C- and N-terminal residue effect on peptide derivatives' antagonism toward the formyl-peptide receptor

Article abstract of DOI:10.1016/S0014-2999(01)01627-2

The biological action of several X-Phe-D-Leu-Phe-D-Leu-Z (X = 3′,5′-dimethylphenyl-ureido; Z = Phe, Lys, Glu, Tyr) analogues was analysed on human neutrophils to evaluate their ability to antagonize formyl-peptide receptors. X-Phe-D-Leu-Phe-D-Leu-Phe analogues obtained as C-terminal olo or amido derivatives and T-Phe-D-Leu-Phe-D-Leu-Phe analogues (T = thiazolyl-ureido) were also analysed. The activities of pentapeptide derivatives were compared with those of X-Phe-D-Leu-Phe-D-Leu-Phe chosen as reference antagonist. Our results demonstrate that X-Phe-D-Leu-Phe-D-Leu-Phe-olo, X-Phe-D-Leu-Phe-D-Leu-Glu and X-Phe-D-Leu-Phe-D-Leu-Tyr are more active antagonists than X-Phe-D-Leu-Phe-D-Leu-Phe. The presence of Lys (X-Phe-D-Leu-Phe-D-Leu-Lys) seems, instead, to inhibit the formyl-peptide receptor antagonist properties. The presence of the N-terminal thiazolyl-ureido group seems to considerably contribute to the receptor antagonist properties of T-Phe-D-Leu-Phe-D-Leu-Phe-OH. The introduction of the C-terminal methyl ester (T-Phe-D-Leu-Phe-D-Leu-Phe-OMe) or amido group (X-Phe-D-Leu-Phe-D-Leu-Phe-NH₂) appears detrimental for the affinity and formyl-peptide receptor antagonist properties of the Phe-D-Leu-Phe-D-Leu-Phe derivatives. The examined peptides inhibit superoxide anion production and lysozyme release more efficaciously than neutrophil chemotaxis.

Full text of DOI:10.1016/S0014-2999(01)01627-2

European Journal of Pharmacology 436 (2002) 187–196
www.elsevier.com/locate/ejphar
C- and N-terminal residue effect on peptide derivatives’ antagonism
toward the formyl-peptide receptor
a
c
Alessandro Dalpiaz a, Maria E. Ferretti b, , Gianni Vertuani , Serena Traniello ,
*
Angelo Scatturin a, Susanna Spisani c
aDepartment of Pharmaceutical Sciences, Ferrara University, via Fossato di Mortara 19, 44100 Ferrara, Italy
bSection of General Physiology, Department of Biology, Ferrara University, via L. Borsari 46, 44100 Ferrara, Italy
cDepartment of Biochemistry and Molecular Biology, Ferrara University, via L. Borsari 46, 44100 Ferrara, Italy
Received 5 July 2001; received in revised form 30 November 2001; accepted 24 December 2001
Abstract
The biological action of several X-Phe-D-Leu-Phe-D-Leu-Z (X = 3V,5V-dimethylphenyl-ureido; Z = Phe, Lys, Glu, Tyr) analogues was
analysed on human neutrophils to evaluate their ability to antagonize formyl-peptide receptors. X-Phe-D-Leu-Phe-D-Leu-Phe analogues
obtained as C-terminal olo or amido derivatives and T-Phe-D-Leu-Phe-D-Leu-Phe analogues (T = thiazolyl-ureido) were also analysed. The
activities of pentapeptide derivatives were compared with those of X-Phe-D-Leu-Phe-D-Leu-Phe chosen as reference antagonist. Our results
demonstrate that X-Phe-D-Leu-Phe-D-Leu-Phe-olo, X-Phe-D-Leu-Phe-D-Leu-Glu and X-Phe-D-Leu-Phe-D-Leu-Tyr are more active
antagonists than X-Phe-D-Leu-Phe-D-Leu-Phe. The presence of Lys (X-Phe-D-Leu-Phe-D-Leu-Lys) seems, instead, to inhibit the formyl-
peptide receptor antagonist properties. The presence of the N-terminal thiazolyl-ureido group seems to considerably contribute to the receptor
antagonist properties of T-Phe-D-Leu-Phe-D-Leu-Phe-OH. The introduction of the C-terminal methyl ester (T-Phe-D-Leu-Phe-D-Leu-Phe-
OMe) or amido group (X-Phe-D-Leu-Phe-D-Leu-Phe-NH2) appears detrimental for the affinity and formyl-peptide receptor antagonist
properties of the Phe-D-Leu-Phe-D-Leu-Phe derivatives. The examined peptides inhibit superoxide anion production and lysozyme release
more efficaciously than neutrophil chemotaxis. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Human, neutrophil; Formyl-peptide receptor; Antagonist; Neutrophil functionality
1. Introduction
neutrophils express a low affinity variant (FPRL1) (Durstin
et al., 1994). The binding of fMLF to its receptor triggers
different functional responses, including adhesion, chemo-
taxis and free radical generation, which contribute to the
physiological defence against bacterial infections and cell
disruption (Prossnitz and Ye, 1997). On the other hand, in
several pathological conditions, the inappropriate activation
of neutrophils can cause tissue damage (Smith, 1994). In
this context, a peptide derivative (Meera et al., 1999) and
cyclosporin A (Spisani et al., 2001) have been proposed as
