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J. Biol. Chem., Vol. 277, Issue 51, 49403-49407, December 20, 2002

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Identification of a Potent and Orally Active Non-peptide C5a Receptor Antagonist^*

Hiroshi Sumichika, Kei Sakata, Noriko Sato, Sanae Takeshita, Seigo Ishibuchi, Mitsuharu Nakamura, Takao Kamahori, Syuji Ehara, Katsuhiko Itoh, Tatsuyuki Ohtsuka, Tomoko Ohbora, Tadashi Mishina, Hirotsugu Komatsu, and Yoichi Naka

From Pharmaceuticals Research Division, Mitsubishi Pharma Co., 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan

Received for publication, September 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The anaphylatoxin C5a is a potent chemotactic factor for neutrophils and other leukocytes, and functions as an important inflammatory mediator. Through a high capacity screening followed by chemical optimization, we identified a novel non-peptide C5a receptor antagonist, N-[(4-dimethylaminophenyl)methyl]-N-(4-isopropylphenyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1- carboxamide hydrochloride (W-54011). W-54011 inhibited the binding of ¹²⁵I-labeled C5a to human neutrophils with a K_i value of 2.2 nM. W-54011 also inhibited C5a-induced intracellular Ca²⁺ mobilization, chemotaxis, and generation of reactive super oxide species in human neutrophils with IC₅₀ values of 3.1, 2.7, and 1.6 nM, respectively. In C5a-induced intracellular Ca²⁺ mobilization assay with human neutrophils, W-54011 did not show agonistic activity at up to 10 µM and shifted rightward the concentration-response curves to C5a without depressing the maximal responses. Examination on the species specificity of W-54011 revealed that it was able to inhibit C5a-induced intracellular Ca²⁺ mobilization in neutrophils of cynomolgus monkeys and gerbils but not mice, rats, guinea pigs, rabbits, and dogs. In gerbils, oral administration of W-54011 (3-30 mg/kg) inhibited C5a-induced neutropenia in a dose-dependent manner. The present report is the first description of an orally active non-peptide C5a receptor antagonist that could contribute to the treatment of inflammatory diseases mediated by C5a.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The complement component C5a is a 74-amino acid peptide generated during the classical, alternative, and lectin pathways of complement activation (1, 2). C5a is a potent chemotactic factor for neutrophils and other leukocytes and is a potent inflammatory mediator. Moreover, C5a causes histamine release from mast cells, smooth muscle contraction, increase in vascular permeability, eliciting of superoxide anion production, enhancement of neutrophil-endothelial cell adhesion, induction of several cytokines (i.e. IL-1, IL-6, IL-8, and TNF-) from leukocytes, and augmentation of the humoral and cell-mediated immune response. C5a exerts these activities by binding to G-protein-coupled C5a receptor (C5aR)¹ on the plasma membrane of target cells (3). These biological activities of C5a are implicated in a variety of diseases such as rheumatoid arthritis (4, 5), systemic lupus erythematosus (6-9), reperfusion injury (10), Alzheimer's disease (11-15), and sepsis (16, 17). The pathogenic action of C5/C5a is also shown in some animal models. For instance, in murine collagen-induced arthritis, an animal model of rheumatoid arthritis (RA), administration of anti-C5 antibody during the disease induction or after the onset suppresses or ameliorates the disease (18). Anti-C5 antibody is also effective in a new RA model (19), K/BxN mouse model induced by anti-glucose 6-phosphate isomerase (GPI) auto-antibodies, which is detected in 64% of RA patients (20-22). Furthermore, the anti-GPI antibody-induced arthritis does not occur in C5aR-deficient mice, indicating that C5a is important in the development of arthritis rather than C5b, which is another part of C5 and is a component of the membrane attack complex.

