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Phoenix Pharmaceuticals, Inc. 
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WRWWWW-NH2 (WRW4)
An Antagonist for FPRL1 |
| Identification of Peptides That Antagonize
Formyl Peptide Receptor-Like 1-Mediated Signaling
Formyl peptide receptor-like 1 (FPRL1) is an important classical
chemoattractant receptor that is expressed in phagocytic cells in
the peripheral blood and brain. Recently, various novel agonists
have been identified from several origins, such as host-derived
molecules. Activation of FPRL1 is closely related to inflammatory
responses in the host defense mechanism and neurodegenerative disorders.
In the present study we identified several novel peptides by screening
hexapeptide libraries that inhibit the binding of one of FPRL1’s
agonists (Trp-Lys-Tyr-Met-Val-D-Met-CONH2 (WKYMVm)) to its specific
receptor, FPRL1, in RBL-2H3 cells. Among the novel peptides, Trp-Arg-Trp-Trp-Trp-Trp-CONH2
(WRWWWW (WRW4) showed the most potent activity in terms
of inhibiting WKYMVm binding to FPRL1. We also found that WRW4 inhibited
the activation of FPRL1 by WKYMVm, resulting in the complete inhibition
of the intracellular calcium increase, extracellular signal-regulated
kinase activation, and chemotactic migration of cells toward WKYMVm.
For the receptor specificity of WRW4 to the FPR family,
we observed that WRW4 specifically inhibit the increase in intracellular
calcium by the FPRL1 agonists MMK-1, amyloid Abeta42 (A42) peptide,
and F peptide, but not by the FPR agonist, fMLF. To investigate
the effect of WRW4 on endogenous FPRL1 ligand-induced
cellular responses, we examined its effect on Abeta42 peptide in
human neutrophils. Abeta42 peptide-induced superoxide generation
and chemotactic migration of neutrophils were inhibited by WRW4,
which also completely inhibited the internalization of A42 peptide
in human macrophages. WRW4 is the first specific FPRL1
antagonist and is expected to be useful in the study of FPRL1 signaling
and in the development of drugs against FPRL1-related diseases.
Bae Y. S., et al. The Journal of Immunology, 2004, 173: 607-614.
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FIGURE 1. The initial screening of the
PS-SPCLs for peptides that inhibit the binding of 125I-labeled
WKYMVm (50 pM) in FPRL1-expressing RBL-2H3 cells. FPRL1-expressing
RBL-2H3 cells (1 x 105 cells/200 µl) were used for the
binding assay. The ligand binding assay was monitored. The
results shown are representative of four independent experiments. |
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FIGURE 2. Effects of several candidate
peptides based on the PS-SPCL screening results with regard
to the inhibition of WKYMVm binding in FPRL1-expressing RBL-2H3
cells. FPRL1-expressing RBL-2H3 cells (1 x 105 cells/200 µl)
were used for binding assay, and various concentrations of
each unlabeled peptide ((RRW4, RHW4, WRW4, and DRW4) were
pretreated before the addition of 125I-labeled WKYMVm (50
pM). Specifically bound 125I-labeled WKYMVm was measured.
The results shown are representative of four independent experiments. |
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FIGURE 3. Flow cytometric analysis of
FPRL1- or vector-expressing RBL-2H3 cells with biotin-WRW4.
