Met-Ile-Phe-Leu derivatives: Full and partial agonists of human neutrophil formylpeptide receptors

Article abstract of DOI:10.1016/S0014-2999(00)00908-0

The biological action of a series of Met-Ile-Phe-Leu analogues was analyzed on human neutrophils, to evaluate their ability to interact with formylpeptide receptors and to induce the related neutrophil responses. Three in vitro assays were carried out: receptor binding, chemotaxis and superoxide anion release. Our results demonstrate that formyl-Met-Ile-Phe-Leu derivatives act as more potent full agonists than formyl-Met-Leu-Phe, the tripeptide normally used as a model chemoattractant for the study of cell functions. On the other hand, the presence of N-ureidoisopropyl substituent in tetrapeptides imparts weak partial agonist properties. It has furthermore been demonstrated that the C-terminal methyl esterification or amination weakly influences the properties of tetrapeptide homologues. Finally, t-Boc-Met-Ile-Phe-Leu derivatives do not appear able to interact with formylpeptide receptors.

Full text of DOI:10.1016/S0014-2999(00)00908-0

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European Journal of Pharmacology 411 2001 327–333
www.elsevier.nlrlocaterejphar
Met-Ile-Phe-Leu derivatives: full and partial agonists of human
neutrophil formylpeptide receptors
Alessandro Dalpiaz a, Angelo Scatturin a,), Gianni Vertuani a, Rita Pecoraro a,
Pier Andrea Borea b, Katia Varani b, Serena Traniello c, Susanna Spisani c
a
Department of Pharmaceutical Science, Ferrara UniÕersity, Õia Fossato di Mortara 19, 44100 Ferrara, Italy
b
Department of Experimental and Clinical Medicine, Pharmacology Section, Ferrara UniÕersity, Õia Fossato di Mortara 19, 44100 Ferrara, Italy
c
Department of Biochemistry and Molecular Biology, Ferrara UniÕersity, Õia L. Borsari 46, 44100 Ferrara, Italy
Received 2 October 2000; received in revised form 20 November 2000; accepted 24 November 2000
Abstract
The biological action of a series of Met-Ile-Phe-Leu analogues was analyzed on human neutrophils, to evaluate their ability to interact
with formylpeptide receptors and to induce the related neutrophil responses. Three in vitro assays were carried out: receptor binding,
chemotaxis and superoxide anion release. Our results demonstrate that formyl-Met-Ile-Phe-Leu derivatives act as more potent full
agonists than formyl-Met-Leu-Phe, the tripeptide normally used as a model chemoattractant for the study of cell functions. On the other
hand, the presence of N-ureidoisopropyl substituent in tetrapeptides imparts weak partial agonist properties. It has furthermore been
demonstrated that the C-terminal methyl esterification or amination weakly influences the properties of tetrapeptide homologues. Finally,
t-Boc-Met-Ile-Phe-Leu derivatives do not appear able to interact with formylpeptide receptors. q 2001 Elsevier Science B.V. All rights
reserved.
Keywords: Neutrophil, human; Formylpeptide receptor; Full agonist, Partial agonist, Neutrophil functionality
1. Introduction
peutic agents on the treatment of inflammation disorders
Ž
Smith, 1994; Behar and Porcelli, 1995; Meera et al.,
Polymorphonuclear neutrophils are phagocytic cells in-
.
1999 Consequently, the study and development of potent
and selective formylpeptide receptor antagonists is of con-
siderable interest.
Agonist or antagonist activities of peptide derivatives
on human neutrophils appear to be dependent on their
primary sequence and on the N- and C-terminal sub-
stituents Freer et al., 1980; Derian et al., 1996; Higgins et
al., 1996; Dalpiaz et al., 1999 . As an example, it has been
reported that the formyl group of CHO-Met-Leu-Phe is
essential for adequate biological activity of agonists Freer
et al., 1980; Derian et al., 1996 . On the other hand, it has
been demonstrated that N-t-Boc or N-ureido-ethyl, n-pro-
pyl and isopropyl substituents in the Met-Leu-Phe chain
induce an antagonist behaviour Higgins et al., 1996 .
The t-Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH derivative has
been reported as displaying an appreciable formylpeptide
receptor antagonist activity on rabbit and human neu-
Ž
volved in the defense against infections Prossnitz and Ye,
.
