TNF-alpha-mediated priming or activation through the neutrophil formyl peptide receptors[END_REF].

The eicosanoid is easily oxidized and by that biologically inactivated [START_REF] Serhan | Anti-inflammatory and proresolving lipid mediators[END_REF][START_REF] Christophe | Phagocyte activation by Trp-Lys-Tyr-Met-Val-Met, acting through FPRL1/LXA4R, is not affected by lipoxin A4[END_REF][START_REF] Forsman | Lipoxin A(4) metabolites/analogues from two commercial sources have no effects on TNF-alpha-mediated priming or activation through the neutrophil formyl peptide receptors[END_REF], and this is true also for many of the peptide agonists [START_REF] Forsman | The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPHoxidase and inactivation of the agonist[END_REF].

Through the use of small molecule libraries screening, a series of non-peptide agonists for the FPRs have been identified during the last couple of years. When investigated, the functional repertoire triggered by small non-peptide agonists such as the Quinazolinonoe (Quin-C1) derivatives that selectively bind FPR2 [START_REF] Zhou | Pharmacological characterization of a novel nonpeptide antagonist for formyl peptide receptor-like 1[END_REF],AG14 that selectively binds FPR1 [START_REF] Schepetkin | High-throughput screening for small-molecule activators of neutrophils: identification of novel Nformyl peptide receptor agonists[END_REF], and pyrazolone that binds both receptors [START_REF] Sogawa | The Pyrazolone Originally Reported to Be a Formyl Peptide Receptor (FPR) 2/ALX-Selective Agonist Is Instead an FPR1 and FPR2/ALX Dual Agonist[END_REF], resembles that of the pro-inflammatory peptides and not that of the anti-inflammatory agonists. Using a chemolibrary screening approach it was recently shown that agonists of FPR1/FPR2 include compounds with wide chemical diversity [START_REF] Kirpotina | Identification of novel small-molecule agonists for human formyl peptide receptors and pharmacophore models of their recognition[END_REF]. Owing to the fact that the function of FPR2 during recent years has been linked not only to innate immune functions but also to chronic inflammation in systemic inflammatory and auto-immune diseases, clinically oriented research focusing on FPR2 has been carried out.

The aim of this study was to identify novel small molecule FPR2 agonists by screening a small molecule library, and of particular interest was to determine the ability of the new agonists, to activate the neutrophil NADPH-oxidase. This could possibly be used to determine if a compound should be regarded as pro-inflammatory or have the potential of being anti-A c c e p t e d M a n u s c r i p t 5 inflammatory. After screening more than 50 000 compounds, 5 structurally diverse FPR2 agonists were selected (from the more then 500 hits that were confirmed as agonists from the screen) and further investigated together with five "hits" identical to earlier described FPR2 agonists. All the agonists were found to activate the neutrophils to produce superoxide. The three most potent agonists, one earlier described pyrazolone compound [START_REF] Burli | Potent hFPRL1 (ALXR) agonists as potential anti-inflammatory agents[END_REF] and two new compound, were further characterized and found to be insensitive to inactivation by oxidants and possess all the functional characteristics of earlier described pro-inflammatory peptide agonists. Upon binding to human neutrophils, they induce granule mobilization, directional cellular migration and production of superoxide anion. Our data also show that these novel agonists preferentially use FPR1, and in contrast to many of the earlier described peptide agonists, they are resistant to oxidation by neutrophil-derived oxygen radicals. We suggest that these small non-peptide compounds could serve as good tools for enhanced understanding of the structure-function relationships of FPRs and for in vitro/in vivo therapeutic studies in which long lived agonist are required.

A c c e p t e d M a n u s c r i p t 6

MATERIAL AND METHODS

Materials.