Inhibition of C5a function has been attempted with anti-C5a antibodies and C5aR antagonists (for review, see Refs. 23, 24). Anti-C5a antibodies inhibit immune complex-induced inflammation and reperfusion injury and are effective in septic primates and rats (25-27). On the other hand, although C5aR antagonists, including non-peptides, small peptides, C5a mutants, and anti-C5aR antibodies, have been studied for the past 2 decades, only a few candidates have been discovered as non-peptide antagonists. These non-peptide antagonists, however, are not so potent (IC₅₀, more than subhundreds nM in ¹²⁵I-rhC5a binding assay) and have not been reported on as to their in vivo activities. As a small peptide antagonist, hexapeptide (MeFKP-D-ChaWr, C089) was first reported in 1994 (28). C089 exhibits an IC₅₀ value of 70 nM in ¹²⁵I-rhC5a binding assay and has been reported recently to inhibit the late airway response in allergic rats and inhibit thrombotic glomerulonephritis in rats (29). The recent reports describe two other types of C5aR antagonists, one of which is a group of F-[OP-D-ChaWR] derivatives (30-34), cyclized peptides of modified C089, and the other is a group of C5a mutants (35-37). Intravenous injection of these C5aR antagonists inhibits immune complex-induced inflammation and reperfusion injury. Based on these findings, the appearance of non-peptide C5aR antagonists that can be used against various human disorders is expected (38). In the present report, we describe the discovery and pharmacological characterization of an orally active non-peptide C5aR antagonist.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Recombinant human C5a (rhC5a) was purchased from Sigma. ¹²⁵I-labeled recombinant human C5a (¹²⁵I-rhC5a) was purchased from Amersham Biosciences. Anti-C5aR monoclonal antibody (clone: S5/1) (39) was purchased from Serotec (Oxford, UK). Fresh isolated whole blood of cynomolgus monkeys, anticoagulated with EDTA, was purchased from Shin-nihonkagaku (Kagoshima, Japan).

Animals-- BALB/c mice (AnNCrj) and Wistar rats (Crj) were purchased from Charles River Japan (Kanagawa, Japan). Mongolian gerbils (MGS/Sea), Hartley guinea pigs (Std), rabbits (KBT JW), and beagle dogs were purchased from Seac Yoshitomi (Fukuoka, Japan), Japan SLC (Shizuoka, Japan), Biotec (Saga, Japan), and Keari (Osaka, Japan), respectively.

Synthesis of Compounds-- N-[(4-dimethylaminophenyl)methyl]-N-(4-isopropylphenyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1-carboxamide hydrochloride (W-54011) was synthesized as follows: a solution of 7-methoxy-1,2,3,4-tetrahydronaphthalen-1-carbonyl chloride (16.3 g, 72.7 mmol) and [(4-dimethylaminophenyl)methyl](4-isopropylphenyl) amine (19.5 g, 72.7 mmol) in CH₂Cl₂ (200 ml) was stirred overnight at room temperature. The reaction mixture was poured into water and extracted two times with CHCl₃. The combined organic layer was washed with brine, dried over anhydrous MgSO₄, and concentrated. The residue was chromatographed over silica gel using a mixture of ethyl acetate and hexane (1:4) as eluent to give a yellow oil (29.9 g, 90.1%). To a solution of the oil (29.9 g, 63.5 mmol) in ethanol (300 ml) was added hydrogen chloride (4.0 M solution in 1,4-dioxane, 17.5 ml), and the resulting solid was filtered, recrystallized from ethanol:water to afford W-54011 (24.2 g, 75.8%) as yellow crystals. The melting point was 147 °C; mass spectrometry (electrospray ionization) was m/z 457 [M + H]⁺. The cyclic C5aR antagonist F-[OP-D-ChaWR] was synthesized according to the method reported by Finch et al. (31).

Cell Line and Neutrophil Isolation-- The human histocytic lymphoma line U-937 was obtained from the American Type Culture Collection. Neutrophils were isolated from whole blood of various species, anticoagulated with 0.2% EDTA. Whole blood was collected from healthy volunteers and animals via vein puncture (human, cynomolgus monkey, dog, and rabbit) or inferior vena cava puncture (guinea pig, rat, gerbil and mouse under anesthesia). The blood was then layered on an equal volume of Lympholyte^®-poly (Cedarlane Laboratories Ltd., Hornby, Canada) in a centrifuge tube and then centrifuged at 500 × g for 30 min at room temperature. After centrifugation, the polymorphonuclear cells were harvested, resuspended in Hanks' balanced salt solution (HBSS, Invitrogen) containing 1% fetal calf serum (FCS), and used as neutrophils. Polymorphonuclear cells in this fraction were >95%.

