Pharmacological characterization of a novel nonpeptide antagonist for formyl peptide receptor-like 1?

Article abstract of DOI:10.1124/mol.107.037564

A series of quinazolinone derivatives were synthesized based on a hit compound identified from a high-throughput screening campaign targeting the human formyl peptide receptor-like 1 (FPRL1). Based on structure-activity relationship analysis, we found tha

Full text of DOI:10.1124/mol.107.037564

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2007/07/31/mol.107.037564.DC1.html
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MOLECULAR PHARMACOLOGY
Vol. 72, No. 4
37564/3260349
Printed in U.S.A.
Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 72:976–983, 2007
Pharmacological Characterization of a Novel Nonpeptide
□
S
Antagonist for Formyl Peptide Receptor-Like 1
Caihong Zhou, Song Zhang, Masakatsu Nanamori, Yueyun Zhang, Qing Liu, Na Li,
Meiling Sun, Jun Tian, Patrick P. Ye, Ni Cheng, Richard D. Ye, and Ming-Wei Wang
The National Center for Drug Screening, Shanghai Institute of Materia Medica (C.Z., Y.Z., Q.L., N.L., M.S., M.-W.W.),
and the Graduate School (S.Z.), Chinese Academy of Sciences, Shanghai, China; and Department of Pharmacology,
College of Medicine, University of Illinois, Chicago, Illinois (M.N., J.T., P.P.Y., N.C., R.D.Y.)
Received April 27, 2007; accepted July 25, 2007
ABSTRACT
A series of quinazolinone derivatives were synthesized based
[
125I]Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm) binding to
on a hit compound identified from a high-throughput screening FPRL1 but not [3H]N-formyl-Met-Leu-Phe to formyl peptide
campaign targeting the human formyl peptide receptor-like 1 receptor. In functional assays using FPRL1-expressing RBL-
(FPRL1). Based on structure-activity relationship analysis, we 2H3 cells, C7 inhibited calcium mobilization and chemotaxis
found that substitution on the para position of the 2-phenyl induced by WKYMVm and C1 and degranulation elicited by C1.
group of the quinazolinone backbone could alter the pharma- C7 also suppressed C1-induced extracellular signal-regulated
cological properties of the compound. The methoxyl substitu- kinase phosphorylation and reduced arachidonic acid-induced
tion produced an agonist 4-butoxy-N-[2-(4-methoxy-phenyl)-4- ear edema in mice. This study represents the first character-
oxo-1,4-dihydro-2H-quinazolin-3-yl]-benzamide (Quin-C1; C1), ization of a nonpeptidic antagonist for FPRL1 and suggests the
whereas a hydroxyl substitution resulted in a pure antagonist, prospect of using low molecular weight compounds as modu-
Quin-C7 (C7). Several partial agonists were derived from other lators of chemoattractant receptors in vitro and in vivo.
substitutions on the para position. C7 partially displaced
The human formyl peptide receptor (FPR) family of che- activate FPRL1 with a reduced affinity (Quehenberger et al.,
moattractant receptors consists of FPR, formyl peptide recep- 1993). Stimulation of FPRL1 elicits a cascade of host defense
reactions against pathogens, including chemotaxis, superox-
ide generation, and exocytosis in human neutrophils. In ad-
dition, it was reported that FPRL1 attenuates HIV-1 infec-
tion by desensitizing and down-regulating the chemokine
receptors CCR5 and CXCR4, which serve as major corecep-
tors for HIV-1, on monocyte surfaces (Li et al., 2001). A recent
study showed that the expression of FPRL1 in mouse C26
cells markedly reduced tumorigenicity in syngeneic mice and
resulted in high levels of humoral immune response to both
FPRL1-containing and wild-type C26 cells (Hu et al., 2005).
The data indicate that FPRL1 also plays a key role in specific
antitumor response. The expression of FPRL1 in activated
microglial cells and its function as a receptor for A␤(1–42), a
42-amino acid form of the ␤-amyloid peptide, suggest that
FPRL1 is closely related to neurodegenerative disorders (Hu
et al., 2005). Investigation in ligand binding, signal transduc-
tion, and functional properties of FPRL1 is expected to facil-
tor-like 1 (FPRL1), and FPRL2. These receptors are ex-
pressed primarily in neutrophils and monocytes and exert
important functions in inflammation and immunity (Le et
al., 2002). The FPRL1 gene was initially cloned in 1992 for its
homology with FPR cDNA (Bao et al., 1992; Murphy et al.,
1992; Ye et al., 1992). The prototype of chemotactic peptide
N-formyl-Met-Leu-Phe (fMLF), an agonist for FPR, can also
This project was supported in part by grants from the Ministry of Science
and Technology of China (2004CB518902 to M.-W.W.), Chinese Academy of
Sciences (KSCX1-SW-11-2 to M.-W.W.), Shanghai Municipality Science and
Technology Development Fund (05DZ22914 and 06DZ22907 to M.-W.W.), and
National Institutes of Health (AI033503 to R.D.Y.).