Ž
1997 . In this context, the CHO-Met-Leu-Phe-OH formyl-
Met-Leu-Phe-OH peptide, derived from bacterial sources
.
or from disrupted mitochondria, was identified as a potent
Ž
chemoattractant for phagocytes Carp, 1982; Marasco et
.
al., 1984 . This discovery led to the identification on
Ž
neutrophils of a G-protein-coupled formylpeptide receptor
.
Ž
which has since been cloned Koo et al., 1983; August et
.
al., 1997 . The binding of CHO-Met-Leu-Phe-OH to
Ž
formylpeptide receptor triggers multiple biochemical re-
sponses by neutrophils, including adhesion, chemotaxis
and free radical generation. These effects constitute the
physiological defense against bacterial infections and tis-
.
Ž
.
Ž
.
sue damage Prossnitz and Ye, 1997 . Formylpeptide re-
ceptor antagonists could have beneficial effects as thera-
Ž
.
trophils Aswanikumar et al., 1977; Dalpiaz et al., 1999 .
Moreover, it has recently been verified that N-ureido-Phe-
D-Leu-Phe-D-Leu-Phe-OH peptide derivatives can enhance
)
Corresponding author. Tel.: q39-0532-291268; fax: q39-0532-
291296.
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.
E-mail address: sca@dns.unife.it A. Scatturin .
0014-2999r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
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.
PII: S0014-2999 00 00908-0

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328
A. Dalpiaz et al.rEuropean Journal of Pharmacology 411 2001 327–333
the noticeable antagonist activity of the t-Boc-homologue
esterification and amination were subsequently examined.
Moreover, the consequences of N-terminal isopropylureido
and t-Boc substitutions—which convert the agonist CHO-
Ž
.
Higgins et al., 1996; Dalpiaz et al., 1999 . All these
pentapeptide derivatives have been reported to be more
Ž
active on human neutrophils than the C-terminal methyl-
Met-Leu-Phe to an antagonist Derian et al., 1996; Higgins
Ž
.
.
ester ones Dalpiaz et al., 1999 .
et al., 1996 —were evaluated. Our conclusions indicate
As for tetrapeptide derivatives, CHO-Met-Ile-Phe-Leu-
OH has been recognised as a full chemotactic agonist on
that formyl and isopropylureido derivatives are respec-
tively more and less potent agonists than CHO-Met-
Leu-Phe. On the other hand, the presence of the t-Boc
substituent appears detrimental for the activity of the tetra-
peptide derivatives.
Ž
.
human monocytes Rot et al., 1987 although its properties
on neutrophils have not been extensively analysed. It could
be of interest to evaluate if t-Boc or N-ureido-aliphatic
substituents in the tetrapeptide chain can induce an antago-
nist behaviour on human neutrophils. In this context, the
present paper reports the synthesis and biological analysis
2. Materials and methods
Ž
.
of nine Met-Ile-Phe-Leu analogues Fig. 1 . In particular,
the t-Boc, formyl and isopropylureido substituents have
been included at the N-terminal of the peptides, which
were all obtained as free acid, methyl ester and amido
derivatives. The biological properties of the peptide deriva-
tives were evaluated in three Ain vitroB assays: receptor
binding, superoxide anion release and chemotaxis. The aim
of the present study was an extensive analysis of the
properties of Met-Ile-Phe-Leu derivatives on human neu-
trophils. Starting from an evaluation of the biological
activity of CHO-Met-Ile-Phe-Leu, the effects of C-terminal
2.1. Peptides
Ž
.
t-Boc-Met-OH Sigma Aldrich-Fluka, Milan, Italy was
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.
coupled to HOSu N-hydroxysuccinimide, Fluka using
X
Ž
.
DCC N,N -dicyclohexylcarbodiimide, Fluka in DMF
Ž
.
N,N-dimethylformamide, Carlo Erba, Milan, Italy at 08C
1 h and kept at room temperature 12 h . The resulting
Ž
.
Ž
.
Ž
.
t-Boc-Met-OSu was coupled to H-Ile-OH Fluka in sodium
bicarbonate solution pH 8 as previously described
Ž
.
.
Ž
Anderson et al., 1964 . The resulting dipeptide t-Boc-
Met-Ile-OH was coupled to H-Phe-Leu-OMe using EDCI
Ž
N-dimethylaminopropyl, N-ethylcarbodiimide, Cal-
.
biochem-Novabiochem, Laufelfingen, Switzerland and
HOBt hydroxybenzotriazole, Fluka in DMF at 08C 1 h
and kept at room temperature 12 h in order to obtain the
tetrapeptide t-Boc-Met-Ile-Phe-Leu-OMe A1 .