Part of the chemical library was selected on the basis of diversity from Biovitrum and iNovica compound collections which consists of 40 000 compounds. The other part (an additional 10 000 compounds) was based on reference compounds collected from the literature ( [START_REF] Burli | Potent hFPRL1 (ALXR) agonists as potential anti-inflammatory agents[END_REF], http://www.wipo.int/pctdb/en/wo.jsp?wo=2005047899), and used as input to computational techniques allowing us to detect compounds with similar properties. In order to do this a pharmacophore was built in Phase [START_REF] Dixon | PHASE: a new engine for pharmacophore perception, 3D QSAR model development, and 3D database screening: 1. Methodology and preliminary results[END_REF] and it consisted of a central acceptor/donor pair flanked on one side of an acceptor and a hydrophobic group and on the other side by one aromatic group and one hydrophobic group (see supplementary Fig 1). A Tuplet hypothesis was then constructed using triplets [START_REF] Abrahamian | Efficient generation, storage, and manipulation of fully flexible pharmacophore multiplets and their use in 3-D similarity searching[END_REF], and used in parallel with the Phase pharmacophore to find matching compounds. The search results were used to select targeted libraries that showed a large proportion of hits in either of the searches. In addition discrete compounds that were found using either method were also selected for the screening, and the additional 10 000 compounds were selected in this way. The purity and identity of the used compounds were verified using NMR spectroscopy, HPLC, and mass spectroscopy. The compounds were dissolved in DMSO at a concentration of 10 mM and stored at -80°C. Isoluminol, TNF-, fMLF, as well as C5a were obtained from Sigma Chemical Co. (St. Louis, Mo., USA). IL-8 was from R&D systems (Minneapolis, MN., USA). The hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) was synthesized and purified by HPLC by Alta Bioscience (University of Birmingham, Birmingham, United Kingdom). Horse radish peroxidase (HRP) was purchased from Boehringer-Mannheim (Mannheim, Germany). Dextran and Ficoll-Paque were from Pharmacia (Uppsala, Sweden). The receptor antagonist Trp-Arg-Trp-Trp-Trp-Trp-CONH 2 (WRW 4 ) was from GenScript Corp (Piscataway,NJ., USA) and cyclosporin H was kindly provided by Novartis Pharma (Basel, Switzerland). The peptides/receptor antagonists The peptide QRLFQVKGRR (gelsolin residues 160-169), prepared by solid phase peptide synthesis and coupled to rhodamine as described [START_REF] Cunningham | Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly[END_REF].

Screening assay with FPR2 transfectants --determination of changes in cytosolic

calcium. CHO-K1 cells stably expressing the human FPR2 and Galpha16 were plated in HAMSF12 with glutamax, 10 % dialyzed FBS 250 mg/l zeocin and 400 mg/l geneticin (GIBCO/Invitrogen AB, Stockholm, Sweden) at a density of 10000 cells/well and incubated at 37 o C and 5 %CO2 over night. After two washes with assay buffer, HBSS with 20 mM HEPES (GIBCO/Invitrogen AB, Stockholm, Sweden) cells were loaded with 3.6 µM Fluo4 AM (Molecular Probes, Eugene, OR, USA) diluted in assay buffer, including 2.5 mM probenecid (Sigma Chemical Co. (St. Louis, Mo., USA) for 60 min, at 37 o C. Prior to measurement cells were washed and incubated in assay buffer for another five minutes.

Compounds, dissolved in DMSO at a concentration of 10 mM were diluted to 0.08 mM in assay buffer. In FLIPR 2 (Molecular Devices Inc., Sunnyvall, CA., USA) cells were stimulated with compounds at a final concentration of 20 µM and changes of cytosolic calcium were measured. Excitation wavelength was 488 nm and a 510-570 nm filter was used for emission measurements. Maximum emission data from each compound stimulation was collected and percentage of response elicited by 2 nM WKYMVm-peptide was calculated.

Compounds that induced a calcium response over 60 % of the response elicited by 2 nM WKYMVm-peptide were defined as hits. All hits were counter screened in CHO-K1 cells transfected with G-alpha16 to exclude unspecific calcium agonists. The ability to activate FPR1 transfectants was determined in a calcium release assay basically as described above. CHO-K1 cells stably expressing the human FPR1 and Galpha16 were plated as described above (but with 800 mg/l of geneticin) at a density of 10 000 cells/well and incubated at 37 o C and 5 % CO2 over night. Cells were labeled with Fluo4 and the change in intracellular calcium ions was determined as described above.

Isolation of human neutrophils

Neutrophil granulocytes were isolated from buffy coats obtained from healthy adults [START_REF] Boyum | Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality[END_REF].

After dextran sedimentation at 1 x g, hypotonic lysis of the remaining erythrocytes, and centrifugation in a Ficoll-Paque gradient, the neutrophils were washed twice and resuspended (1 x 10 7 /ml) in KRG. The cells were stored on melting ice and used within 120 min of preparation.