anti-inflammatory agents on the basis of their ability to
inhibit neutrophil-derived lysosome enzymes and reactive
oxygen species. Moreover, annexin I peptides have been
recently reported as novel, endogenous fMLF receptor
ligands able to induce anti-inflammatory effects (Walther
et al., 2000). It has been demonstrated that mice with a
disrupted formyl-peptide receptor gene display an impaired
antibacterial immunity (Gao et al., 1999), indicating that an
inflammatory response of neutrophils towards chemotactic
peptides also plays a role in immune responses. The im-
Polymorphonuclear neutrophils are phagocytic cells in-
volved in the defence against infections (Prossnitz and Ye,
1997). Evidence has shown that the peptide derivative
formyl-Met-Leu-Phe-OH (fMLF), produced by bacterial
sources or by disrupted mitochondria, is a potent stimulator
of neutrophils (Carp, 1982; Marasco et al., 1984) and it is
consequently used as a model chemoattractant for the study
of phagocyte functions (Prossnitz and Ye, 1997; August et
al., 1997). These researches led to the identification, on
neutrophil membrane, of a G-protein-coupled formyl-pep-
tide receptor which has since been cloned (Koo et al., 1983;
Boulay et al., 1990; August et al., 1997). In addition to this
receptor, which shows high affinity toward fMLF, human
*
Corresponding author. Tel.: +39-532-291-481; fax: +39-532-207-143.
E-mail address: clm@dns.unife.it (M.E. Ferretti).
0014-2999/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0014-2999(01)01627-2

188
A. Dalpiaz et al. / European Journal of Pharmacology 436 (2002) 187–196
munomodulatory activity of cyclosporins, proposed as can-
cer chemotherapeutics, has been recently related to the
inhibition of formyl-peptide receptor functions (Tiberghien
et al., 2000). The development of fMLF receptor antagonists
can be of considerable interest to better understand the role
of drugs with immunomodulatory activity and potential
effects in inflammation-related disorders.
D-Leu-Phe-D-Leu-Phe shows formyl-peptide receptor antag-
onist activity on human neutrophils (Aswanikumar et al.,
1977; Toniolo et al., 1990). It was also suggested that the
tert-butyloxycarbonyl group in peptide derivatives is essen-
tial for imparting antagonist activity on rabbit and human
neutrophils even if it causes a loss of binding potency (Freer
et al., 1980; Derian et al., 1996). Higgins et al. (1996) have
reported that N-ureido-Phe-D-Leu-Phe-D-Leu-Phe deriva-
tives display an appreciable antagonist activity on human
neutrophils on the basis of their observation that these
peptides inhibit [3H]fMLF binding and superoxide anion
(Oꢀ2 ) production. We have recently confirmed and extended
these studies, demonstrating that N-ureido-Phe-D-Leu-Phe-
D-Leu-Phe peptide derivatives are able to antagonize multi-
ple neutrophil functions evoked by fMLF, i.e. chemotaxis,
Oꢀ2 production and secretagogue activity (Dalpiaz et al.,
1999; Scatturin et al., 1999). Moreover, they displace
[3H]fMLF from its binding sites and reduce fMLF effective-
ness in enhancing the cytosolic level of Ca2 +, a second
messenger involved in fMLF actions. The extent of inhib-
ition of Ca2 + intracellular enhancement, as well as of Oꢀ2
production and enzyme release by N-ureido-Phe-D-Leu-Phe-
D-Leu-Phe peptide derivatives, appears related to their af-
Although the structural requirements for agonist inter-
action with formyl-peptide receptor have been extensively
studied (Freer et al., 1982; Derian et al., 1996; Higgins et al.,
1996; Dalpiaz et al., 2001), the information about formyl-
peptide receptor antagonists appears to be relatively scarce.
As a matter of fact, it has been reported that cyclosporine H
(De Paulis et al., 1996), pyrazolidinedione derivatives
(Levesque et al., 1991), spinorphin (Yamamoto et al.,
1997), chenodeoxycholic acid (Chen et al., 2000) and
peptide derivatives (Higgins et al., 1996) inhibit the fMLF
binding to its receptor and some related cellular effects, but
detailed information about the structural basis of this action
is available for relatively few compounds (Levesque et al.,
1992; Wenzel-Seifert et al., 1998; Dalpiaz et al., 1999).
As for the peptide derivatives, it was reported that the
tert-butyloxycarbonyl (t-Boc) peptide derivative t-Boc-Phe-
Fig. 1. Chemical formulas of the N-xilen-ureido pentapeptide derivatives analysed. X = 3V,5-dimethylphenyl-ureido; amino acids are indicated by one-letter
codes.