¨
Ž
.
.
Ž
.
Ž
Ž
.
The dipeptide H-Phe-Leu-OMe was prepared coupling
t-Boc-Phe-OH to H-Leu-OMe using the previous coupling
reaction and the removal of t-Boc group was performed by
treatment with a 1:1 mixture of trifluoroacetic acid–chlo-
Ž
.
roform at room temperature. The tetrapeptide A1 was in
part saponified with 1 N NaOH and in part dissolved in
Ž
methanol at 08C in the presence of NH3 gas Bodanszky
.
and Bodanszky, 1984 in order to obtain the C-terminal
free acid A2 and the C-terminal amido A3 homologues,
Ž
.
Ž
.
respectively.
Ž
.
Tetrapeptide A1 was moreover used as amino de-
blocked for the synthesis of alkyl-ureido peptide B1 by
Ž
.
reaction with isopropyl-N-succimidyl carbamate as previ-
Ž
.
ously described Dalpiaz et al., 1999 . The peptide deriva-
Ž
.
tive B1 was in part saponified and in part used to obtain
the C-terminal free acid B2 and the amido B3 homo-
Ž
.
Ž
.
logues, respectively.
For the synthesis of compounds C1 , C2 , C3 the
tetrapeptide derivative A1 was unprotected by treatment
Ž
. Ž . Ž
.
Ž
.
with 98% formic acid. The resulting formate salt of the
Ž
peptide was directly treated with EEDQ N-ethoxy-
.
carbonyl-2-ethoxy-1,2-dihydroquinoline, Fluka and kept
Ž
.
at room temperature for 3 h Lajoie and Kraus, 1984 . The
resulting peptide derivative C1 was treated, using the
Ž
.
previous described reactions, in order to obtain the free
Ž
.
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.
acid C2 and C-terminal amido C3 homologues.
Fig. 1. Chemical formulas of the nine tetrapeptide derivatives analysed.

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A. Dalpiaz et al.rEuropean Journal of Pharmacology 411 2001 327–333
329
All peptides were purified by silica gel column and
were analyzed by reverse phase liquid chromatography on
a Vydac C18 column with acetonitrile gradient elution. The
compounds were characterized by mass spectrometry anal-
ysis, melting point determination and polarimetric mea-
surements.
free radioactivity were separated by filtering the assay
mixture through Whatman GFrC glass-filters using a Mi-
Ž
.
cro Mate 196 Cell Harvester Packard Instrument . The
Ž
filter-bound radioactivity was counted on Top Count ef-
ficiency 57% with Micro Scint-20 30 ml in 96-well
plates . All the values obtained are means of three inde-
.
Ž
.
Stock solutions, 10y2 M of CHO-Met-Leu-Phe Sigma,
Ž
pendent experiments performed in duplicate.
y3
.
St. Louis MO, USA and 10
M of tetrapeptide deriva-
tives, were prepared in dimethylsulfoxide DMSO, Sigma
Ž
.
2.4. Random locomotion and chemotaxis
and diluted using Krebs–Ringer-phosphate containing 0.1%
Ž
.
wrv glucose KRPG , pH 7.4, before use. KRPG was
made up as a stock solution of the following composition:
NaCl, 40 grl; KCl, 1.875 grl; Na2 HPO4.2H2O, 0.75 grl;
KH2 PO4, 0.125grl; NaHCO3, 1.25grl; glucose, 10 grl.
This solution was five times working strength. 1 mM
MgCl2 and CaCl2 were supplemented to the buffer before
the biological test. At the concentrations used, DMSO did
not interfere with any of the biological assays performed.
Random locomotion and chemotaxis were evaluated
Ž
using a 48-well microchemotaxis chamber BioProbe, Mi-
.
Ž
lan, Italy , with a 3-mm pore size membrane filter Milli-
pore, Milan, Italy , estimating the distance in micrometers
.
that the leading-front of the cell migrated, and using the
Ž
.
method of Zigmond and Hirsch 1973 . Chemoattractant
activity was determined by adding CHO-Met-Leu-Phe or
the tetrapeptide derivatives to the lower compartment of
the chemotaxis chamber and it was expressed in terms of
2.2. Cell preparation
Ž
.
chemotactic index C.I. , which is the ratio:
migrationtowardtestattractantymigrationtowardthebuffer
Ž
.