Cultured FPRL1-expressing (A) or vector-expressing (B) RBL-2H3
cells (1 x 107/ml) were labeled with 10 µM biotin-WRW4
in the absence (bold solid line) or the presence (solid line)
of 30 µM WKYMVm. The samples were further incubated
with streptavidin-FITC, fixed with 0.2% paraformaldehyde,
and analyzed in FACSCalibur (BD Biosciences). The shaded area
indicates unstained cells. |
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FIGURE 4. Effect of WRW4 on the WKYMVm-induced
[Ca2+]i increase in FPRL1-expressing RBL-2H3 cells. Cells
were stimulated with vehicle or WRW4 (10 µM) and then
with WKYMVm (10 nM) or the Ag DNP-HSA (1 µg/ml). The
changes in 340/380 nm were monitored. The results are representative
of three independent experiments (A). Cells were stimulated
with various concentrations of WRW4 before adding 10 nM WKYMVm
or 1 µg/ml DNP-HSA. The results shown are the mean ±
SE of four independent experiments (B). |
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FIGURE 5. Effect of WRW4 on WKYMVm-stimulated
ERK phosphorylation in FPRL1-expressing RBL-2H3 cells. FPRL1-expressing
RBL-2H3 cells were treated with various concentrations of
WRW4 for 5 min, then stimulated with vehicle, 10 nM WKYMVm,
or 1 µg/ml DNP-HSA for 5 min (A). Phosphorylated ERK
was determined by immunoblot analysis with anti-phospho-ERK
Ab (A). ERK phosphorylation was quantified by densitometry.
Results are presented as the mean ± SE of at least
six independent experiments (B). |
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FIGURE 6. Effect of WRW4 on WKYMVm-induced
cellular chemotaxis in FPRL1-expressing RBL-2H3 cells. Cultured
FPRL1-expressing RBL-2H3 cells (1 x 106 cells/ml RPMI 1640)
were added to the upper wells of a 96-well chemotaxis chamber
for 4 h at 37°C. The numbers of migrated cells were determined
by counting in a high power field (x400). Various concentrations
of WRW4 or WKYMVm were used in the assays (A). Several concentrations
of WRW4 or 10 µM LFMYHP were added before the chemotaxis
assay using 10 nM WKYMVm (B). Results are presented as the
mean ± SE of three independent experiments, each performed
in duplicate. |
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FIGURE 7. Specific inhibition of the
FPRL-1-induced [Ca2+]i increase by WRW4 in human neutrophils.
Fura-2-loaded human neutrophils were treated with vehicle
or WRW4 (10 µM), then stimulated with A42 (40 µM)
or fMLF (1 µM). Changes in 340/380 nm were monitored.
The results shown are representative of three independent
experiments (A). Neutrophils were stimulated with vehicle
or WRW4 (10 µM), then stimulated with MMK-1 (1 µM),
A42 (40 µM), F peptide (30 µM), LXA4 (1.4 µM),
fMLF (1 µM), or ATP (500 µM) (B). Changes in 340/380
nm were monitored, and the calibrated fluorescence ratio was
converted to [Ca2+]i. Results are presented as the mean ±
SE of three independent experiments, each performed in duplicate
(B). |
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FIGURE 8. Effect of WRW4 on Abeta42-induced
superoxide generation and chemotaxis in human neutrophils.
Human neutrophils (1 x 106 cells/100 µl) were stimulated
with various concentrations of WRW4 or Abeta42 (A). Cells
were preincubated with several concentrations of WRW4 or with
10 µM control peptide (LFMYHP) for 1 min before adding
40 µM Abeta42 peptide (B). Cytochrome c reduction was
monitored. The results shown are representative of four independent
experiments (A and B). Chemotaxis assays were performed with
various concentrations of WRW4 or Abeta42 (C). Several concentrations
of WRW4 or 10 µM control peptide (LFMYHP) were pretreated
before the chemotaxis assay using 40 µM Abeta42 (D).
The data are presented as the mean ± SE of three independent
experiments, each performed in duplicate (C and D). |
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FIGURE 9. Effect of WRW4 on the internalization
of Abeta42 peptide in human macrophages. Human macrophages
were cultured on chamber slides and incubated with 10 µM
Abeta42 at 37°C for various times in the absence or the
presence of 10 µM WRW4. The cells were then rinsed,
permeabilized, and stained with anti-Abeta42 Ab or control
Ab. The samples were further incubated with FITC-conjugated
goat anti-mouse IgG. Abeta42 staining was examined under a
confocal microscope. |
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Ac2-26 is a ligand for Formylated Peptide Receptor
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| Monitoring
Formylated Peptide Receptor with Fluorescent Peptide |
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keywords: WRW, WRW4, WKYMVm,WKYMVM,Antagonist,
WRW, WRW4, WKYMVm, WKYMVM, Antagonist |
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