¹²⁵I-rhC5a Binding Assay-- This assay was performed in 96-well filtration plates (Multiscreen MADV NOB, Millipore, Bedford, MA) in a total volume of 100 µl. Human neutrophils (1 × 10⁵ cells/well) were incubated with ¹²⁵I-rhC5a (200 pM) and varying concentrations of test compounds in the binding buffer (50 mM HEPES, 5 mM MgCl₂, 1 mM CaCl₂, 0.5% bovine serum albumin, and 0.02% NaN₃, adjusted to pH 7.2 with NaOH) at 4 °C for 2 h. Filters were washed four times by vacuum filtration with 300 µl/well cold binding buffer, then dried, punched, and measured for radioactivity in a gamma counter (Packard Cobra, GMI, Inc., Albertville, MI). The nonspecific binding was defined by the binding in the presence of 20 nM unlabeled rhC5a.

Intracellular Ca²⁺ Mobilization Assay-- Neutrophils (5 × 10⁶ cells/ml) were loaded with 5 µM Fura-2 AM (Dojindo, Kumamoto, Japan) for 40 min at 37 °C. After two washes, the cells were suspended at a concentration of 1 × 10⁶ cells/ml in HBSS containing 1% FCS. The studies were conducted by a microtiter plate-based assay using a functional drug screening system (FDSS6000, Hamamatsu Photonics, Shizuoka, Japan) in black wall 96-well plates (Corning, Acton, MA) in a total volume of 140 µl. The cells (1 × 10⁵ cells/100 µl/well) and varying concentrations of test compounds (20 µl/well) in HBSS containing 1% FCS were plated in black wall 96-well plates. The plates were placed into a FDSS6000, and then the changes in fluorescence were monitored at 37 °C at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm. 10 s after the start of monitoring, stimulators (20 µl/well) were added. Calculation of Ca²⁺ concentration was performed using a K_d for Ca²⁺ binding of 224 nM (40). The maximal change in Ca²⁺ concentration after stimulator addition was quantitated.

Chemotaxis Assay-- Neutrophils were resuspended in RPMI 1640 (Invitrogen) containing 25 mM HEPES and 0.1% BSA at a concentration of 5 × 10⁶ cells/ml and loaded with 5 µM Calcein-AM (Funakoshi, Tokyo, Japan) for 30 min at 37 °C. After three washes, the cells were resuspended at a concentration of 1 × 10⁶ cells/ml in RPMI 1640 containing 0.1% bovine serum albumin. The cells (1 × 10⁵ cells/200 µl/well) and varying concentrations of test compounds (200 µl/well) in RPMI 1640 containing 25 mM HEPES and 0.1% bovine serum albumin were placed into chemotaxicells (3-µm pore size, Kurabou, Osaka, Japan) within 24-well plates containing 100 pM rhC5a (300 µl/well). The plates were incubated for 90 min at 37 °C and 5% CO₂. After removing the chemotaxicells, migrated cells were lysed by adding 100 µl/well cell lysing solution (10% SDS, 0.01 N HCl), and the fluorescence was measured using a fluoro-plate reader (Cyto FlourII, Perseptive Biosystems, Framingham, MA) at excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Reactive Super Oxide Species (ROS) Generation Assay-- The studies were conducted by a microtiter plate-based assay using a microplate luminometer (Microlumat Plus LB96V, Berthold, Bad Wildbad, Germany) in white wall 96-well plates (Berthold) in a total volume of 200 µl. The cells (1 × 10⁵ cells/150 µl/well) and varying concentrations of test compounds (25 µl/well) in HBSS containing 1% FCS and 1 mM luminol (Wako, Osaka, Japan) were plated in white wall 96-well plates. After the plates were incubated in a Microlumat at 37 °C for 5 min, rhC5a (25 µl/well) was added at a final concentration of 3 nM, and the luminescence was monitored for 15 min. The maximal change in luminescence was quantitated as ROS generation.