Cells were obtained from heparinized 10 Urml pe-
ripheral blood of fasting healthy subjects. Twenty milliliters
of blood was supplemented with 12 ml of a solution
C.I.s
migrationtowardthebuffer
Migration in the presence of buffer alone was 30 mm"4
Ž
.
consisting of 6% by weight Dextran T70 and was settled
onto a 50 ml polypropylene tube at room temperature for
45 min. The turbid upper supernatant containing leuko-
cytes was carefully removed and layered onto 10 ml of
Ficoll-Paque Pharmacia, density gradient for lymphocyte
isolation , centrifuged at 250=g for 20 min at room
Ž
.
S.E. ns6 . All the values obtained are means of six
independent experiments performed in duplicate.
2.5. Superoxide anion production
Ž
.
Oy2 production was measured by the superoxide dismu-
temperature. The pellet containing neutrophils was further
Ž
Ž
.
purified by erythrocytes hypotonic lysis 0.86% NH4Cl for
tase 0.5 mgrml -inhibitable reduction of ferricytochrome
.
Ž
.
10 min . The cells were washed twice and resuspended
c
Torrini et al., 1996 modified for microplate-based
using KRPG, pH 7.4, at a final concentration of 50=106
cellsrml and used immediately. The percentage of neu-
trophils was 98–100% pure and G99% viable, as deter-
mined by the Trypan blue exclusion test. The study was
approved by the local Ethics Committee, and informed
consent was obtained from all participants.
assays. The tests were carried out in a final volume of 200
ml containing 4=105 neutrophils, 100 nmol of cy-
Ž
.
tochrome c Sigma and KRPG. At zero time, different
amounts ranging from 10
derivative were added, and the plates were incubated in a
microplate reader Ceres 900, Bio-TeK instruments with
the compartment temperature set at 378C.
y12
to 10y5 M of each peptide
.
Ž
Ž
.
2.3. Receptor binding assays
Absorbance was recorded at wavelengths of 550 and
468 nm. Differences in absorbance at the two wavelengths
were used to calculate nanomoles of Oy2 produced, using a
molar extinction coefficient for cytochrome c of 18.5
mMy1 cmy1. This value has been calculated by the differ-
ence between the molar extinction coefficients from re-
Binding assays were carried out essentially according to
Ž
.
Ž
.
Dalpiaz et al. 1999 and Spisani et al. 1996 . Briefly
6
Ž
.
neutrophils 5=10 were incubated in 200 ml of KRPG,
supplemented with 1 mM MgCl2 and CaCl2 , at 378C for
15 min at 378C and for 90 min at 48C, according to
previous time course analysis. Displacement experiments
duced ´550 s27.6=103 My1 cmy1 and oxidized ´550
Ž
.
Ž
s 9.1 = 103 My1 cmy1 states of cytochrome
c
.
w3
x
Ž
.
were performed in the presence of 10 nM H CHO-Met-
Margoliash and Frohwirt, 1959 . Neutrophils were prein-
cubated in the presence of 5 mgrml cytochalasin B Sigma
Ž
Ž
.
Leu-Phe specific activitys71.5 Cirmmol; NEN Re-
.
search Products, Du Pont de Nemours, Milan, Italy using
for 5 min prior to activation by CHO-Met-Leu-Phe or the
tetrapeptide derivatives. Results were expressed as net
nanomoles. The net nanomoles were calculated using the
formula: stimulated neutrophilsyresting neutrophils alone.
All the values obtained are means of six independent
experiments performed in duplicate.
at least six different concentrations of cold drugs added
w3
x
with H CHO-Met-Leu-Phe before addition of neutrophils
to the experimental system. Non-specific binding was de-
fined as binding in the presence of 100 mM CHO-Met-
Leu-Phe and was about 20% of total binding. Bound and

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330
A. Dalpiaz et al.rEuropean Journal of Pharmacology 411 2001 327–333
obtained with each individual peptide derivatives. EC50
values were calculated from the mean of the individual
dose–response curves fitting using response data increas-
ing with concentration of peptide derivatives. All data
were analysed using the non-linear regression curve fitting
Ž
computer program Graph Pad Prism Graph Pad, San
.
Diego, CA, USA .
Analysis of variance was performed with SigmaStat
Ž
.
version 2.0, Jandel Scientific software . Difference was
considered statistically significant at P values -0.05.