C.Z. and S.Z. contributed equally to this work.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.107.037564.
□S The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: FPR, formyl peptide receptor; FPRL1, formyl peptide receptor-like 1; SAR, structure-activity relationship; HTS, high-throughput
screening; WKYMVm, Trp-Lys-Tyr-Met-Val-D-Met-NH2; WRW4, Trp-Arg-Trp-Trp-Trp-Trp-NH2; fMLF, N-formyl-Met-Leu-Phe; DMEM, Dulbecco’s
modified Eagle’s medium; FBS, fetal bovine serum; BSA, bovine serum albumin; NF-␬B, nuclear factor-␬B; ERK, extracellular signal-regulated
kinase; DMSO, dimethyl sulfoxide; AA, arachidonic acid; GPCR, G protein-coupled receptor; HIV-1, human immunodeficiency virus-1; Quin-C1,
4-butoxy-N-[2-(4-methoxy-phenyl)-4-oxo-1,4-dihydro-2H-quinazolin-3-yl]-benzamide.
976

Quinazolinone Antagonist for FPRL1
977
itate the understanding of this receptor as a new therapeutic ylacetamide catalyzed by acetic acid) using molecular sieve as dehy-
drate reagent.
target.
Among the FPR family of receptors, FPRL1 has the broad-
Ligand Binding Assay. Ligand binding assay was performed as
described previously (Yan et al., 2006). RBL-FPRL1 cells (ϳ109) were
harvested and washed twice with phosphate-buffered saline. Cell
membrane was prepared with BioNeb Cell Disruption System (Glas-
Col, Terre Haute, IN). Various concentrations of compounds were
incubated together with RBL-FPRL1 cell membrane preparation,
0.16 nM [125I]WKYMVm (PerkinElmer Life and Analytical Sciences,
Kd ϭ 0.32 nM), and FlashBlue GPCR beads (100 ␮g/well) to give a
final volume of 0.1 ml. The plates were incubated at 4°C for 12 h and
est spectrum of ligands (Migeotte et al., 2006). Except for one
lipid (lipoxin A4), all identified FPRL1 ligands are small
peptides and include host-derived agonist LL-37, natural or
synthetic peptides (humanin, MMK-1, and WKYMVm), and
peptides derived from HIV-1 envelope proteins (Le et al.,
1999; Yang et al., 2000). These pharmacological properties of
FPRL1 imply that it is a potential target for therapeutic
intervention. However, identification of antagonists for this centrifuged for 3 min at 2500g before counting on a MicroBeta
scintillation counter (PerkinElmer Life and Analytical Sciences). To
test the binding affinity for FPR, RBL-FPR cells (1 ϫ 105) were
seeded onto 24-well plates and incubated for 48 h. After being
washed twice with blocking buffer (RPMI 1640 medium supple-
mented with 25 mM HEPES and 0.1% BSA, pH 7.5), cells were
incubated with blocking buffer for 2 h and then sequentially with 30
nM [3H]fMLF and different concentrations of C7 or unlabeled fMLF
in binding buffer (phosphate-buffered saline with 10% BSA) for
another 2 h. Radioactivity was measured as above.
receptor has met with difficulties. A new peptide (WRW-
WWW; WRW4) has been identified as an antagonist for
FPRL1 by screening a hexapeptide library (Bae et al., 2003).
Because of inherent limitations of peptides as therapeutic
agents, it is desirable to develop synthetic, nonpeptidic li-
gands for receptors. Not long ago, we initiated a high-
throughput screening (HTS) campaign to identify FPRL1
ligands from a synthetic compound library. After vigorous
screening and structure modification, a substituted quinazo-
Reporter Assay. HeLa cells expressing NF-␬B-Luc/FPRL1
linone compound (Quin-C1; 4-butoxy-N-[2-(4-methoxy-phe- (HeLa-␬B-FPRL1) were seeded onto 96-well plates at a density of
1.5 ϫ 104 cells/well. After cells became adherent, they were serum-
nyl)-4-oxo-1,4-dihydro-2H-quinazolin-3-yl]-benzamide) was
starved in DMEM for 16 h before screening assay. Different concen-
discovered. This compound (C1) displayed selective agonistic
trations of compounds were added to the cells for 5 h, and the
effects on FPRL1 (Nanamori et al., 2004). As an ongoing
expressed luciferase activity was determined in an EnVision 2101
effort to study the structure-activity relationship (SAR) of the
multilabel reader (PerkinElmer Life and Analytical Sciences) using
original compound, we designed, synthesized, and character-
the Steady-Glo Luciferase Assay solutions.
ized a series of substituted quinazolinone derivatives as mod-
ulating agents for FPRL1. Our results indicate that a hy-
droxyl substitution on the para position of the 2-phenyl group
of the quinazolinone backbone resulted in a pure antagonist,
Quin-C7 (C7), which displayed inhibitory effects on FPRL1.