Gerbil Neutropenia Assay-- The C5a-induced neutropenia was based on the observation that neutrophils transiently disappear from circulation after systemic infusion of chemoattractants such as C5a or leukotrien B4 (32, 37, 41). Male mongolian gerbils (6-12 weeks) were orally treated with W-54011 (3, 10, 30 mg/kg) suspended in 0.5% hydroxypropylmethylcellulose 4 h before rhC5a injection. The animals were anesthetized with pentobarbital (Abbott Laboratories, Abbot Park, IL), and the skin was incised to expose the jugular veins for rhC5a injection and blood collection using syringes. RhC5a was injected at time 0. Blood was sampled at -1, 1, 3, and 5 min into EDTA-treated tubes (Capiject, Terumo Medical, Somerset, NJ) and counted using Technicon H*1E Hematology System (Technicon, Macon, GA). To determine the levels of C5a-induced neutropenia, data were expressed as percentages of the neutrophil counts 1 min before rhC5a injection.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To identify a non-peptide C5aR antagonist, a high capacity radioligand screening was configured using ¹²⁵I-rhC5a and C5aR-expressing U-937 cells. In consequence of the followed chemical optimization, we identified a novel non-peptide C5aR antagonist, N-[(4-dimethylaminophenyl)methyl]-N-(4-isopropylphenyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1-carboxamide (W-54011, Fig. 1). W-54011 inhibited ¹²⁵I-rhC5a competitive binding to human neutrophils with a K_i value of 2.2 nM and was more potent than anti-C5aR antibody (S5/1) with a K_i value of 8.1 nM and F-[OP-D-ChaWR] with a K_i value of 15 nM (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Structure of W-54011

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Inhibition of 125I-rhC5a binding to human neutrophils by C5a, W-54011, anti-C5a R antibody and F-[OP-D-ChaWR]. Human neutrophils were incubated with 0.2 nM 125I-rhC5a in the absence and presence of a range of concentrations of C5a, W-54011, anti-C5a R antibody, or F-[OP-D-ChaWR], and specific binding was determined. Data are means ± S.E. of multiple experiments (n = 3).

To determine whether W-54011 was a functional antagonist for human C5aR or not, we measured the ability of this compound to inhibit rhC5a-induced intracellular Ca²⁺ mobilization, chemotaxis, and generation of ROS in human neutrophils. W-54011 inhibited these responses with IC₅₀ values of 1.6-3.1 nM and was more potent than F-[OP-D-ChaWR] (Table I) as was observed in ¹²⁵I-rhC5a competitive binding. Further, at concentrations up to 10 µM, W-54011 had no effect on intracellular Ca²⁺ mobilization in human neutrophils, indicating that this compound is a full antagonist (data not shown). Next, to determine the selectivity for the C5aR, W-54011 was tested for the ability to inhibit Ca²⁺ mobilization stimulated with other G-protein-coupled receptor (GPCR) ligands, such as fMLP, platelet-activating factor, and IL-8. At concentrations up to 10 µM, W-54011 did not affect Ca²⁺ mobilization stimulated with submaximally effective concentrations of fMLP (1 nM), platelet-activating factor (0.3 nM), and IL-8 (0.1 nM) (data not shown). This result demonstrates that W-54011 is highly specific for C5aR.

The mechanism of the antagonistic effect of W-54011 was examined with human neutrophils by Schild analysis (42). The concentration-response curves for Ca²⁺ mobilization induced by rhC5a were determined in the presence of increasing concentrations of W-54011 (Fig. 3). W-54011 shifted rightward the concentration-response curves to C5a without depressing the maximal responses. This result indicates that W-54011 has a competitive antagonist-like function, but the response curves at low concentrations (0.3-3 nM) of W-54011 were not parallel to those at the other concentrations.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of W-54011 on the concentration-dependent curves of rhC5a-induced Ca2+ mobilization in human neutrophils. Ca2+ mobilization in human neutrophils was induced by a range of rhC5a concentrations in the absence and presence of W-54011 at the concentrations indicated. Data are expressed as the maximal changes of the intracellular Ca2+ level. The results shown are representative from three separate studies.

The species selectivity of W-54011 was examined in rhC5a-induced intracellular Ca²⁺ mobilization of neutrophils in various species. The responses of neutrophils to rhC5a were different among these species (Fig. 4A, mice, rats, guinea pigs, rabbits, and dogs; data not shown). The W-54011 was able to inhibit the response in cynomolgus monkeys and gerbils with IC₅₀ values of 1.7 and 3.2 nM, respectively (Fig. 4B) but not in mice, rats, guinea pigs, rabbits, and dogs (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Species selectivity of rhC5a and W-54011 in rhC5a-induced intracellular Ca2+ mobilizations of neutrophils in various species. A, Ca2+ mobilization in neutrophils of human, cynomolgus monkey, and gerbil was induced by a range of rhC5a concentrations. Data are expressed as the maximal changes of the intracellular Ca2+ level. Data are means ± S.E. of multiple experiments. B, Ca2+ mobilization in neutrophils of human, cynomolgus monkey, and gerbil was induced by rhC5a concentrations of 0.1, 1, and 10 nM, respectively in the absence and presence of a range of W-54011 concentrations. Data are means ± S.E. of multiple experiments.