3. Results
3
w
x
Fig. 2 shows the results from H CHO-Met-Leu-Phe
displacement experiments carried out at 378C and 48C
using CHO-Met-Leu-Phe, N-formyl- and N-isopropy-
lureido tetrapeptide derivatives. The CHO-Met-Ile-Phe-Leu
and N-isopropylureido-Met-Ile-Phe-Leu derivatives appear
to bind with respectively higher and lower affinity toward
formylpeptide receptors than CHO-Met-Leu-Phe. The IC50
values are presented in Table 1. In particular, the affinities
Ž
.
Fig. 2. Competition experiments of CHO-Met-Leu-Phe fMLF , CHO-
Ž
.
Ž
.
fMIFL and N-isopropylureido ipuMIFL tetrapeptide derivatives for
Ž
of CHO-Met-Ile-Phe-Leu tetrapeptides 3.6 nMFIC50
F
w3
x
specific H fMLF binding carried out at 378C and 48C on human
neutrophils. These are single representative experiments carried out in
duplicate.
.
5.6 nM at 378C and 2.9 nMFIC50 F6.1 nM at 48C are
one order of magnitude higher than CHO-Met-Leu-Phe
Ž
.
affinity 41 nM at 378C and 45 nM at 48C . On the other
hand, the IC50 values of N-isopropylureido-Met-Ile-Phe-
Ž
Leu derivatives 3200 nMFIC50 F6500 nM at 378C and
.
2.6. Data analysis
2900 nMFIC50 F6000 nM at 48C indicate that their
affinities are two orders of magnitude lower than that of
CHO-Met-Leu-Phe. The IC50 values of tetrapeptide deriva-
tives reported in Table 1 do not appear to change consider-
ably with the different C-terminal substituents. Very weak
affinities have been found for t-Boc derivatives: their IC50
The cold drug concentrations displacing 50% of la-
Ž
.
belled ligand IC50 were obtained by computer analysis of
displacement curves.
w
For determination of EC50 values EC50 sconcentration
Ž
.
of peptide required to produce 50% of the maximum effect
values exceeded 1 mM at 378C and 48C data not shown .
Chemotactic responses of human neutrophils to CHO-
Met-Leu-Phe, CHO-Met-Ile-Phe-Leu and N-isopropy-
x
as determined from the concentration–effect curve , data
were expressed as a percentage of the maximal response
Table 1
Ž
.
Ž
.
Ž
.
Receptor binding and related effects chemotaxis and superoxide anion production of CHO-Met-Leu-Phe fMLF , CHO- fMIFL and N-isopropylureido
Ž
.
ipuMIFL tetrapeptide derivatives on human neutrophils
Peptide
Receptor binding
Chemotaxis
Oy2 superoxide release
Ž
.
Ž
.
Ž
.
Ž
.
IC50 nM , 378C
IC50 nM , 48C
EC50 nM
Efficacy
EC50 nM
Efficacy
fMLF
41"3
45"4
0.049"0.003
4.7"0.4 nM
3.3"0.2 nM
1.25"0.11
0.60"0.05
0.53"0.04
0.58"0.05
1.29"0.12
1.23"0.10
1.32"0.12
23"2
43"5
27"3
30"3
37"4
48"6
44"5
42"5
Ž
.
B1 ipuMIFL-OMe
6500"400
5200"300
3200"200
5.6"0.3
4.7"0.2
3.6"0.2
6000"500
4800"400
2900"200
6.1"0.3
5.2"0.3
2.9"0.2
2000"100
2100"100
1150"60
1.20"0.05
1.59"0.08
2.1"0.1
Ž
.
B2 ipuMIFL-OH
Ž
.
B3 ipuMIFL-NH2
7.6"0.5 nM
Ž
.
C1 fMIFL-OMe
0.0056"0.0003
0.0065"0.0005
0.0030"0.0002
Ž
.
C2 fMIFL-OH
Ž
.
C3 fMIFL-NH2
Efficacy data of chemotaxis are expressed as chemotactic index; efficacy data of superoxide anion production are expressed as net nanomoles of
Oy2 r1=106 cellsr5 min. "S.E. are reported. Differences between results obtained with ipuMIFL derivatives or fMIFL derivatives are not systematically
significant for each response.
Receptor binding values are means of three independent experiments performed in duplicate.
Chemotaxis and superoxide Oy2 anion release values are means of six independent experiments performed in duplicate.