Neutrophil NADPH-oxidase activity

Neutrophil superoxide anion production was determined using an isoluminol-enhanced chemiluminescence (CL) system [24,[START_REF] Dahlgren | Measurement of respiratory burst products generated by professional phagocytes[END_REF]. The CL activity was measured in a 6-channel Biolumat LB 9505 (Berthold Co, Wildbad, Germany) using disposable 4-ml polypropylene tubes with a 1-ml reaction mixture. Tubes containing isoluminol (2x10 -5 M), horseradish peroxidase (HRP, 4 U), and neutrophils (2x10 5 /ml) were allowed to equilibrated for 5 minutes at 37°C, after which 0.1 ml of stimuli was added and the light emission was recorded continuously. All experiments (with resting as well as primed cells) were performed with neutrophils in suspension.

A c c e p t e d

M a n u s c r i p t 9 By a direct comparison of the superoxide dismutase (SOD) inhibitable reduction of cytochrome C and SOD inhibitable CL, 7.2x10 7 counts were found to correspond to a production of 1 nmol of superoxide (a millimolar extinction coefficient for cytochrome C of 21.1 was used; details about the CL technique is given in [START_REF] Dahlgren | Measurement of respiratory burst products generated by professional phagocytes[END_REF][START_REF] Dahlgren | Respiratory burst in human neutrophils[END_REF]).

Chemotaxis assay

Neutrophil migration was determined using a 96-well microplate chemotaxis chambers with hydrophobic filters of pore size 3 m (ChemoTx; Neuro Probe Inc., Gaithersburg, MD, USA) according to the instructions given by the manufacturer. In short, compounds and the positive control, N-formyl-Met-Leu-Phe, fMLF (10 -8 M) were added to wells in the lower chamber.

All dilutions were made in KRG buffer supplemented with 0.3% BSA. Cell suspensions (30 l) neutrophils (10 6 /ml) were placed on top of the filters and allowed to migrate for 90 min at 37C. Cell migration was examined under microscope. For quantification, the content of myeloperoxidase was assessed in the lysates of transmigrated cells by adding a peroxidase substrate o-phenylenediamine.

Cell surface receptor exposure by FACS analysis

To determine the effect of compounds on cell surface CR3 exposure, cells were incubated with various concentrations of neutrophil activators at 37 o C for 15 min. After which the cells were labelled with a PE-conjugated CR3 antibody on ice for 30 min. The amount of specifically bound antibody after washing off excess of unbound antibody was determined by flow cytometry.

The release of myeloperoxidase (MPO), an azurophil granule marker of importance for inactivation of chemoattractants [START_REF] Forsman | The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPHoxidase and inactivation of the agonist[END_REF], was determined as described earlier [START_REF] Betten | Oxygen radical-induced natural killer cell dysfunction: role of myeloperoxidase and regulation by serotonin[END_REF] A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 11

RESULTS

Identification of novel non-peptide molecules that interact with FPR2

We used a small molecule screening set approach to identify novel agonists for formyl peptide receptor 2 (FPR2). Transfected CHO cells stably expressing FPR2 were loaded with Fluo4 AM and then stimulated with 50 000 compounds in 384 format FLIPR and analyzed for increase in the concentration of cytosolic calcium. The screen was performed at 20 µM to identify only agonists.

Out of the 50 K compounds screened, more then 500 was confirmed as agonist hits and five compounds representing different chemical series were selected for further profiling. In addition, five earlier described FPR2 agonists (compound 1,2,3,4 and 7 in Fig. 1) were also profiled. All 10 compounds were found to trigger an intracellular Ca 2+ response in FPR2 expressing cells. The EC 50 values for the different compounds were determined and were found to vary from 4 x 10 -9 to 2 x 10 -7 M (Fig 1). Based on the activity, they could be divided into three groups. The EC 50 value for compound 8 and 9 was higher (in the 10 -7 M range) than the corresponding values for compound 1, 2, 3, 6 and 7 (in the 10 -8 M range), whereas the most potent compounds (the compounds 4, 5 and, 10) were active in the nM concentration range (Fig 1).

The potential pro-inflammatory activity of FPR2 binding compounds derived from the small molecule library was investigated using human neutrophils as model cells and the NADPHoxidase mediated superoxide production as readout system.