The in vivo effect of W-54011 was examined in the C5a-induced neutropenia model of mongolian gerbils. Intravenous injection of 100 µg/kg rhC5a caused a rapid and transient neutropenia, which reached nadir at 1 min after the rhC5a injection and returned to baseline within 5 min. When W-54011 (3-30 mg/kg) was orally administered 4 h before rhC5a injection, it inhibited the neutropenia in a dose-dependent manner (Fig. 5). This inhibition by W-54011 was also observed when it was administered 1 or 8 h before rhC5a injection but not 24 h (data not shown). Because the model described above was based on the reaction between different species, human C5a and gerbil C5aR, it is unclear whether the compound inhibits the interaction between gerbil C5a and C5aR. So, we next used gerbil zymosan-activated serum (gZAS) as a source of gerbil C5a. Intravenous injection of gZAS caused a rapid and transient neutropenia in a dose-dependent manner similar to that of rhC5a (Fig. 6A). When W-54011 (10, 30 mg/kg) was orally administered 4 h before gZAS (5-fold dilution, 2 ml/kg) injection, it inhibited this neutropenia (Fig. 6B).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of W-54011 on rhC5a-induced neutropenia in gerbils. Gerbils were orally treated with W-54011 (3, 10, 30 mg/kg) 4 h before rhC5a (100 µg/kg, intravenous) injection. Vehicle control was treated with vehicles of W-54011 and rhC5a. Neutropenia control was treated with a vehicle of W-54011 before rhC5a injection. Blood was sampled at -1, 1, 3, and 5 min after rhC5a injection, and neutrophils were counted. Data are expressed as percentages of the neutrophil counts 1 min before rhC5a injection and are shown as means ± S.E. (n = 6-8). *, p < 0.05; **, p < 0.01, significantly different from neutropenia control (Dunnett's multiple comparison test). ##, p < 0.01, significantly different from vehicle control (Student's t test).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Neutropenia induced by gZAS and effects of W-54011 on gZAS-induced neutropenia in gerbils. A, gerbils were treated with gZAS (3-, 10-, and 30-times dilution, 2 ml/kg, intravenous). Blood was sampled at -1, 1, 3, 5 min after gZAS injection, and neutrophils were counted. B, gerbils were orally treated with W-54011 (10, 30 mg/kg) 4 h before gZAS (5 times dilution, 2 ml/kg, intravenous) injection. Vehicle control was treated with vehicles of W-54011 and gZAS. Neutropenia control was treated with a vehicle of W-54011 before gZAS injection. Blood was sampled at -1, 1, 3, 5 min after gZAS injection, and neutrophils were counted. Data are expressed as percentages of the neutrophil counts 1 min before gZAS injection and are shown as means ± S.E. (n = 4). ++, p < 0.01, significantly different from vehicle control (Dunnett's multiple comparison test). *, p < 0.05; **, p < 0.01, significantly different from neutropenia control (Dunnett's multiple comparison test); ##, p < 0.01, significantly different from vehicle control (Student's t test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present report, we represented a potent and orally active non-peptide C5aR antagonist, named W-54011, for the first time. W-54011 inhibited C5a binding and C5a-induced functions in human neutrophils with IC₅₀ values of less than 5 nM and was more potent than F-[OP-D-ChaWR], which has been considered to be the most potent small C5aR antagonist. The C5aR antagonistic activity of W-54011 was species-specific and C5aR-specific. W-54011 also exhibited C5a antagonistic activity in vivo (gerbils).