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A. Dalpiaz et al.rEuropean Journal of Pharmacology 411 2001 327–333
331
lureido-Met-Ile-Phe-Leu derivatives are shown in Fig. 3.
The dose–response curves rise to a peak and then decline
to zero, with ligand concentrations higher than the opti-
mum value. This behaviour appears typical of chemoattrac-
Ž
.
tants Rot et al., 1987; Vertuani et al., 1987 . It can be
observed that the optimum concentration value for chemo-
tactic response is around 10y9 M for CHO-Met-Leu-Phe,
whereas the efficacy values are around 10y10 and 10y7 for
CHO-Met-Ile-Phe-Leu and N-isopropylureido-Met-Ile-
Phe-Leu derivatives, respectively. Moreover, the chemo-
tactic index peaks, attributed to CHO-Met-Leu-Phe and
CHO-tetrapeptides, are similarly high. On the other hand,
the peaks obtained for N-isopropylureido-derivatives indi-
cate that their maximal responses are reduced in compari-
son to the other peptides. The results are quantitatively
reported in Table 1, which includes EC50 and efficacy
values of the chemotactic index for the compounds re-
ported in Fig. 3. EC50 values of CHO-Met-Ile-Phe-Leu
Ž
.
Fig. 4. Superoxide anion production of CHO-Met-Leu-Phe fMLF ,
Ž
.
Ž
.
CHO- fMIFL and N-isopropylureido ipuMIFL tetrapeptide derivatives
toward human neutrophils. Data are expressed as net nmolr1=106
cellsr5 min. These are single representative experiments carried out in
duplicate.
peaks range within three orders of magnitude. In parti-
cular, optimal responses for CHO-Met-Ile-Phe-Leu deri-
vatives are obtained around 10y8 M, whereas for CHO-
Met-Leu-Phe and N-isopropylureido-Met-Ile-Phe-Leu
Ž
.
tetrapeptides 3 pMFEC50 F6.5 pM are one order of
Ž
.
magnitude lower than CHO-Met-Leu-Phe value 49 pM .
As for efficacy, the chemotactic index values of these
compounds are distributed in a narrow range around 1.25.
These results indicate that the CHO-tetrapeptide deriva-
tives are full agonists, like CHO-Met-Leu-Phe, albeit more
potent. Moreover, their ability to induce chemotaxis does
not significantly change with the different C-terminal sub-
derivatives, such responses are obtained around 10y6
M
and near 10y5 M, respectively. The peaks of moles of Oy2 ,
attributed to CHO-Met-Leu-Phe and CHO-tetrapeptides,
have similar height, whereas the peaks of N-isopro-
pylureido derivatives appear shorter. Table 1 includes the
quantitative results from superoxide anion production ex-
periments. It can be observed that the EC50 values of
Ž
.
stituents. Similarly, the EC50 3.3 nMFEC50 F7.6 nM
Ž
.
Ž .
cantly lower than the value of CHO-Met-Leu-Phe 23 nM ,
and efficacy 0.53FefficacyF0.60 values of N-isopro-
pylureido derivatives are not markedly influenced by these
C-terminal substitutions, whereas the same derivatives ap-
pear to be weak partial agonists with respect to CHO-Met-
Leu-Phe.
CHO-tetrapeptides 1.20FIC50 F2.10 nM are signifi-
Ž
.
whereas their efficacy ranges around that of CHO-Met-
y
Ž
.
Leu-Phe value 44 nmol of O2 . It can be concluded that,
as far as superoxide anion production is concerned, CHO-
tetrapeptide derivatives are more potent full agonists than
No significant chemotactic response is induced by any
Ž
.
Ž
t-Boc-Met-Ile-Phe-Leu derivatives data not shown in the
CHO-Met-Leu-Phe. On the other hand, the EC50 1.15F
y13
range of concentration investigated from 10
to 10y5
EC50 F2.1 mM and efficacy 27FefficacyF37 nmol
Ž
.
Ž
Oy2 values of N-isopropylureido derivatives indicate that
.
.
M .
Fig. 4 shows the ability of CHO-Met-Leu-Phe, CHO-
Met-Ile-Phe-Leu and N-isopropylureido-Met-Ile-Phe-Leu
derivatives to trigger superoxide anion production in hu-
man neutrophils. Also in this case, dose–response curves
present a peak. The concentrations corresponding to the
these compounds are weak partial agonists.
The ability of all tetrapeptide derivatives reported in
Table 1 to trigger Oy2 production does not considerably
change with the different C-terminal substituents.