The non-peptide FPR2 agonists activate neutrophils to produce superoxide anions

The abilities of the 10 compounds identified as FPR2 agonists to induce a respiratory burst activity in resting and TNF- primed neutrophils were determined. All compounds induced a A c c e p t e d M a n u s c r i p t 12 neutophil respiratory burst and with respect to the potency they grouped in the same way as described above. The compounds 8 and 9 induced a respiratory burst only in TNF-a primed cells and in concentrations higher that 1µM, whereas the compounds 1, 2, 3, 6 and 7 triggered also resting cells to produce superoxide anions (data not shown). The three compounds (4, 5 and 10) that had the highest activity in the calcium assay, were the most potent also in triggering a respiratory burst, and a robust superoxide production was induced in resting cells (Fig2). The response induced in TNF- primed cells were strongly increased with all compounds including the three most potent ones (data not shown). We decided to characterize the activities induced in neutrophils by the three most active compounds 4, 5 and, 10, in more detail, but it should be noticed that although the magnitude of superoxide production differed between the compounds they all showed the same kinetics. This is shown for the three most potent compounds in Fig 2 . The time course was characterized by a very short lag phase followed by a rapid increase of superoxide release and a peak of activity was reached after around one minute. This is a response that is typical for most chemoattractants that bind to a neutrophil GPCR and activate the oxidase, and one such, fMLF response, is shown for

comparison (Fig 2 inset).
The magnitudes of the responses induced by compound 4 and compound 5 at a concentration of 1µM were in the same level as that induced by WKYMVM (10 -7 M) and fMLF (10 -7 M) , two very potent agonists for FPR2 and FPR1, respectively (shown for fMLF; Fig2 inset). The

The compounds 4, 5, and 10 are not inactivated by reactive oxygen species generated by neutrophils

Neutrophil derived reactive oxygen species are highly reactive not only with bacterial proteins during bacterial clearance process, they could also oxidize and inactive small molecules very rapidly. Accordingly, we recently showed that the FPR agonists fMLF and WKYMVM are rapidly inactivated by ROS producing neutrophils in a process that is dependent on the azurophil granule protein MPO [START_REF] Forsman | The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPHoxidase and inactivation of the agonist[END_REF][START_REF] Karlsson | A methodological approach to studies of desensitization of the formyl peptide receptor: Role of the read out system, reactive oxygen species and the specific agonist used to trigger neutrophils[END_REF]. To determine the sensitivity of the new compounds for neutrophil derived oxygen radicals, we added the compounds 4, 5, or 10 at high concentrations (10µM) to neutrophils. After an incubation period of 10 min, the cells were removed by centrifugation and the agonists remaining in the supernatant after interaction with the neutrophils were used to trigger a new/fresh sample of cells to generate ROS. A high concentration (10µM) of the agonists was chosen for inactivation, making it possible to use a small volume (0.1ml) of each supernatant, and add that to a new/fresh sample of cells.

Inactivation of a compound would then result in a reduced cellular response induced by a cell free supernatant (with 10 µM from the beginning), compared to the corresponding control pre-incubated without any cells. the activities induced by the three compounds were, however, basically unchanged after interaction with the neutrophils (Fig 3A, shown for compound 5;Table 1) whereas the triggering effect of fMLF in a comparable system was reduced by more than 75% and a complete inactivation of WKYMVM (Fig 3B ;Table 1) was obtained.

We know from earlier studies that the inactivation is dependent on the granule enzyme MPO [START_REF] Forsman | The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPHoxidase and inactivation of the agonist[END_REF], but no MPO secretion was induced by any of the compounds used (data not shown), suggesting that the basal level MPO is sufficient. Moreover, the neutrophil activity induced by a cell free supernatant, originating from a mixture of cells, WKYMVM (an FPR2 agonist) and, compound 5 (primarily an FPR1 agonist; see below) was totally inhibited by A c c e p t e d M a n u s c r i p t 14 cyclosporine H (an FPR1 antagonist), showing that when one sensitive and one stable agonist are added to the cells simultaneously, only one of them "survives".

Formyl peptide receptor1 (FPR1) is the preferred receptor for compound 4, 5 and, 10.

Neutrophils express in addition to FPR2 also the closely related receptor FPR1 and these receptors have the ability to recognize a large range of structurally unrelated agonists that are either specific or overlapping. An agonist that can bind and activate both receptors sometimes has a preference for either of the receptors [START_REF] Karlsson | The peptide Trp-Lys-Tyr-Met-Val-D-Met activates neutrophils through the formyl peptide receptor only when signaling through the formylpeptide receptor like 1 is blocked. A receptor switch with implications for signal transduction studies with inhibitors and receptor antagonists[END_REF], meaning that one of the receptors is not in use unless the preferred receptor is blocked by a specific antagonist or inhibitor. In order to determine which of the receptors that is used by compound 4,5 and, 10, we used a couple of earlier characterized receptor antagonists and a receptor specific inhibitor. The receptor specific inhibitors used were cyclosporine H [START_REF] Wenzel-Seifert | Cyclosporin H is a potent and selective formyl peptide receptor antagonist. Comparison with N-t-butoxycarbonyl-L-phenylalanyl-Lleucyl-L-phenylalanyl-L-leucyl-L-phenylalanine and cyclosporins A[END_REF][START_REF] Stenfeldt | Boc-MLF and Boc-FLFLF are antagonists that preferentially inhibit activity triggered through the formyl peptide receptor[END_REF] and the PBP10 [START_REF] Stenfeldt | Boc-MLF and Boc-FLFLF are antagonists that preferentially inhibit activity triggered through the formyl peptide receptor[END_REF][START_REF] Fu | The two neutrophil members of the formylpeptide receptor family activate the NADPHoxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide[END_REF][START_REF] Forsman | The FPR2-induced rise in cytosolic calcium in human neutrophils relies on an emptying of intracellular calcium stores and is inhibited by a gelsolin-derived PIP2-binding peptide[END_REF],