Calcium Mobilization Assay. Calcium mobilization assay was
performed as described previously (Yan et al., 2006). In brief, RBL-
FPRL1 cells were detached and collected by centrifugation, loaded
with 5 ␮M Fluo-4/AM (Invitrogen) in Hanks’ balanced salt solution
supplemented with 2.5 mM probenecid for 45 min, and then washed
twice with Hanks’ balanced salt solution. Cell suspensions were
adjusted to a density of 5 ϫ 106 cells/ml and seeded onto 96-well
plates (100 ␮l/well). Cells were reattached by centrifugation and
then analyzed for calcium mobilization using FlexStation (Molecular
Devices, Sunnyvale, CA) with excitation wavelength at 485 nm and
emission wavelength at 525 nm. For antagonist mode, cells were
incubated with or without test compounds for 15 min before the
addition of WKYMVm (2 nM) or C1 (5 ␮M). For detailed character-
ization of RBL-FPRL1 cells, calcium mobilization assays were con-
ducted on a spectrofluorometer (Photon Technology Inc., Law-
renceville, NJ), using Indo-1 as indicator and procedures described
previously (Nanamori et al., 2004)
Materials and Methods
Materials. WKYMVm was synthesized at GL Biochem (Shang-
hai) Ltd. (Shanghai, China). WRW4 and MMK-1 were made at HD
Biosciences Co. (Shanghai, China). 125I-WKYMVm (Bolton-Hunter-
labeled), [3H]fMLF, and FlashBlue GPCR scintillating beads were
obtained from PerkinElmer Life and Analytical Sciences (Waltham,
MA). Steady-Glo Luciferase Assay Solutions were purchased from
Promega Corporation (Madison, WI). Fluo-4/acetoxymethyl ester,
Dulbecco’s modified Eagle’s medium (DMEM) culture medium, and
trypsin were bought from Invitrogen (Carlsbad, CA). Fetal bovine
serum (FBS) was purchased from HyClone Laboratories (Logan,
UT). The anti-ERK1/2 and anti-phospho-ERK antibodies were pro-
cured from Cell Signaling Technologies (Danvers, MA). Other re-
agents were supplied by Sigma Chemical Co. (St. Louis, MO).
Cell Culture. The human cervical carcinoma cell line HeLa was
transfected with pNF-␬B-Luc reporter plasmid that contains five
copies of NF-␬B binding sequence (Stratagene, La Jolla, CA) and a
human FPRL1 cDNA expression vector in pSFFV.neo vector as re-
ported previously (Nanamori et al., 2004; Tian et al., 2005). The
transfected cells were maintained in DMEM supplemented with 10%
FBS. Rat basophilic leukemia cell line RBL-2H3 expressing either
the human FPRL1 (RBL-FPRL1) or human FPR (RBL-FPR) was
described previously and was maintained in DMEM supplemented
with 20% FBS (He et al., 2000).
Chemotaxis. WKYMVm and C1-induced migration of cells was
assessed in a 48-well microchemotaxis chamber (Neuro Probe, Cabin
Compound Synthesis. The quinazolinone series compounds
were synthesized according to the method described previously
(Mayer et al., 1997), and the synthetic route is shown in Fig. 1.
Anthranilic acid derivative 5 was obtained subsequently via reduc-
tion of compound 4 with zinc and acetic acid in CH2Cl2. Moderate to
high yield was achieved through refluxing compound 5 with different
substituted benzaldehydes in a mixed solvent (CH2Cl2/N,N-dimeth-
Fig. 1. The synthetic route of quinazolinone C. Reagents and conditions:
a, 1: NaOH, MeOH, 0°C, 20 min; 2: n-BuBr, MeOH, reflux, 5 h; b,
N2H4.H2O, MeOH, reflux, 6 h; c, 2-nitrobenzoyl chloride, Et3N, CH2Cl2,
0°C to room temperature, overnight; d, Zn, AcOH, CH2Cl2, 0°C to room
temperature, 4 h; e, substituted benzaldehyde, AcOH/N,N-dimethyl-
acetamide/CH2Cl2 (5:5:90), 4A molecular sieve, reflux, 12 h.

978
Zhou et al.
John, MA) as described previously (Nanamori et al., 2004). In brief, one backbone might be a possible determinant for its func-
WKYMVm (10 nM, 30 ␮l) or C1 (100 nM, 30 ␮l) were placed in the
lower chamber, and RBL-FPRL1 cells (50 ␮l at 1 ϫ 106 cells/ml) were
preincubated with or without test compounds for 15 min and then
loaded in the upper chamber, which was separated from the lower
chamber by a polycarbonate filter (pore size, 8 ␮m). After incubation
at 37°C for 4 h, the filter was removed, fixed, and stained with
Diff-Quick staining solutions (IMEB Inc., San Marcos, CA). Chemo-
taxis was quantified by counting migrated cells in five randomly
chosen high-power fields (400ϫ).
tional property. Thus, more derivatives of C series were made
(Fig. 2B) in accordance with the method described previously
(Mayer et al., 1997). Amino-substituted compound (C11) was
obtained by reduction of its corresponding nitro substituted
compound (C10).