No Oy2 production is triggered by any of the t-Boc-
Ž
.
Met-Ile-Phe-Leu derivatives data not shown in the range
y12
to 10y5 M .
.
Ž
of concentration investigated from 10
4. Discussion
We synthesised a series of Met-Ile-Phe-Leu derivatives
with the aim of evaluating their affinity and their agonist
or antagonist power toward formylpeptide receptors of
human neutrophils. The biological properties of tetrapep-
Ž
.
tides Fig. 1 were compared with those of CHO-Met-Leu-
Phe. Previous studies have demonstrated that CHO-Met-
Ile-Phe-Leu-OH acts as a full chemotactic agonist on
Ž
.
Ž
.
Fig. 3. Chemotactic index of CHO-Met-Leu-Phe fMLF , CHO- fMIFL
Ž
.
and N-isopropylureido ipuMIFL tetrapeptide derivatives toward human
neutrophils. These are single representative experiments carried out in
duplicate.
Ž
.
human monocytes Rot et al., 1987 : the IC50 value of the
same compound, obtained by competition experiments us-

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332
A. Dalpiaz et al.rEuropean Journal of Pharmacology 411 2001 327–333
ing fluoresceinated CHO-Met-Leu-Phe-Lys, was 9"2 nM.
As reported in Table 1, the IC50 values we obtained on
human neutrophils for the binding of the same compound
to formylpeptide receptors were 4.7"0.2 nM at 378C and
5.2"0.2 nM at 48C. To the best of our knowledge, this is
the first report demonstrating a significantly higher affinity
of CHO-Met-Ile-Phe-Leu with respect to CHO-Met-Leu-
anion production in human neutrophils, even if they show
Ž
.
a weak partial agonist behaviour Table 1 . It is therefore
important to note that the presence of the N-isopro-
pylureido substituent reduces the affinity and the efficacy
of CHO-Met-Ile-Phe-Leu derivatives to formylpeptide re-
ceptors but does not cause the transformation of the te-
trapeptides from agonists to antagonists.
Ž
Ž
.
Phe IC50 s41"3 nM at 378C and IC50 s45"5 nM at
As observed from affinity and activity data Table 1 ,
the agonist properties of Met-Ile-Phe-Leu derivatives on
human neutrophils do not seem noticeably influenced by
C-terminal methyl esterification or by the conversion to
the corresponding amide. A similar behaviour on human
neutrophils has been described for CHO-Met-Leu-Phe
.
48C, Table 1 .
At physiological temperatures, receptor processing
events such as its internalization can influence the results
of the binding studies Hoffman et al., 1996a,b . For this
reason, the displacement experiments have been performed
not only at 378C, but also at 48C. In fact, at this latter
Ž
.
Ž
.
derivatives Dentino et al., 1991 and it is shared with
N-isopropylureido-Met-Ile-Phe-Leu derivatives but not
with other formylpeptide receptor agonists or antagonists.
In fact, it has been reported that the presence of the methyl
ester function at the carboxy terminus of ethyl-Met-Leu-Phe
and n-Boc-Met-Leu-Phe, respectively, induces a signifi-
cant enhancement and increase of formylpeptide receptor
Ž
temperature the receptor is not internalized Jesaitis et al.,
.
1984 . The results reported in Fig. 2 and in Table 1
suggest that at both incubation temperatures the com-
pounds have the same effectiveness in displacing
H CHO-Met-Leu-Phe from its binding site. A similar
w3
x
result was previously obtained using CHO-Met-Leu-Phe-
Ž
.
Ž
.
OMe derivatives Fabbri et al., 2000 .
affinity on human neutrophils Derian et al., 1996 . It has
been reported that the methyl ester derivative of CHO-
Met-Leu-Phe is more potent than the parent compound on
Figs. 2–4 allow us to observe that CHO-Met-Ile-Phe-
Leu derivatives act as full agonists more potent than
CHO-Met-Leu-Phe. In fact, they show efficacy properties
similar to those of CHO-Met-Leu-Phe and higher affinity
Ž
.
rabbit neutrophils Iqbal et al., 1984; Dentino et al., 1997 .
The properties of Met-Ile-Phe-Leu derivatives seem to
conform to formylpeptide receptor models proposed and
Ž
.
toward formylpeptide receptors Table 1 .