antagonists/inhibitors for FPR1 and FPR2, respectively (Fig 4). The FPR2 specific inhibitor PBP10, is a peptide derived from one of the PIP 2 binding domains present in the cytoskeletal protein gelsolin. The FPR1 specific antagonist cyclosporine H completely abolished the neutrophil response both to compound 4 and 5 as well as to compound 10 (table 2 and shown for compound 5 in Fig 4). No inhibitory effect was obtained with the FPR2 specific inhibitor PBP10, but when the FPR1 antagonist was replaced by the specific FPR2 antagonist WRWWWW a partial inhibition was observed (data not shown). Taken together, these data suggest that all three compounds (4,5 and 10) activate neutrophils preferentially through FPR1 rather than through FPR2.

Using transfected CHO cells stably expressing FPR1, we could show that the three compounds (4, 5 and 10) trigger an intracellular Ca 2+ response also in these cells (table 2

Receptor desensitization and hierarchy

To further elucidate the receptors involved in the cellular responses induced by three novel compounds used, we performed receptor desensitization experiments using agonist with known receptor specificity. It is well established that neutrophils challenged with one GPCR agonist are non-responsive to a second dose of the same agonist, a process known as homologous desensitization. Accordingly, compounds 4, 5 and 10 induced a homologues desensitization in neutrophils (data not shown). Cells that were desensitized with the FPR1 agonist fMLF were desensitized not only to this agonist, but also to the compound 4, 5 and 10 and the desensitized state was there when the order for agonist addition was reversed, i.e., cells triggered with any of the three compounds were desensitized to fMLF (table 2 and shown for compound 5 in Fig 6A). Neutrophils challenged with any of the three compounds were also desensitized to the FPR2 specific agonist WKYMVM, but when the order was reversedfirst WKYMVM and then one of the compoundsthe cells were only partially desensitized to the three compounds (Fig 6B). This suggests that all three compounds prefer FPR1 over FPR2.

Neutrophils may also be desensitized heterologously, meaning that an occupied receptor not only desensitize itself but also an adjacent receptor with another agonist specificity. This type of desensitization also disclose a receptor hierarchy, illustrated by the fact that an occupation of FPR1 leads to a desensitization of this receptor as well as CXCR (the IL-8 receptor), whereas occupation of CXCR does not desensitize FPR1 [START_REF] Heit | An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients[END_REF][START_REF] Fu | The mechanism for activation of the neutrophil NADPH-oxidase by the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8[END_REF]. To further explore hierarchy, we determined the desensitization patterns with compound 4, 5 and, 10, three compounds and the intermediate receptor agonist CXCR as well as the end type receptor C5aR. We show that neutrophils challenged with any of the three compounds were nonresponsive to a second stimulation with IL-8 whereas cells first triggered with IL-8 were fully responsive to compound 4, 5 and 13 (table 2 and shown for compound 5 in Fig 7A) Neutrophils activated with either of the three compounds were non-responsive also to C5a whereas no such desensitization is obtained when the order of agonist addition is reversed (table 2 and shown for compound 5 in Fig 7B). This type of desensitization pattern is obtained also when fMLF replaces compound 4, 5 and, 10, i.e., fMLF could desensitize neutrophils to C5a, but not vice versa (data not shown). The precise molecular mechanism underlying this cross/heterologous-desensitization is not yet known.