A. Dalpiaz et al. / European Journal of Pharmacology 436 (2002) 187–196
189
finity toward the formyl-peptide receptor. We have also
demonstrated that the C-terminal free acid peptide deriva-
tives appear to be more active on human neutrophils than
the methyl ester ones (Dalpiaz et al., 1999; Scatturin et al.,
1999). The latter observation was the stimulus for our
further investigations into the effect of a hydrophilic envi-
ronment in the C-terminal position of N-ureido-pentapeptide
derivatives. This paper therefore reports the synthesis and
analysis of the biological properties of several X-Phe-D-Leu-
Phe-D-Leu-Z [X = xilen-ureido; Z = Phe (F), Lys (K), Glu
(E), Tyr (Y)] analogues with potential formyl-peptide recep-
tor antagonist properties (Fig. 1). In particular, we have
compared X-FDLFDLK-OH (L= Leu), X-FDLFDLE- OH
and X-FDLFDLY-OH activities with respect to those of X-
FDLFDLF-OH, which thus represents a reference com-
pound in view of its previously demonstrated ability to act
as a formyl-peptide receptor antagonist (Dalpiaz et al.,
1999). In this context Lys, Glu or Tyr have been inserted
in the C-terminal position of the free acid peptide chain with
the aim of verifying the ability of these amino acids, more
hydrophilic than Phe, to influence the antagonist behaviour
of the ligands to which they belong. The derivatives X-
FDLFDLF-olo and X-FDLFDLF-NH2 (Fig. 1) were also
analysed.
Fig. 2) have been synthesised and analysed with the aim
to investigate the effects derived by a new N-terminal
group.
The biological properties of the pentapeptide analogues
were verified on human neutrophils by means of several in
vitro assays: receptor binding and evaluation of their ability
to inhibit fMLF-induced chemotaxis, Oꢀ2 production and
enzyme release.
2. Materials and methods
2.1. Peptides
The peptides were synthesised by conventional methods
in the solution. Amino acid derivatives were obtained from
commercial sources or synthesised according to the liter-
ature (Bodanszky and Bodanszky, 1984). The peptide deriv-
atives 3V,5V-dimethylphenyl-ureido-(Phe-D-Leu)2-Phe-OMe
and 3V,5V-dimethylphenyl-ureido-(Phe-D-Leu)2-Phe-OH were
obtained as previously described by Dalpiaz et al. (1999).
The protecting groups tert-butyloxycarbonyl (t-Boc) and
tert-butyl ester (OtBu) were removed by treatment with a
mixture of trifluoroacetic acid and CH2Cl2 (1:1 v/v). The
benzyloxycarbonyl (Z) protecting group was removed by
catalytic hydrogenation in methanol in the presence of 10%
palladium on carbon and equivalent HCl using a Parr
apparatus at about 60 psi. The methyl ester group was
hydrolysed with 2 N NaOH and methanol (1:1 v/v). The
active carbamate, thiazol-2-yl-carbamic acid 2,5-dioxo-pyr-
rolidin-1-yl ester, was prepared by stirring for 24 h at room
The xilen-ureido substituent (X) has been included at
the N-terminal of all the peptide analogues on the basis of
our previous demonstration that this group confers appre-
ciable formyl-peptide receptor antagonist properties to the
Phe-D-Leu-Phe-D-Leu-Phe derivative (Dalpiaz et al., 1999;
Scatturin et al., 1999). Moreover, the derivatives T-FDL
FDLF-OMe and T-FDLFDLF-OH (T = thiazolyl-ureido,
Fig. 2. Chemical formulas of the N-thiazolyl-ureido pentapeptide derivatives analysed. T = thiazolyl-ureido; amino acids are indicated by one-letter codes.