The binding site of W-54011 is not fully investigated, but we considered that it binds to C5aR by the following three observations. 1) In Ca²⁺ mobilization assay, the inhibitory activity of W-54011 remained after W-54011-treated cells were washed briefly in assay buffer to remove this drug (data not shown). This observation indicates that W-54011 reacts with C5aR-expressing cells, but not C5a. 2) W-54011 does not inhibit IL-8-, PAF-, or fMLP-induced Ca²⁺ mobilization. This observation indicates that W-54011 does not inhibit signal machineries commonly used by GPCRs. 3) The antagonistic action of W-54011 was exhibited only in the neutrophils of human, cynomolgus monkeys, and gerbils, but not in those of other examined species. The homology of C5aRs is more than 90% between human and Rhesus monkeys (43) and is about 60-70% between humans and mice (44), rats (45), rabbits (46), dogs (47), or guinea pigs (48), suggesting that W-54011 binds to a region that is conserved in the C5aRs of humans and cynomolgus monkeys and does not exist in those of other species. Although the C5aR sequence of gerbils has not been defined, it is considered that the binding site of W-54011 is conserved similarly to that of humans. On the other hand, in the Ca²⁺ mobilization assay, W-54011 shifted rightward the concentration-response curves to C5a without depressing the maximal responses. The response curves were not parallel between the low concentrations (0.3-3 nM) of W-54011 and the high concentrations (10-300 nM), although the curves were parallel among the high concentrations (10-300 nM) of W-54011. These results suggest that W-54011 inhibits competitively the action of C5a at high concentrations, but not at low concentrations. However, the binding mechanism of W-54011 remains to be resolved.

Over the past two decades, many efforts have been exerted to discover C5aR antagonists, then C-terminal mimic peptides of C5a and C5a mutants were identified, but potent and non-peptide antagonists had not been discovered. On this point, Wong (24) and Pellas and Wennogle (23) review in detail. Briefly, bezodiazepine derivatives and spiroindane-bearing hydantoin derivatives discovered by de Laszlo et al. have affinity to C5aR, but they are partial agonists (49). Lanza et al. also reported substituted 4,6-diaminoquinolines as C5aR antagonists, but their activity is not so potent (IC₅₀, 2 µg/ml in C5a binding assay) (50). In addition to these compounds, Astles et al. reported phenylguanidine derivatives as C5aR antagonist with an IC₅₀ value of 0.8 µM (51), but these compounds seem to be cytotoxic.

Thus, non-peptide C5a binding inhibitors reported previously are not so potent, and some are partial agonists. Moreover, most of these compounds are positively charged. That reason is considered since C5a is a highly cationic polypeptide. C5a binding to its receptor is predominantly through two-site binding of charge-charge interactions (3, 52, 53), one of which is an interaction of the positive charged N-terminal disulfide-linked core of C5a and the negative charged N-terminal domain of its receptor, and the other, which is an interaction of the positive charged C-terminal tail of C5a and the interhelical region of its receptor and which is essential for the functional response to C5a. Based on this information, we dared to pick up an uncharged compound to discover a new type of C5a receptor antagonist after we screened our chemical libraries by binding assay using ¹²⁵I-labeled C5a according to the similar method for C5a inhibitors described previously. In addition to this point, we used whole cells of stimulated U-937 cells as a source of C5aR in the binding assay instead of the plasma membrane, which have been used in previously reported C5a inhibitors. Moreover, we used two screening systems for chemical optimization, a binding assay system the same as for the first screening and a ROS assay system with human neutrophils. Although it is not clear whether the former contributed to the discovery of potent C5aR antagonists, the latter is thought to be important for this study for the following reasons: structure activity relationships obtained during chemical optimization did not completely coincide between ROS assay using intact human neutrophils and binding assay using human tumor line, U-937 cells (data not shown). Namely, it is thought to be important to use intact human cells.

We discovered a potent and orally active non-peptide C5aR antagonist. Since C5a is implicated in a variety of diseases, it is anticipated that an orally active non-peptide C5aR antagonist may have potential as novel therapeutics.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-45-963-4286; Fax: 81-45-963-3326; E-mail: sumichika.hiroshi@mg.m-pharma.co.jp.

Published, JBC Papers in Press, October 15, 2002, DOI 10.1074/jbc.M209672200

	ABBREVIATIONS

The abbreviations used are: C5aR, C5a receptor; RA, rheumatoid arthritis; GPI, glucose 6-phosphate isomerase; rhC5a, recombinant human C5a; FCS, fetal calf serum; HBSS, Hanks' balanced salt solution; ROS, reactive oxide species; GPCR, G-protein-coupled receptor; gZAS, gerbil zymosan-activated serum.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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