The compounds 4, 5, and, 10 are neutrophil chemoattractants

Having identified three novel neutrophil FPR1 agonists and shown that they have the ability to activate the neutrophil NADPH-oxidase to generate superoxide anions, we next investigated whether they could induce other cellular functions that are induced by most peptide agonists that are recognized by these receptors. Using a trans-well chamber assay system we could show that cells migrated to the lower chamber where the novel compounds were present suggesting that these substances are chemotactic (

The compounds 4, 5, and 10 induce granule mobilization

Neutrophil activation by chemoattractants is associated with an induction of granule secretion, and such a process leads to a mobilization to cell surface of new receptors. To investigate the ability of these compounds in triggering granule mobilization, we examined the degree of surface exposure of CR3, a marker protein localized in easily mobilizable granules in neutrophils (i.e. secretory vesicles, gelatinase granules, and to some extent specific granules), upon stimulation with various concentrations of the compounds 4, 5, and 10. The chemoattractants fMLF and WKYMVM were used as controls and they both induced a pronounced increased in CR3 exposure, in a concentration dependent manner (Fig 9 inset).

Similarly, pre-incubation of cells with either compound resulted in a large increase of CR3 exposure and this increase was also found to be concentration dependent (Fig 9). Compound 4 was found to be the most potent whereas compound 5, the most potent in triggering the production of ROS, was the least potent in mobilizing CR3 (Fig 9).

Discussion

Agonists as well as antagonists for FPR2 have been suggested to be of therapeutic value [START_REF] Zhang | Circulating mitochondrial DAMPs cause inflammatory responses to injury[END_REF][START_REF] Dufton | Therapeutic anti-inflammatory potential of formyl-peptide receptor agonists[END_REF], and in addition, if they in one way or another selectively stimulate/inhibit neutrophil functions, they may be used also as research tools [START_REF] Ye | International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family[END_REF]. In order to find agonists with this potency, we screened a library of small compounds and several ligands for FPR2 with varying EC 50 values were identified. The identification of small-molecule ligands represent an attractive approach to analyze structure and function of FPRs, and in line with this several research articles using the same approach have been published during the last couple of years [START_REF] Schepetkin | High-throughput screening for small-molecule activators of neutrophils: identification of novel Nformyl peptide receptor agonists[END_REF][START_REF] Arterburn | Discovery of Selective Probes and Antagonists for G-Protein-Coupled Receptors FPR/FPRL1 and GPR30[END_REF][START_REF] Young | Highthroughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands[END_REF][START_REF] Young | Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes[END_REF]. Of the compounds that we used and found to activate human neutrophils to produce/release superoxide anions, 5 (4 unique and one enantiomer) have been described earlier. Compound 1 and 2 in this study (Fig 1) are enantiomers of an earlier described FPR2 agonist (Acadia C7; http://www.wipo.int/pctdb/en/wo.jsp?wo=2005047899), that has been shown to dose dependently prevent hyperalgesia induced by carrageenan in a rat model. It should be noticed that large differences have been described for receptor agonists and their enantiomers [START_REF] Frohn | New 'chemical probes' to examine the role of the hFPRL1 (or ALXR) receptor in inflammation[END_REF], but in our hands compound 1 and 2 were equally potent, but not among the most potent neutrophil activators in. Compound 3 used in this study is a substituted quinazinolione (QuinC1), that has been described to be chemotactic and induce degranulation in neutrophils [START_REF] Nanamori | A novel nonpeptide ligand for formyl peptide receptor-like 1[END_REF], but be unable to stimulate superoxide production [START_REF] Zhou | Pharmacological characterization of a novel nonpeptide antagonist for formyl peptide receptor-like 1[END_REF]. We show using a more sensitive technique to detect ROS formation that compound 3 activates the NADPHoxidase in resting as well as primed cells, and we could from experiments with receptor antagonists confirm that FPR2 is the preferred receptor (data not shown). It might well be that ligands may behave differently in functional assays because of their intrinsic ability to induce different receptor conformational changes, but we have not looked into this issue since compound 3 was not among the most potent neutrophil activators and thus not further investigated. Compound 4 is a pyrazolone originally described as an FPR2 agonist that dose A c c e p t e d M a n u s c r i p t 19 dependently inhibit chemotaxis of human neutrophils in response to fMLF and IL8 [START_REF] Burli | Potent hFPRL1 (ALXR) agonists as potential anti-inflammatory agents[END_REF]. This is in accordance with earlier studies suggesting that FPR2 is a down-regulating receptor when interacting with the lipoxygenase-derived eicosanoid lipoxin A 4 or peptides derived from the N-terminus of the calcium regulated protein annexin I [START_REF] Serhan | Anti-inflammatory and proresolving lipid mediators[END_REF][START_REF] Perretti | Exploiting the Annexin A1 pathway for the development of novel anti-inflammatory therapeutics[END_REF]. We found that compound 4 (Fig 1) activates neutrophils preferentially through FPR1 (see below) which could explain the down-regulating effects on fMLF and IL8 induced chemotaxis, and we also found this compound to be one of the three most potent agonists in our ROS production assay, and based on the potency of activity, the three most potent activators were further characterized.