190
A. Dalpiaz et al. / European Journal of Pharmacology 436 (2002) 187–196
temperature 2-aminothiazol (5 mmol; from Fluka) and N,
NI-disuccinimidyl carbonate (10 mmol; from Novabio-
chem) in anhydrous acetonitrile (150 ml) (Takeda et al.,
1983). The xilen-ureidic derivatives were obtained by the
reaction of respective N-deprotected peptides with 3,5-
dimethyl-phenylisocyanate in N,N-dimethylformamide in
the presence of triethylamine. Using N-ethyl-N V-(3-di-
methylaminopropyl) carbodiimide hydrochloride and hy-
droxybenzotriazole, t-Boc-Phe-OH was coupled (on 28
mmol scale) to HClꢁH-D-Leu-OMe in N,N-dimethylforma-
mide containing N-methylmorpholine at 0 ꢀC (1 h). After
12 h at room temperature, the dipeptide t-Boc-Phe-D-Leu-
OMe (1) was obtained. The same coupling method was
employed in further condensation procedures. In order to
obtain the pentapeptides 3V,5V-dimethylphenyl-ureido-(Phe-
D-Leu)2-Lys-(HꢁHCl)-OH (9) and 3V,5V-dimethylphenyl-ure-
ido-(Phe-D-Leu)2-Tyr-OH (10), dipeptide 1 was then hydro-
lysed to t-Boc-Phe-D-Leu-OH (2). The same peptide was
condensed (on a 5-mmol scale) with the two derivatives
HClꢁH-Lys-(Z)-OMe and HClꢁH-Tyr-OMe, producing the
tripeptides t-Boc-Phe-D-Leu-Lys-(Z)-OMe (3) and t-Boc-
Phe-D-Leu-Tyr-OMe (4), respectively. Tripeptides 3 and
4, N-deprotected, were then condensed (on a 2.9-mmol
scale) with dipeptide 2, obtaining two pentapeptides: t-
Boc-(Phe-D-Leu)2-Lys-(Z)-OMe (5), which was purified
by chromatography on a silica gel column eluted with
ethyl acetate–methanol (9:1), and t-Boc-(Phe-D-Leu)2-Tyr-
OMe (6). Successively, pentapeptides 5 and 6 were N-
deprotected and added (on the 0.8-mmol scale) to the
appropriate isocyanate, obtaining two xilen-ureidic deriv-
atives: 3V,5V-dimethylphenyl-ureido-(Phe-D-Leu)2-Lys-(Z)-
OMe (7), which was purified in the same way as compound
5, and 3V,5V-dimethylphenyl-ureido-(Phe-D-Leu)2-Tyr-OMe
(8). Peptide 7 was hydrogenated (0.4 mmol) and succes-
sively hydrolysed (0.3 mmol), producing the final peptide
9, purified by stirring in ethyl ether. Peptide 8 was hydro-
lysed (0.35 mmol) to obtain the final peptide 10, which was
crystallised from ethyl acetate and petroleum ether. Dipep-
tide 1 was N-deprotected and condensed (on the 2.46-mmol
scale) with dipeptide 2 to obtain the tetrapeptide t-Boc-
(Phe-D-Leu)2-OMe (11). Peptide 11, after the cleavage of
the t-Boc-group, was added to isocyanate (on the 1.4-mmol
scale) to obtain 3V,5V-dimethylphenyl-ureido-(Phe-D-Leu)2-
OMe (12). Peptide 12 was successively hydrolysed (1.34
mmol) and condensed (on the 0.73-mmol scale) with de-
rivative HClꢁH-Glu-(OtBu)-OMe, obtaining pentapeptide
3V,5V-dimethylphenyl-ureido-(Phe-D-Leu)2-Glu-(OtBu)-OMe
(13). Peptide 13, which was hydrolysed (0.6 mmol) and
cleft on the side chain of the Glu residue (0.5 mmol),
produced the final peptide 3V,5V-dimethylphenyl-ureido-
(Phe-D-Leu)2-Glu-OH (14) that was purified by stirring in
ethyl ether. Tetrapeptide 12, after hydrolysis, was condensed
(on the 0.23-mmol scale) with derivative HClꢁH-Phe-NH2 to
produce the final peptide 3V,5V-dimethylphenyl-ureido-(Phe-
D-Leu)2-Phe-NH2 (15) which was purified in the same way
as compound 14. The final pentapeptide 3V,5V-dimethyl-
phenyl-ureido-(Phe-D-Leu)2-Phe-olo (19) was synthesised
starting from dipeptide 2, which was condensed (on the
4.5-mmol scale) with derivative HClꢁH-Phe-OMe to ob-
tain the tripeptide t-Boc-Phe-D-Leu-Phe-OMe (16). The
methyl ester group of peptide 16 (3.71 mmol) was reduced
to the primary alcoholic group in ethanol/H2O (1:1 v/v)
containing NaBH4 (14.84 mmol). The solution was stirred
at room temperature for 1 h and heated at reflux for 5 h,
giving t-Boc-Phe-D-Leu-Phe-olo (17). Tripeptide 17, after
the cleavage of the t-Boc-group, was coupled (on the 1.71-
mmol scale) to dipeptide 2, producing the pentapeptide t-
Boc-(Phe-D-Leu)2-Phe-olo (18). Peptide 18 was N-de-
protected (1.16 mmol) and added (on the 0.73-mmol scale)
to 3,5-dimethyl-phenylisocyanate to obtain final peptide
19, which was crystallised from ethyl acetate and petro-
leum ether. The derivative T-FDLFDLF-OMe (20) was pre-
pared by stirring for 48 h thiazol-2-yl-carbamic acid 2,5-
dioxo-pyrrolidin-1-yl ester (3 mmol) with the corresponding
N-deprotected pentapeptide (1.5 mmol; Dalpiaz et al.,
1999) in anhydrous dioxane containing N-methylmorpho-
line (1.5 mmol). The derivative T-FDLFDLF-OH (21) was
obtained from peptide 20 by treatment with NaOH in
methanol (on the 1-mmol scale) and purified by stirring
in ethyl ether. The structure of the purified peptides was
1
confirmed by amino acid analysis and H NMR spectrom-
etry. The molecular weight of peptides was determined
using a mass spectrometer Hewlett-Packard MALDI TOF
model G2025A. All peptides were purified by silica gel
column and were analysed by reverse phase liquid chro-
matography on a Vydac C18 column with acetonitrile gra-
dient elution. The compounds were characterised by mass
spectrometry analysis, melting point determination and po-
larimetric measurements.