To determine the substituted position, four compounds
were synthesized first. There was a methoxy group at the 4Ј
position in C1 and a 2Ј,4Ј-disubstitution in C2 and C4. C6
contains no substitution in the aromatic ring. These modifi-
Phosphorylation of Mitogen-Activated Protein Kinases. Ac-
tivation of the p44/p42 mitogen-activated protein kinases (ERK1/2) cations produced drastically different effects: among these
was determined essentially as described previously (Nanamori et al.,
2004). In brief, cells were cultured in six-well plates and serum-
starved overnight before agonist stimulation. Some samples were
pretreated with the antagonist (C7) for 15 min before agonist (C1)
stimulation for 5 min. The reaction was terminated by adding 300 ␮l
of ice-cold SDS-polyacrylamide gel electrophoresis loading buffer
[15% (v/v) glycerol, 125 mM Tris-Cl, pH 6.8, 5 mM EDTA, 2% (w/v)
SDS, 0.1% bromphenol blue, and 1% ␤-mercaptoethanol]. Samples
were sonicated to disperse DNA contents. After boiling, samples
were analyzed by SDS-polyacrylamide gel electrophoresis and West-
compounds, C2, C4, and C6 lost agonist activity entirely in
the calcium mobilization assay (Supplementary Fig. S1).
However, C1 showed a stronger agonist activity than that of
the hit compound (Table 1).
Next, compounds with different substitutions at the 4Ј
position were synthesized to study the impacts of the substi-
tuted groups. C1 (4Ј-methoxy), C5 (4Ј-methyl), and C10 (4Ј-
nitro) exhibited strong agonist activity (Table 1). Substitu-
tions with bulky groups, such as isobutoxy (C9) or n-butoxy
ern blot using anti-ERK1/2 and anti-phospho-ERK1/2 antibodies at (C12), resulted in decreased or loss of bioactivity. Bioactivity
1:1000 dilution. Horseradish peroxidase-conjugated anti-rabbit an-
tibody (1:3000) was used as secondary antibody. The resulting im-
munocomplex was visualized by SuperSignal West Pico Chemilumi-
nescence (Pierce, Rockford, IL).
Ear Edema Assay. Male BALB/c mice weighing 20 to 24 g
(Shanghai SLAC Laboratory Animals Co., Shanghai, China) were
used in the experiment. The animals were housed in an environmen-
tally (25°C) and air humidity (60%)-controlled room with a 12-h
light/dark cycle and kept on a standard laboratory diet and drinking
water ad libitum. Mice were fasted for 18 h with free access to water
and divided into groups of four to seven animals. The study was
conducted according to the procedures approved by the institutional
animal care committee.
also decreased when the nitro was changed to amino (C11)
and lost when changed to N,N-dimethyamino group (C3). Of
particular interest is the complete reversal of bioactivity
when the methoxy group was substituted with a hydroxyl
Inflammation was induced by arachidonic acid (AA) as described
previously with minor modifications (Rao et al., 1993). In brief, AA
(0.25 mg in 20 ␮l of 5% DMSO and 95% acetone) was topically
applied onto both surfaces of the right ear of each mouse. Left ear
(control) received solvent treatment. C7, at various doses dissolved
in 1% DMSO, 19% polyethylene glycol 400, and 80% normal saline,
was administered intraperitoneally (200 ␮l) 0.5 h before AA appli-
cation. Two control groups were used: one was treated with vehicle,
and the other received dexamethasone (1 mg). Inflammation was
induced for 3 h after AA application, and the animals were sacrificed
by cervical dislocation. An 8-mm section from each ear was removed
with a metal punch and weighed immediately. Ear edema was de-
termined by subtracting the weight of the left ear from that of the
right ear. The rate of edema (percentage) was calculated by dividing
the weight difference between the left and right ear with the left ear
weight and multiplied by 100.
Results
Characterization of Quinazolinone Derivatives as
FPRL1 Ligands. In a previous study, we conducted an HTS
of 15,760 synthetic and 400 natural compounds. Among
these, three compounds were found to be potential FPRL1
agonists. Based on one hit compound, whose core structure is
shown in Fig. 2A, we developed the first nonpeptidic ligand
Quin-C1 for FPRL1 (Nanamori et al., 2004). To search for
FPRL1 antagonists, four series of quinazolinone derivatives
(C, M, W, and O) were synthesized (Fig. 2A). Few compounds
in the M, W, and O series showed activity. The preliminary
SAR analysis of C series compounds indicated that the sub-
Fig. 2. Chemical structures of hit compound (C8) and its derivatives. A,
structure of the hit compound identified from high-throughput screening
and its four series derivatives (C, M, W, and O). B, structures of C series
derivatives at R1. Compound C1 is the previously reported FPRL1 agonist
Quin-C1. Derivatives were designed based on hit compound C8 by chang-
ing the substitution on the phenyl group at the 2 position while keeping
stitution of the phenyl group at the 2 position of quinazolin- the basic quinazolinone skeleton.