Many FPR agonists can trigger their own inactivation when interacting with neutrophils (reported here for fMLF and WKYMVM) and the activity of the MPO-hydrogen peroxide system is the basis for the loss of biological activity [START_REF] Forsman | The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPHoxidase and inactivation of the agonist[END_REF][START_REF] Clark | Chemotactic factors trigger their own oxidative inactivation by human neutrophils[END_REF]. Agents sensitive to this type of inactivation is not restricted to chemotactic factors [START_REF] Reumaux | Priming by tumor necrosis factoralpha of human neutrophil NADPH-oxidase activity induced by anti-proteinase-3 or anti-myeloperoxidase antibodies[END_REF][START_REF] Hsieh | Antimyeloperoxidase antibodies enhance phagocytosis, IL-8 production, and glucose uptake of polymorphonuclear neutrophils rather than anti-proteinase 3 antibodies leading to activation-induced cell death of the neutrophils[END_REF], but the three most potent agonists in our ROS production assay (4, 5 and, 10), were all stable in relation to the radicals generated.

Cyclosporin H is a well established antagonist that abrogates FPR1-dependent responses but not those related to FPR2 [START_REF] Stenfeldt | Boc-MLF and Boc-FLFLF are antagonists that preferentially inhibit activity triggered through the formyl peptide receptor[END_REF]. The NADPH-oxidase response induced by the three most potent neutrophil activators, compounds 4, 5, and 10 were all inhibited by cyclosporin H, suggesting that they activate the neutrophil oxidase through FPR1 rather than FPR2. Although it is clear that the three compounds can bind and activate both FPR1 and FPR2, it is obvious that in neutrophils FPR1 is the preferred receptor. This conclusion is based on the following: cross-desensitization was obtained with cells desensitized with fMLF, i.e., the fMLF response was downregulated by the three compounds; the receptor specific inhibitor PBP10 (an FPR2 inhibitor) was without effects whereas the receptor specific antagonist cyclosporin H inhibited the neutrophil response to the three compounds. The fact that the FPR2 antagonist WRW 4 partly inhibited the activity by compound 4, 5 an 10 is most probably due to a cross reactivity A c c e p t e d M a n u s c r i p t 20 of the antagonist with FPR1, when the agonist used is present in low concentration or has a lower affinity for the receptor than fMLF. PBP10 is a more specific FPR2 inhibitor, and the fact that this was without effect suggests that FPR1 is the preferred receptor for all three compounds.

We show that all compounds trigger an activation of the neutrophil NADPH-oxidase in neutrophils primed with TNF-. These cells are primed, not only in response to the new compounds, but to various inflammatory mediators, chemoattractants, and other ligands [START_REF] Almkvist | Newcastle disease virus neuraminidase primes neutrophils for stimulation by galectin-3 and formyl-Met-Leu-Phe[END_REF][START_REF] Bylund | Lipopolysaccharide-induced granule mobilization and priming of the neutrophil response to Helicobacter pylori peptide Hp(2-20), which activates formyl peptide receptor-like 1[END_REF][START_REF] Bylund | Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils[END_REF][START_REF] Karlsson | Galectin-3 activates the NADPHoxidase in exudated but not peripheral blood neutrophils[END_REF]. The primed response may be a regulatory mechanism, granting a cellular response (such as release of toxic oxygen radicals) only at sites where it is functional and necessary, e.g., in inflamed or infected areas. Although the molecular mechanism(s) responsible for induction of the primed state is unclear, the exposure of new receptors is an attractive model as the molecular basis for an augmented response. We know from an earlier study that intracellular organelles are mobilized to the cell surface during TNF- priming [START_REF] Forsman | Lipoxin A(4) metabolites/analogues from two commercial sources have no effects on TNF-alpha-mediated priming or activation through the neutrophil formyl peptide receptors[END_REF], resulting in an increased exposure of various receptors including CR3, FPR1, and FPR2. The molecular background to the primed response to the new peptides may, thus, be the result of an increased exposure of these receptors.