Stock solutions, 10 ꢀ 2 M of fMLF (Sigma, St. Louis,
MO, USA) and pentapeptide analogues, were prepared in
dimethylsulfoxide (DMSO, Sigma) and diluted in Krebs–
Ringer phosphate containing 0.1% w/v glucose (KRPG, pH
7.4) before use.
KRPG was made up as a stock solution of the following
composition: NaCl, 40 g/l; KCl, 1.875 g/l; Na2HPO4ꢁ2H2O,
0.6 g/l; KH2PO4, 0.125 g/l; NaHCO3, 1.25 g/l; and glucose,
10 g/l. This solution was five times the working strength.
MgCl2 and CaCl2 (1 mM) were supplemented to the buffer
before the biological test. All reagents were of the purest
grade and commercially available.
Pentapeptide derivatives were assayed for their ability to
cause leakage of the cytoplasmic marker lactic dehydrogen-
ase but none of them did (data not reported). At the con-
centrations used, DMSO did not interfere with any of the
biological assays performed.
2.2. Cell preparation
Cells were obtained from the blood of healthy subjects,
and the neutrophils were purified employing the standard
techniques of dextran sedimentation (Pharmacia, Uppsala,

A. Dalpiaz et al. / European Journal of Pharmacology 436 (2002) 187–196
191
Sweden), centrifugation on Ficoll-Paque (Pharmacia) and
hypotonic lysis of contaminating red cells. The cells were
washed twice, resuspended in KRPG, pH 7.4, at a final
concentration of 50 ꢂ 106 cells/ml and used immediately.
The percentage of the neutrophils was 98–100% pure and
99% viable as determined by the Trypan blue exclusion test.
instruments) with the compartment T set at 37 ꢀC. Absorb-
ance was recorded at wavelengths of 550 and 468 nm.
Differences in absorbance at the two wavelengths were used
to calculate the nanomoles of O2ꢀ produced using a molar
extinction coefficient for cytochrome c of 18.5 mM ꢀ 1
cm ꢀ 1. Neutrophils were preincubated in the presence of 5
mg/ml cytochalasin B (Sigma) for 5 min prior to activation
by fMLF. The actual control of O2ꢀ generation produced by
1 mM fMLF was 45 F 4 nmol/1 ꢂ106 cells/5 min.
2.3. Receptor binding assays
Neutrophils (5 ꢂ 106 ) were incubated in 200 ml of KRPG
at 37 ꢀC for 15 min according to previous time course
experiments. Displacement experiments were performed
using at least eight different concentrations of cold drugs
in the presence of 10 nM [3H]fMLF (specific activity = 71.5
Ci/mmol, NEN Research Products, Du Pont de Nemours,
Milan, Italy). Non-specific binding was measured in the
presence of 10 mM fMLF. Separation of bound from the free
radioligand was performed by rapid filtration through What-
man GF/B filters, which were washed with the ice-cold
buffer. Filter-bound radioactivity was measured by scintil-
lation spectrometry after the addition of 4 ml of Packard
Emulsifier Safe.
The release of neutrophil granule enzymes was evaluated
by determining the lysozyme activity (Torrini et al., 1996)
which is modified for microplate-based assays. Cells
(3 ꢂ 106) were incubated in microplate wells in the presence
of 1 mM fMLF for 15 min at 37 ꢀC. The plates were then
centrifuged for 5 min at 400 ꢂ g and the lysozyme was
quantified nephelometrically by the rate of lysis of the cell
wall suspension of Micrococcus lysodeikticus (Sigma). Cells
were preincubated in the presence of 5 mg/ml cytochalasin B
for 15 min prior to activation by fMLF. Reaction rate was
measured with a microplate reader at 465 nm. Enzyme
output was expressed as a net percentage of the total enzyme
content released by 0.1% Triton X-100. Spontaneous release
was less than 10% and total enzyme activity was 85 F 1 mg/
1 ꢂ107 cells/min.
The cold drug concentrations displacing 50% of labelled
ligand (IC50) were obtained by the computer analysis of
displacement curves. All data were analysed using the non-
linear regression curve fitting computer program Graph Pad
Prism (Graph Pad, San Diego, CA, USA). All the values
obtained are means of three independent experiments per-
formed in duplicate.