Quinazolinone Antagonist for FPRL1
979
group (C7). This substitution resulted in an antagonist for C1. Our results also indicate that C7 could reduce
FPRL1 (see below).
WKYMVm-stimulated degranulation by up to 57% (data not
shown) and C1-induced degranulation in a dose-dependent
manner (Supplement Fig. S2) in RBL-FPRL1 cells.
C7 Inhibits WKYMVm-Stimulated Calcium Mobiliza-
tion. Although C7 showed a higher binding affinity than C9,
which exhibited bioactivity in reporter assay, no agonist ac-
tivity was detected in both reporter and calcium mobilization
assays (Table 1). Because the reporter assay involves the
activation of several signaling pathways and is distal from
the receptor, we conducted additional studies to study the
proximal signaling events induced by the activated receptor.
Further characterization was conducted with C7 and C12 in
calcium mobilization assays. Calcium mobilization results
from FPRL1-activated PLC␤, which generates the second
messengers diacyl glycerol and inositol 1,4,5-trisphosphate.
The RBL-FPRL1 cell line, used in this and other functional
studies described in this article, was generated through sta-
ble expression of the human FPRL1 cDNA. Reverse tran-
scription-polymerase chain reaction analysis showed that it
does not contain transcript for human FPR and FPRL2 (Fig.
3A). In calcium mobilization assays (Fig. 3B), the cell line
responded strongly to WKYMVm (100 nM); it responded to
C1 at a higher concentration (100 ␮M) but was sensitive to
the FPRL1-selective agonist MMK1 (100 nM). However,
RBL-FPRL1 only weakly responded to fMLF (100 nM), a
high-affinity agonist for FPR and low-affinity agonist for
FPRL1. F2L, an agonist for FPRL2, did not induce calcium
mobilization (data not shown). These results confirmed that
the observed calcium mobilization was mediated by FPRL1
but not the structurally related FPR or FPRL2. As shown in
Fig. 4, C7 antagonized WKYMVm-stimulated calcium mobi-
lization in a dose-dependant manner, whereas C12 inhibited
WKYMVm-stimulated calcium mobilization no more than
20% at concentrations up to 100 ␮M.
C7 and Its Analogs Compete with WKYMVm for
Binding to RBL-FPRL1 Cells. To study the binding prop-
erties of C7 and other C series compounds to FPRL1, com-
petitive binding assays using [125I]WKYMVm were per-
formed with membranes prepared from RBL-FPRL1 cells. C7
and selected analogs competed effectively for binding to RBL-
FPRL1 (Fig. 6). Among the analogs studied, C1 and C5 are
more potent than C7, exhibiting IC50 values of 92 Ϯ 1 and
174 Ϯ 61 nM, respectively. C7 could not completely displace
[
125I]WKYMVm binding to FPRL1 at 100 ␮M, the highest
concentration used in the assay because of limited solubility
of the compound. The IC50 value of 6653 Ϯ 859 nM (Table 1)
was derived from maximal but not complete displacement of
the radioligand. Although C7 is less potent than C1 in the
competitive binding assay, it specifically interacts with
FPRL1 but not FPR. In binding assays using FPR-trans-
fected RBL cells, C7 at concentrations up to 100 ␮M could not
effectively compete with [3H]fMLF for binding to the RBL-
FPR cells (Fig. 7).
C7 Inhibits Agonist-Induced ERK Phosphorylation.
FPRL1-mediated activation of the mitogen-activated protein
kinases, ERK1 and ERK2, was determined in C1-stimulated
RBL-FPRL1 cells based on activation-associated phosphory-
lation (Payne et al., 1991). Maximal activation was observed
at ϳ5 min after agonist stimulation. When the cells were
pretreated with C7, the agonist-induced phosphorylation of
ERK1 and ERK2 was reduced (Fig. 8). The inhibitory effect of
C7 was evident at 3 ␮M and higher. At 100 ␮M, C7 reduced
phosphorylated ERK to its base level.