Ž
It has been noted that the presence of N-terminal t-Boc
or branched aliphatic substituents convert the agonist
CHO-Met-Leu-Phe to weak antagonists for human neu-
discussed by several authors Freer et al., 1982; Edmund-
.
son and Ely, 1985; August et al., 1997; Mills et al., 1998 .
Accordingly, the N-formyl group has been suggested to
form a hydrogen bond with a phenolic hydroxyl group of a
tyrosine residue. Methionine in ligand position 1 appears
optimal for the binding whereas position 2 is less critical.
An aromatic residue like phenylalanine is optimal in posi-
tion 3. It was also observed that the receptor has sufficient
AroomB to accommodate even a fourth amino acid residue.
Moreover, the results obtained from the present work
allow us to suggest that C-terminal methyl esterification,
or the conversion to the corresponding amide, weakly
influences the formylpeptide receptor agonist properties of
peptide derivatives.
In conclusion, the CHO-Met-Ile-Phe-Leu peptide inter-
actions with human neutrophils have been characterized by
receptor binding, chemotaxis and superoxide anion produc-
tion assays. The results provide new information relative to
structure–activity relationships and formylpeptide recep-
tor: C-terminal modification has little effect upon biologi-
cal activity and the substitution of the N-formyl group
with the N-isopropylureido one induces their conversion to
weak partial agonists. The t-Boc-Met-Ile-Phe-Leu deriva-
tives do not appear to be capable of interacting with
human neutrophil formylpeptide receptors.
Ž
.
trophils Derian et al., 1996; Higgins et al., 1996 . Besides,
while CHO-Phe-D-Leu-Phe-D-Leu-Phe has been reported
to be a potent chemoattractant, its homologue t-Boc-Phe-
D-Leu-Phe-D-Leu-Phe has been shown to be an antagonist
Ž
for rabbit and human neutrophils Aswanikumar et al.,
.
1977; Dalpiaz et al., 1999 . These observations were the
stimulus for us to synthesise the tetrapeptide derivatives,
including N-terminal t-Boc and isopropylureido sub-
Ž
.
stituents Fig. 1 , with the aim of verifying whether their
presence can influence the biological properties of Met-Ile-
Phe-Leu derivatives. The affinity toward formylpeptide
receptors of tetrapeptides appears to be strongly affected
by N-terminal substituents. As shown in Fig. 2, the capac-
ity of N-isopropylureido-derivatives to inhibit 10 nM
w3
x
H CHO-Met-Leu-Phe binding is drastically reduced with
respect to that of N-formyl derivatives. In particular, as
reported in Table 1, the IC50 values of N-isopropylureido-
derivatives are three orders of magnitude higher than those
of the formyl ones. Moreover, the presence of the N-termi-
nal t-Boc group seems to extinguish the affinity toward
formylpeptide receptors of these tetrapeptides: their IC50
values have been estimated as greater than 1 mM. On the
other hand, the agonist CHO-Met-Ile-Phe-Leu derivatives
do not become antagonists in the presence of N-isopro-
pylureido substituents. In fact, the results reported in Figs.
3 and 4 allow us to observe that N-isopropylureido-deriva-
tives are able to induce chemotaxis and trigger superoxide
Acknowledgements
This research was supported by Italian Ministry of
University, Scientific and Technological Research

(
)
A. Dalpiaz et al.rEuropean Journal of Pharmacology 411 2001 327–333
333
and antagonists toward the neutrophil fMLF receptor. J. Med. Chem.
39, 1013–1015.
Ž
Ž
.
MURST and Italian Association for Cancer Research
.
AIRC . The text has been independently revised, for
Hoffman, J.F., Linderman, J.J., Omann, G.M., 1996a. Receptor up-regu-
lation, internalization, and interconverting receptor states. Critical
components of a quantitative description of N-formyl peptide receptor
dynamics in the neutrophil. J. Biol.Chem. 271, 18394–18404.
Hoffman, J.F., Keil, M.L., Riccobene, T.A., Omann, G.M., Linderman,
J.J., 1996b. Interconverting receptor states af 48C for the neutrophil
N-formylpeptide receptor. Biochemistry 35, 13047–13055.
Iqbal, M., Balaram, P., Showell, H.J., Freer, R.J., Becker, E.L., 1984.
Conformationally constrained chemotactic peptide analogs of high
biological activity. FEBS Lett. 165, 171–174.
English style and language, by Mrs. Linda Bruce and Mr.
Larry Costache. The authors thank the Blood Bank of
Ferrara for supplying fresh blood.
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