Antagonist activity was determined by measuring the
pentapeptide’s ability to inhibit chemotaxis, O2ꢀ production
or granule enzyme release as activated by fMLF. The data of
antagonists (percentage of activity) were obtained by com-
paring the chemotactic index, nanomoles of O2ꢀ or the
percentage of lysozyme release in the absence (100%) and
in the presence of pentapeptides. The antagonist concen-
trations inhibiting the fMLF-induced O2ꢀ production or
granule enzyme release by 50% (IC50) were obtained by
the computer analysis of inhibition curves. All data were
2.4. Neutrophil functions
Random locomotion and chemotaxis were evaluated
using a 48-well microchemotaxis chamber (BioProbe,
Milan, Italy), which estimates the distance in micrometers
which the leading front of the cell migrated, and using the
method of Zigmond and Hirsch (1973). Chemoattractant
activity was determined by adding each peptide derivative
to the lower compartment of the chemotaxis chamber, and it
was expressed in terms of chemotactic index (CI) which is
the ratio:
Table 1
IC50 values for the displacement by the pentapeptide derivatives of 10 nM
[3H]fMLF from human neutrophils and IC50 values for inhibition by the
same compounds of superoxide anion production and enzyme release
induced by 1 mM fMLF
Ligand
IC50
IC50ꢀ
IC50
(binding) [nM] (O2 production) (enzyme release)
[mM]
[mM]
migration toward test attractant ꢀ migration toward the buffer
CI ¼
migration toward the buffer
X-FDLFDLF-OH
528 F 40
2.3 F 0.2
H10 *
5.0 F 0.3
H10 *
X-FDLFDLF-OMe 11310 F 600
X-FDLFDLF-NH2
X-FDLFDLF-olo
X-FDLFDLK-OH
X-FDLFDLE-OH
X-FDLFDLY-OH
T-FDLFDLF-OH
6240 F 400
327 F 20
807 F 50
256 F 15
306 F 18
94 F 6
H10 *
H10 *
Migration in the presence of the buffer alone was 30 mm F 4
S.E. (n = 15) and the CI of 10 nM fMLF was 1.15 F 0.1 S.E.
(n = 15).
1.2 F 0.1
3.3 F 0.2
1.1 F 0.1
1.5 F 0.1
0.80 F 0.07
H10 *
2.1 F 0.1
8.6 F 0.5
2.7 F 0.2
3.4 F 0.2
1.41 F 0.06
H10 *
O2ꢀ production was measured by the superoxide dismu-
tase–inhibitable reduction of ferricytochrome c (Torrini et
al., 1996) modified for microplate-based assays. The tests
were carried out in a final volume of 200 ml containing
4 ꢂ 105 neutrophils, 100 nmol of cytochrome c (Sigma) and
KRPG. At time zero, 1 mM fMLF was added and the plates
were incubated in a microplate reader (Ceres 900, Bio-TeK
T-FDLFDLF-OMe 5100 F 350
Each IC50 value represents an average of three (binding) or six (neutrophil
functions) independent experiments performed in duplicate, F S.E.M.
* Completely unable at concentrations ranging from 10 ꢀ 10 to 10 ꢀ 5 M
to significantly inhibit superoxide anion production and granule enzyme
release produced by 1 mM fMLF.

192
A. Dalpiaz et al. / European Journal of Pharmacology 436 (2002) 187–196
Fig. 3. Competition experiments of the pentapeptide derivatives analysed for specific [3H]fMLF binding carried out at 37 ꢀC on human neutrophils. These are
single representative experiments carried out in duplicate.
analysed using the non-linear regression curve fitting com-
puter program Graph Pad Prism, and the obtained values are
means of six independent experiments performed in dupli-
cate.
receptor (Dalpiaz et al., 1999). As shown in Fig. 3, all
analysed compounds progressively inhibited [3H]fMLF
binding although with varying effectiveness. Table 1 reports
the IC50 values for 10 nM [3H]fMLF displacement by the
pentapeptides. It can be observed that the presence of C-
terminal methyl ester (X-FDLFDLF-OMe, IC50 = 11310
nM; T-FDLFDLF-OMe, IC50 = 5100 nM) or amido groups
(X-FDLFDLF-NH2, IC50 = 6240 nM) is detrimental for the
affinity of peptide analogues toward the formyl-peptide
receptor. On the contrary, the presence of the C-terminal
olo group exerts a beneficial effect (X-FDLFDLF-olo,
IC50 = 327 nM). As for the substitution of the Phe residue
in the peptide chain, an enhancement of the affinity toward
the formyl-peptide receptor is observed with Glu (X-
FDLFDLE-OH, IC50 = 256 nM) or Tyr (X-FDLFDLY-OH,
IC50 = 306 nM), whereas a reduction is obtained with Lys
(X-FDLFDLK-OH, IC50 = 807 nM). Finally, the presence of
the N-terminal thiazolyl-ureido group induces a good im-
provement of the affinity toward the formyl-peptide receptor
(T-FDLFDLF-OH, IC50 = 94 nM).
2.5. Statistical analysis
Analysis of variance was performed with SigmaStat
(version 2.0, Jandel Scientific software). The difference
was considered statistically significant at P values of < 0.05.
3. Results
In a first series of studies, we carried out [3H]fMLF
displacement experiments by incubating human neutrophils
in the presence of increasing concentrations of pentapeptide
derivatives. X-FDLFDLF-OH (IC50 = 528 nM, Table 1) was
utilised as a reference compound in view of its ability to
appreciably antagonize fMLF binding to the formyl-peptide
Fig. 4. Effect of the pentapeptide derivatives on chemotaxis activated by 10 nM fMLF. Data are expressed as percentage of CI. Pentapeptides were added to
neutrophils 10 min before the incubation step for chemotaxis. Each value represents the mean of six separate experiments carried out in duplicate.