C7 Suppresses Chemotaxis in C1 and WKYMVm-
Stimulated RBL-FPRL1 Cells. In chemotaxis assay, C7
inhibited C1-induced cell migration in a dose-dependent
manner (Fig. 5). Given the structural similarity between
these two compounds, the antagonistic effect was expected. It
is interesting that C7 also suppressed chemotaxis induced by
WKYMVm, a highly potent FPRL1 agonist with no struc-
tural resemblance to C7. Our earlier study suggested a par-
tial overlap between C1 and WKYMVm in FPRL1 binding
(Nanamori et al., 2004). The data shown in Fig. 5 could be
Comparing C7 with WRW4 for Their Antagonistic
Activities. To date, WRW4 is the only characterized peptide
with selectivity for FPRL1 (Bae et al., 2004). The antagonis-
tic effect of C7 was compared with that of WRW4 in calcium
mobilization assay. WKYMVm-induced calcium mobilization
in RBL-FPRL1 cells was dose-dependently inhibited by C7
(Fig. 9A). Likewise, WRW4 displayed inhibitory effect in the
calcium mobilization assay (Fig. 9B). An analysis of the re-
sults demonstrated that C7 had antagonist efficacy (90.6%)
partially explained with the homologous structures of C7 and comparable with that of WRW4, although its potency was not
TABLE 1
Bioactivities detected with the quinazolinone C derivatives
The compounds were evaluated for their binding abilities to FPRL1 in competition with ͓125I͔WKYMVm. Agonist activities were assessed in HeLa cells stably transfected
with human FPRL1 gene and a pNF-␬B-Luc reporter plasmid. Calcium responses were examined in RBL-2H3 cells stably transfected with human FPRL1 genes
(RBL-FPRL1). Efficacy was expressed as a percentage of the maximal response that elicited by 10 ␮M (in the reporter assay) or 1 ␮M (in the calcium mobilization assay)
WKYMVm. Each value represents means Ϯ S.E.M. of three independent experiments.
Reporter Assay
Calcium Mobilization
EC50
FPRL1
Binding,
IC50
Compound
EC50
Efficacy
Efficacy
nM
nM
%
␮M
%
Hit (C8)
C1
745 Ϯ 104
92 Ϯ 1
472 Ϯ 9
15 Ϯ 4
46 Ϯ 10
N.A.
8064 Ϯ 487
89 Ϯ 8
1352 Ϯ 201
N.A.
1.3 Ϯ 0.2
54.3 Ϯ 0.1
88.5 Ϯ 9.1
87.6 Ϯ 5.0
N.A.
12.34 Ϯ 0.83
0.47 Ϯ 0.02
1.48 Ϯ 0.15
N.A.
43 Ϯ 3
90 Ϯ 4
74 Ϯ 4
N.A.
C5
174 Ϯ 61
6653 Ϯ 859
Ͼ15,000
261 Ϯ 59
Ͼ15,000
Ͼ15,000
0.4 Ϯ 0.1
C7
C9
52.7 Ϯ 4.2
86.7 Ϯ 11.5
31.1 Ϯ 3.9
N.A.
N.A.
N.A.
C10
1.10 Ϯ 0.17
N.A.
N.A.
0.0011 Ϯ 0.0001
55 Ϯ 3
N.A.
N.A.
100
C11
C12
WKYMVm
100
N.A., no activity (efficacy Ͻ15%, or potency Ͼ20 ␮M).

980
Zhou et al.
Fig. 4. Antagonist activity of compounds on WKYMVm-stimulated cal-
cium mobilization. Different concentrations of C7 or C12 were added into
RBL-FPRL1 cells 30 min before induction with 2 nM WKYMVm. The
results (means Ϯ S.E.M.) are expressed as relative activity compared
with the maximal [Ca2ϩ] release induced by 2 nM WKYMVm.
Fig. 5. Effects of C7 on C1 (100 nM) or WKYMVm (10 nM)-induced
chemotaxis in RBL-FPRL1 cells. Various concentrations of C7 were
added before the chemotaxis assay using C1 or WKYMVm as agonists.
Data (means Ϯ S.E.M.) are expressed as a percentage of the maximal
chemotaxis and are representative of two independent experiments.
Fig. 3. Identification of FPRL1, but not FPR and FPRL2, in RBL-FPRL1
and characterization of its specificity. A, gel electrophoresis data showing
polymerase chain reaction products obtained from RBL-FPRL1-derived
cDNA. Only the FPRL1 transcript was detected. The specificity of the
primers used is confirmed in reverse transcription-polymerase chain
reaction using control cDNAs for human FPRL, FPRL1, and FPRL2.
Polymerase chain reaction was conducted for 30 cycles. Glyceraldehyde-
3-phosphate dehydrogenase was used as an internal control for the qual-
ity of the reverse-transcriptase product from RBL-FPRL1. B, calcium
mobilization assay demonstrating the responsiveness of RBL-FPRL1 to
C1 (100 ␮M), WKYMVm (W-pep), the FPRL1-selective peptide MMK1,
and fMLF, a low-affinity peptide ligand for FPRL1. All peptide agonists
were used at 100 nM. Representative tracings from three are shown.
Fig. 6. Competitive binding of selected
C series derivatives to
[
125I]WKYMVm with FPRL1. Cell membrane was prepared from RBL-
FPRL1 cells. Various concentrations of compounds were incubated to-
gether with RBL-FPRL1 cell membrane preparation, 0.16 nM
[
125I]WKYMVm, and FlashBlue GPCR beads. The plates were incubated
at 4°C for 12 h and centrifuged for 3 min at 2500g before counting on a
MicroBeta scintillation counter. The results (means Ϯ S.E.M.) are ex-
pressed as the percentage of specific binding from three independent
experiments.