Our study demonstrates several important functional features of the three compounds characterized: i) they are neutrophil chemoattractants; ii) they mobilize intracellular stores containing the integrin receptor for complement factor iC3b; iii) they are potent activators of the NADPH-oxidase, especially in primed neutrophils; iv) they trigger neutrophils through FPR1 and v) they are not inactivated by the reactive oxygen species generated. Availability of potent, cheep, easy to handle, and stable activators of FPRs will undoubtedly be of great help for screening of antagonists for these receptors. The described non-peptide ligands for FPR1 and 2 may also serve as the nucleus for further structural modifications leading to the discovery of more potent and efficacious agonists. This type of work has been performed

A c c e p t e d M a n u s c r i p t 21 already, using the pyrazolone originally described as an FPR2 agonist, and an even more potent FPR2 agonist has been described together with a 100 fold less active enatiomer [START_REF] Frohn | New 'chemical probes' to examine the role of the hFPRL1 (or ALXR) receptor in inflammation[END_REF].

The selective activation property of Quin-C1 (our compound 3) has also been suggested as an interesting starting structure for future development of small non-peptide molecules that may be clinically useful antagonists for FPR2. The two compounds not earlier described, compound 5 and 10, and found to be agonist for both FPR2 and FPR1, may also be used as starting structures for the development of clinically useful compounds that should be valuable tools also is future studies to reveal structural functional relationships for FPR ligands and their receptors. The chemical structure for the ten compounds identified in the screening assay as FPR2 agonists. The EC 50 values for the compounds, determined through their abilities to induce a Ca 2+ response in cells stably expressing FPR2, are also given.

  dimethyl sulfoxide to 10 -2 M and stored at -80 o C until use. Further dilutions were made in Krebs-Ringer phosphate buffer containing glucose (10mM), Ca 2+ (1mM), and Mg 2+ (1.5mM) (KRG; pH 7.3).

A c c e p t e d M a n u s c r i p t 8 Selected

 8 hits were confirmed in calcium release analysis in FPR2 and Galpha16 transfected cells described above in a dose dependent manner from 20 µM to 3.4 pM in 11 steps.

  EC 50 values for activation of the neutrophil oxidase were in the range 0.1-0.2 µM for both compound 4 and 5. Even though the EC 50 value for activation of the oxidase with compound 10 was in the same range as the other two, the maximal amount of radicals produced was lower (Fig2).

  A c c e p t e d M a n u s c r i p t

13

 13

  and shown for compound 5 in Fig 5), confirming the results obtained with normal neutrophils.

15

 15

  hierarchy that is in line with the known desensitization model in which the FPRs are superior to CXCR. For comparision, the desensitization pattern obtained when fMLF replaces compound 4, 5 and, 10, and showing that fMLF could desensitize neutrophils to IL-8 but not vice versa, is also shown (Fig7C).

  Fig 8). The positive control peptides fMLF and WKYMVM (at 10 nM concentration) triggered migration under similar conditions (shown for fMLF inFig 8). Around 3 % of the cells were recovered in the lower compartment when this contained buffer alone, and this value thus represent spontaneous cellular migration (Fig8). The potency by which compound 5 induced cellular migration was similar to that of the positive control fMLF, but the concentration required to induce this migration was much higher (1µM compared to 10nM). The two other compounds (4 and 10) were less potent.

17

 17

  Fig 1

Fig 2

 2 Fig 2

Compound 5 Fig 4 Fig 5 A

 545 Fig 4

Fig 6 Receptor desensitization induced by compound 5 ,Fig 7

 657 Fig 6

A

  c c e p t e d M a n u s c r i p t addition of the agonists are indicated by arrows and the amount of superoxide is expressed in arbitrary units. For comparison the response in neutrophils first challenged with fMLF (10 -7 M; in C first arrow; solid line) and then triggered with IL8 (second arrow) is also shown.The agonist order was also reversed; the cells were firs challenged with IL8 (broken line in C) and then with fMLF (10 -7 M final concentration; second arrow in C).Abscissa; Time of study (min); ordinate; superoxide production given as light emission and expressed in cpmx10 -6 .

Fig 8

 8 Fig 8

Fig 9 Complement receptor 3 (

 93 Fig 9

  s c r i p t

Acknowledgements

This work was supported by the Swedish Research Council, the Swedish Society for Medical Research, the Medical Faculty of Göteborg, The Swedish government under the ALFagreement, King Gustaf the Vth 80-year foundation and the IngaBritt and Arne Lundberg research foundation. Torbjörn Morein, Marie Andersson och Katrin Bergdahl at Biovitrum are acknowledged for help in adopting the calcium release assay to the screening platform and Marguerite Luthman for fruitful discussions about signaling.