A. Dalpiaz et al. / European Journal of Pharmacology 436 (2002) 187–196
193
Fig. 5. Effect of the pentapeptide derivatives on superoxide anion production triggered by 1 mM fMLF. Pentapeptides were added to neutrophils 10 min before
the incubation step for neutrophil functionality. These are single representative experiments carried out in duplicate.
In the first stage of functional studies, we verified whether
the compounds under investigation were able to induce
chemotaxis, trigger O2ꢀ production or lysozyme release per
se. The results indicated that the peptides have no intrinsic
agonist activity for human neutrophils (not shown).
release, respectively, triggered by 1 mM fMLF. As can be
seen, all compounds were scarcely efficacious up to 10 ꢀ 7 M,
while at higher concentrations, they exert a strong and dose-
dependent inhibition of neutrophil responses. The IC50 val-
ues, which were obtained by the analysis of the curves
illustrated in Figs. 5 and 6, are reported in Table 1. As in
the case of binding experiments, the peptide analogues X-
FDLFDLF-olo, X-FDLFDLE-OH, X-FDLFDLY-OH and T-
FDLFDLF-OH were more potent antagonists than X-PDLPD
LF-OH, whereas X-PDLPDLK-OH showed a weaker antag-
onist power.
In the second stage, we verified the antagonist activity of
these peptides on the same neutrophil responses, once again
utilising X-FDLFDLF-OH as a reference compound. In Fig.
4, the effect of increasing doses (from 10 ꢀ 12 to 10 ꢀ 5 M) of
peptides X-FDLFDLF-OH, X-FDLFDLF-olo, X-FD
LFDLK-OH, X-FDLFDLE-OH, X-FDLFDLY-OH and T-
FDLFDLF-OH on the chemotactic response evoked by 10
nM fMLF is illustrated. As shown, a dose-dependent reduc-
tion of CI was observed, and inhibitions near 50% were
obtained at concentrations ranging from 10 ꢀ 6 to 10 ꢀ 5 M.
Figs. 5 and 6 report the curves describing the effects
exerted by the same peptides on O2ꢀ production or lysozyme
The peptide derivatives X-FDLFDLF-OMe, X-FDLFD
LF-NH2 and T-FDLFDLF-OMe, at concentrations ranging
from 10 ꢀ 12 to 10 ꢀ 5 M, were almost completely unable to
inhibit either the chemotaxis evoked by 10 nM fMLF or the
O2ꢀ production and granule enzyme release evoked by 1 mM
fMLF (not shown).
Fig. 6. Effect of the pentapeptide derivatives on lysozyme release induced by 1 mM fMLF. Pentapeptides were added to neutrophils 10 min before the
incubation step for neutrophil functionality. These are single representative experiments carried out in duplicate.

194
A. Dalpiaz et al. / European Journal of Pharmacology 436 (2002) 187–196
4. Discussion
derivatives with the free acid C-terminal group (X-FD
LFDLF-OH, X-FDLFDLK-OH, X-FDLFDLY-OH and X-
FDLFDLE-OH) and the olo derivative (X-FDLFDLF-olo)
show an appreciable affinity toward the formyl-peptide
receptor, whereas the analogues with the C-terminal methyl
ester (X-FDLFDLF-OMe and T-FDLFDLF-OMe) or amido
groups (X-FDLFDLF-NH2) scarcely bind to it. Thus, the
carbonyl group does not appear to be directly involved in
this interaction. These observations suggest, therefore, that
an appreciable affinity to the formyl-peptide receptor could
be obtained either by the presence of a negative charge in
the C-terminal region of the peptide derivatives (as in the
case of COO ꢀ moiety) or via a hydrogen bond accepted by
the receptor (as in the case of the olo moiety).
The various neutrophil functions are evoked when ago-
nists, such as fMLF, interact with specific membrane
receptors and activate multiple transduction pathways
(Prossnitz and Ye, 1997). Many drugs exist which are able
to inhibit neutrophil responses, acting by impairing some of
the different steps of these transduction pathways (Berg-
´
strand et al., 1992; Mocsai et al., 1997). A major limitation
in their use as therapeutic agents for the treatment of in-
flammation-related diseases is that these drugs are not
selective since other cellular responses could be inhibited
at the same time.
Gao et al. (1999) have demonstrated that the absence of
fMLF receptor impairs the antibacterial immunity in mice.
Nevertheless, the development of receptor antagonists of
neutrophil stimulators that are able to transiently inhibit
cellular responses should improve the knowledge about
leukocyte chemoattractant functions and could be of clinical
relevance.
It is interesting to observe that the presence of the N-
terminal thiazolyl-ureido greatly improves the affinity of T-
FDLFDLF-OH toward the formyl-peptide receptor. In fact,
the IC50 value obtained for this compound (94 nM) appears
near to that previously reported for the endogenous ligand
fMLF (IC50 = 41 nM; Dalpiaz et al., 2001).