Quinazolinone Antagonist for FPRL1
981
as good as WRW4. When C1 was used as agonist, the efficacy
C7 Inhibits Arachidonic Acid-Induced Ear Edema.
of C7 in the suppression of calcium release was similar to The in vivo effect of C7 was determined in an ear edema
that of WRW4 (65.9 versus 59.6%; Fig. 9, C and D).
model (Rao et al., 1993). BALB/c mice were treated with AA
(right ears) or solvent (left ears). In testing groups, different
concentrations of C7 were given intraperitoneally to mice
0.5 h before AA application. A positive control group was
included in which mice received dexamethasone instead of
C7. Three hours later, animals were sacrificed, and ear
edema was determined as described under Materials and
Methods. As shown in Fig. 10, C7 dose-dependently inhibited
AA-induced ear edema. At concentrations of 1 and 5 mg, the
inhibitory effects of C7 approached to that of dexamethasone
(1 mg).
Discussion
We reported previously the identification of Quin-C1 (C1),
the first synthetic, nonpeptidic agonist for FPRL1 identified
through an HTS campaign (Nanamori et al., 2004). Quin-C1
is one of the quinazolinone derivatives prepared from a core
structure identified from 16,160 compounds. A preliminary
SAR study suggests that substitutions at the 4Ј position of
the 2-phenyl group of quinazolinone backbone play a key role
for the bioactivity of the compounds, ranging from full ago-
nists to full antagonists. These results indicate that small
substitution groups at the 4Ј position can exhibit modulatory
effects. In this study, we describe the functional character-
ization of Quin-C7 (C7), which contains a hydroxyl group at
the 4Ј position and displays antagonistic effects in a variety
of functional assays. In competitive binding assays, C7
showed binding affinity for FPRL1 lower than that of C1, C5,
and C10 but higher than the binding affinities of C9, C11,
and C12. This binding property is highly selective for FPRL1
because it did not compete the interaction between FPR and
[3H]fMLF (Fig. 7). It is notable that C7 was unable to com-
Fig. 7. Competitive binding of C7 with [3H]fMLF. RBL-FPR cells (1 ϫ
105) were seeded onto 24-well plates and incubated for 48 h. After brief
washing twice, cells were incubated with the blocking buffer (RPMI 1640
supplemented with 25 mM HEPES and 0.1% BSA, pH 7.5) for 2 h and
then treated with different concentrations of C7 or unlabeled fMLF
together with 30 nM [3H]fMLF for an additional 2 h. Radioactivity was
measured by a MicroBeta scintillation counter. Data (means Ϯ S.E.M.)
were collected from three to four independent experiments.
Fig. 8. Effects of C7 on C1-induced phosphorylation of ERK. RBL-FPRL1
cells were pretreated with different concentrations of C7 or with vehicle
control (DMSO of same concentration). After 15 min, the cells were
stimulated with C1 (1 ␮M) for 5 min. Phosphorylation of ERK1 and ERK2
was determined as described under Materials and Methods. A represen-
tative set of blots is shown.
Fig. 9. Antagonist activity of C7 on
WKYMVm or C1-stimulated calcium
mobilization. WRW4 was used as a
control. Different concentrations of C7
or WRW4 were added into RBL-
FPRL1 cells 30 min before induction
with 2 nM WKYMVm (A and B) or 5
␮M C1 (C and D). Data are expressed
as relative activity compared with the
maximal [Ca2ϩ] release induced by 2
nM WKYMVm or 5 ␮M C1. Results
were means Ϯ S.E.M. of three inde-
pendent experiments.

982
Zhou et al.
pletely displace [125I]WKYMVm in competitive binding as- to the function of the compounds. However, disubstitutions
says. Because of the limitation of its solubility, C7 could not such as those in C2 and C4 and bulky substitutions such as
be used for more than 100 ␮M in our assays. The binding the ones in C3 and C12 produced no bioactivity in the calcium
data suggest that C7 is a partial antagonist for WKYMVm mobilization assay. This latter finding indicates that the
binding to FPRL1. As discussed below, WKYMVm and C7 binding pocket for the C series compounds is relatively small
may occupy partially overlapping binding sites on FPRL1 and cannot accommodate bulky groups or disubstitutions.
because of the difference in their structures. The fact that C7 The observation that these compounds bound poorly to
was more effective in the inhibition of C1-induced than FPRL1 supports the notion that a part of the binding pocket
WKYMVm-induced responses (Figs. 8 and 9) clearly reflects for the C series compounds provides very limited space.
the structural similarities between C7 and C1. As shown in
Our preliminary SAR study also points to the importance
Fig. 4, the inhibitory effect of C7 is specific for FPRL1 and not for proper contact between the small substitution group at
caused by cytotoxicity, because C12, a compound of the same the 4Ј position and the receptor’s binding site. Such an inter-
structural group, was neither toxic nor bioactive when tested action is crucial to the bioactivity of the compounds. Small
in the same system. Moreover, the RBL-FPRL1 cell line used substitution groups such as the methoxy, methyl, and nitro
in this study was an engineered cell line that expresses groups are excellent for the agonistic activity, whereas a
human FPRL1 but not human FPR or FPRL2 (Fig. 3A). The larger substitution group in C8 may be responsible for the
selectivity of the cell line was confirmed in calcium mobiliza- reduced agonistic activity. Relative potency, both agonistic
tion assays, which displayed pharmacological properties of and antagonistic, may also require proper spacing between
FPRL1 with functional responses to MMK1 and WKYMVm the contact sites such that compounds with an oxygen placed
but only weakly to fMLF (Fig. 3B). Together, our data sup- at the 4Ј position of the phenol ring (C1 and C7) proves to be
port the conclusion that C7 is a selective antagonist for most efficacious. However, C9 and C12 are much less effec-
FPRL1. Our attempt to generate an RBL cell line expressing tive probably because of the larger size of the substitution
human FPRL2 was not successful, probably because of the groups.
reported tendency of this receptor to express intracellularly
(Migeotte et al., 2006). Therefore, the possibility that C7 acts tion of short- and long-term inflammation. In this study, we
on FPRL2 cannot be ruled out at this time. have shown that C7 effectively inhibited AA-induced ear
Chemoattractant receptors play a key role in the regula-
The binding pockets of FPRL1 for its ligands have not been edema, suggesting an in vivo effect of C7 in the suppression
fully characterized. Based on the broad spectrum of ligand of inflammation. Several possibilities exist for this anti-in-
specificity, it is predicted that multiple binding sites exist in flammatory effect. First, C7 suppresses AA-induced ear
FPRL1 that allow its interaction with both peptides (e.g., edema through a blockade of FPRL1, offsetting the proin-
A␤1–42, MMK-1, SAA) and a lipid (lipoxin A4). Our previous flammatory effect of a FPRL1 agonist released by AA-treated
characterization revealed a partial interference between cells. Supporting this possibility is our in vitro result indi-
WKYMVm and C1 in binding assays (Nanamori et al., 2004). cating C7 as a selective antagonist for FPRL1. However, the
This may result from a partial overlap of the two binding exact agonist(s) produced by AA-stimulated cells are not
sites or an allosteric effect of one ligand that alters the known at present, because FPRL1 has a particularly broad
binding of the other ligand. The C series compounds charac- ligand selectivity and can respond to a variety of proinflam-
terized in this work are derivatives of C1, and they probably matory peptides (Migeotte et al., 2006). Second, C7 directly
share the same binding site with C1. The observation that acts on FPRL1 as an anti-inflammatory ligand in a manner
C6, which contains no substitutions in the phenol ring and similar to that of lipoxin A4. There are indeed similarities
lost its bioactivity in the calcium mobilization assay, suggests between the two agents because neither was able to perform
that small substitution groups at the 4Ј position are critical like a typical agonist yet exhibited anti-inflammatory prop-
erties. The mechanism underlying the anti-inflammatory ef-
fect of lipoxin A4 has not been fully understood, but induction
of suppressor of cytokine signaling 2 was shown to contribute
to this effect (Machado et al., 2006). It would be interesting to
determine whether C7 can also induce suppressor of cytokine
signaling 2. Finally, it remains a possibility that C7 acts on a
target molecule other than FPRL1. Although there is no
direct evidence supporting the presence of another C7 target,
the fact that C7 can reduce ear edema as effectively as
dexamethasone is of interest and suggests the potential of
developing an anti-inflammatory agent based on this lead
compound.
As one of the primary chemoattractant receptors in neu-
trophils and monocytes, FPRL1 has a particularly broad
Fig. 10. Inhibition of arachidonic acid-induced ear edema by C7. BALB/c
ligand selectivity. However, progress has been slow in the
mice were treated intraperitoneally with vehicle (1% DMSO, 19% poly-
identification of its antagonist. WRW4 was the first peptidic
ethylene glycol 400, and 80% normal saline; Ctrl), dexamethasone (DEX,
antagonist specifically targeting FPRL1, and its potency for
maximal antagonism in the low micromolar range is reason-
ably good. Although C7 was approximately 36- to 50-fold less
potent than WRW4 in similar functional assays, it could
attain comparable suppression efficacy in the calcium mobi-
1 mg), or various concentrations of C7 30 min before application of AA
(0.25 mg in 20 ␮l of a solution containing 5% DMSO and 95% acetone).
Three hours later, ear edema was determined as described under Mate-
rials and Methods and expressed as ear edema rate. Data shown are
means Ϯ S.E.M. based on one experiment with four to seven mice in each
group.

Quinazolinone Antagonist for FPRL1
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Acknowledgments
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Address correspondence to: Dr. Ming-Wei Wang, the National Center for
Drug Screening, 189 Guo Shou Jing Road, Shanghai 201203, China. E-mail:
mwwang@mail.shcnc.ac.cn