Several peptide as well as non-peptide formyl-peptide
receptor antagonists have been developed but relatively few
information are available about the structural requirements
for such antagonism. As an example, cyclosporin H was
reported as a potent formyl-peptide receptor antagonist (De
Paulis et al., 1996) and very recently, it was demonstrated
that it acts as an inverse agonist (Wenzel-Seifert et al.,
1998). A pyrazolidinedione derivative was proposed as a
selective and potent competitor of [3H]fMLF binding (Lev-
esque et al., 1992), and more recently, it was demonstrated
that the endogenous ligands spinorphin (Yamamoto et al.,
1997) or the chenodeoxycholic acid (Chen et al., 2000)
inhibits the fMLF binding to its receptor some related
cellular responses. The complete knowledge of how these
ligands interact with the formyl-peptide receptor requires a
more detailed analysis. As for the peptide derivatives, we
have recently demonstrated that the C-terminal-free acid N-
ureido-pentapeptide derivatives appear to be more active
antagonists on human neutrophils than the methyl ester ones
(Dalpiaz et al., 1999; Scatturin et al., 1999). On the basis of
this observation, we synthesised the N-ureido-pentapeptide
derivatives X-FDLFDLF-olo and X-FDLFDLF-NH2 with
the aim of further investigating the role of the C-terminal
groups in the interaction with the formyl-peptide receptor.
Moreover, the analogues X-FDLFDLK-OH, X-FDLFDLY-
OH and X-FDLFDLE-OH were obtained with the purpose
of evaluating the influence of amino acids that are more
hydrophilic than Phe in the C-terminal position on the
pentapeptide antagonist behaviour. The analysis of the in-
fluence of a new N-terminal group (thiazolyl-ureido) was
performed by using the derivatives T-FDLFDLF-OMe and
T-FDLFDLF-OH.
The antagonist power of the pentapeptide derivatives
appears similarly influenced by the molecular features of
the C- and N-terminal groups. In fact, among the compounds
analysed, only the analogues devoid of a hydroxyl moiety
are unable to significantly antagonize the fMLF effects on
human neutrophils as far as chemotaxis, Oꢀ2 production and
lysozyme release are concerned.
The substitution of Phe with more hydrophilic amino
acids at the C-terminal of the pentapeptide chain seems to
produce weak effects on the affinity and antagonist power
toward the formyl-peptide receptor. Nevertheless, it can be
observed that the presence of the Lys residue slightly at-
tenuates the affinity and antagonist power toward the
formyl-peptide receptor, whereas the insertion of Glu or
Tyr residues produces opposite effects (Table 1). It can be
concluded that amino acids with long and positive-charged
side chain, such as Lys, are not qualified for the last position
in the pentapeptide chain in view of their interaction with
the formyl-peptide receptor. On the other hand, it must be
underlined that T-FDLFDLF-OH shows the highest affinity
and antagonist potency for the formyl-peptide receptor
among the peptide derivatives analysed.
The data reported here indicate that the peptides under
examination inhibit Oꢀ2 production and lysozyme release
more efficaciously than neutrophil chemotaxis (Figs. 4–6).
Similar results have previously been obtained utilising
different pentapeptide analogues (Dalpiaz et al., 1999). In
a previous study carried out in order to evaluate the agonist
activity of fMLF analogues that are able to selectively
induce chemotaxis or Oꢀ2 production, we put forward the
hypothesis that the formyl-peptide receptor undergoes con-
formational changes, depending on the type of cellular
response that it must evoke: when exposed to the high
fMLF doses required for Oꢀ2 production, it could in fact
assume a conformation different from that suitable for evo-
king chemotaxis in response to low fMLF concentrations
As far as the C-terminal group is concerned, our results
allow us to hypothesize that the presence of a hydroxyl
function is essential to impart affinity to the formyl-peptide
receptor. In fact, as reported in Table 1, only pentapeptide

A. Dalpiaz et al. / European Journal of Pharmacology 436 (2002) 187–196
195
K., Traniello, S., Spisani, S., 2001. Met-Ile-Phe-Leu derivatives: full
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that the transduction pathway underlying the chemotactic
response is different from those responsible for Oꢀ2 produc-
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1997; Li et al., 2000). On the other hand, the existence of at
least three or six formyl-peptide receptor subtypes for
human and mouse species, respectively, has been demon-
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reported that some of these receptor subtypes show different
affinity values for fMLF and induce or not chemotactic
responses requiring different optimal fMLF concentrations
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hypothesize that the different antagonist activity exerted by
the pentapeptide derivatives towards the various neutrophil
responses could be the consequence of their interaction with
different states and/or with different subtypes of the formyl-
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Acknowledgements
This research was supported by the Italian Ministry of
University, Scientific and Technological Research (MURST)
and the Italian Association for Cancer Research (AIRC). The
authors thank Mrs. Linda Bruce, a qualified mother tongue
teacher, for the English revision of the text and Banca del
sangue of Ferrara for supplying fresh blood.
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