Báo cáo Y học: Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN Design of a potent endozepine antagonist pot

Synthesis, conformational analysis and biological activity of cyclic
analogs of the octadecaneuropeptide ODN
Design of a potent endozepine antagonist
Je
´
ro
ˆ
me Leprince
1
, Hassan Oulyadi
2
, David Vaudry
1
, Olfa Masmoudi
1
, Pierrick Gandolfo
1
, Christine Patte
1
,
Jean Costentin
3
, Jean-Luc Fauche
`
re
4
, Daniel Davoust
2
, Hubert Vaudry
1
and Marie-Christine Tonon

1
1
Institut Fe
´
de
´
ratif de Recherches Multidisciplinaires sur les Peptides (IFRMP 23), Laboratoire de Neuroendocrinologie Cellulaire et
Mole
´
culaire, Institut National de la Sante
´
et de la Recherche Me
´
dicale Unite
´
413, CNRS, Universite
´
de Rouen, Mont-Saint-Aignan, France;
2
IFRMP 23, Laboratoire de Re
´
sonance Magne
´
tique Nucle
´
aire, Institut de Recherche en Chimie Organique Fine, CNRS Unite
´
Mixte de
Recherches 6014, Universite
´

de Rouen, Mont-Saint-Aignan, France;
3
IFRMP 23, Laboratoire de Neuropsychopharmacologie, CNRS Unite
´
Mixte de Recherches 6036, Universite
´
de Rouen, Rouen, France;
4
Institut de Recherches SERVIER, Suresnes, France
The octadecaneuropeptide (ODN; QATVGDVNTDRPG
LLDLK) and its C-terminal octapeptide (OP; RPGLLDLK),
which exert anxiogenic activity, have been previously shown
to increase intracellular calcium concentration ([Ca
21
]
i
)in
cultured rat astrocytes through activation of a metabotropic
receptor positively coupled to phospholipase C. It has also
been found that the [
D-Leu5]OP analog possesses a weak
antagonistic activity. The aim of the present study was to
synthesize and characterize cyclic analogs of OP and
[
D-Leu5]OP. On-resin homodetic backbone cyclization of
OP yielded an analog, cyclo
128
OP, which was three times
more potent and 1.4-times more efficacious than OP to
increase [Ca

21
]
i
in cultured rat astrocytes. Cyclo
128
OP also
mimicked the effect of both OP and ODN on polyphos-
phoinositide turnover. Conversely, the cyclo
128
[D-Leu5]OP
analog was totally devoid of agonistic activity but
suppressed the effect of OP and ODN on [Ca
21
]
i
and
phosphoinositide metabolism in astrocytes. The structure of
these cyclic analogs has been determined by two-dimen-
sional
1
H-NMR and molecular dynamics. Cyclo
128
OP
exhibited a single conformation characterized by a g turn
comprising residues Pro2–Leu4 and a type III b turn
encompassing residues Leu5–Lys8. Cyclo
128
[D-Leu
5
]OP

was present as two equimolar conformers resulting from
cis/trans isomerization of the Arg–Pro peptide bond. These
pharmacological and structural data should prove useful for
the rational design of non peptidic ODN analogs.
Keywords: solid-phase peptide synthesis; cyclic peptides;
structure-activity relationship; astrocytes; cytosolic calcium
concentration.
Diazepam-binding inhibitor (DBI) is an 86-amino-acid
polypeptide that has been originally isolated from rat brain
extracts as an endogenous ligand of benzodiazepine (BZ)
receptors [1]. Proteolytic cleavage of DBI generates several
biologically active peptides including the triakontatetra-
neuropeptide TTN (DBI
17250
) [2] and the octadecaneuro-
peptide ODN (DBI
33250
) [3] which are collectively
designated by the term endozepines [4]. Intracerebroven-
tricular injection of endozepines provokes anxiogenic effects
[5], induces proconflict behavior [1,6], reverses the anti-
conflict action of diazepam [1] and inhibits food intake [7].
The mechanism of action of endozepines is not fully
understood. It has been initially proposed that these peptides
act as inverse agonists of central-type benzodiazepine
receptors [6] thus inhibiting the activity of the GABA
A
-
chloride channel complex [8]. Subsequently, endozepines
were found to interact with peripheral-type BZ receptors and

to stimulate cholesterol transport into mitochondria [9].
More recently, it has been shown that, in rat astrocytes, ODN
activates a metabotropic receptor positively coupled to
phospholipase C, leading to an increase in cytosolic
calcium concentration [10,11]. Structure-activity relation-
ship studies have shown that the C-terminal octapeptide of
ODN (OP; ODN
11218
) is the minimum sequence retaining
full calcium-mobilizing activity [12]. The Ala-scan of OP
has revealed that replacement of the Leu6 residue
suppresses the activity of the peptide. It has also been
found that the [
D-Leu5]OP analog exhibits a weak
antagonistic activity [12].
Correspondence to H. Vaudry, European Institute for Peptide Research,
Laboratory of Cellular and Molecular Neuroendocrinology, Institut
National de la Sante
´
et de la Recherche Me
´
dicale Unite
´
413, Unite
´
Associe
´
e au Centre National de la Recherche Scientifique, University of
Rouen, 76821 Mont-Saint-Aignan, France. Fax: 1 33 235 14 6946,
Tel.: 1 33 235 14 6624, E-mail:

Note: all optically active amino acids are of the
L configuration unless
otherwise noted.
(Received 17 April 2001, revised 30 July 2001, accepted 21 September
2001)
Abbreviations: ODN, octadecaneuropeptide; OP, octapeptide; DBI,
diazepam-binding inhibitor; BZ, benzodiazepine; TTN,
triakontatetraneuropeptide; HMP, 4-hydroxymethyl-phenoxymethyl-
copolystyrene-1%-divinylbenzene resin; HBTU, O-benzotriazol-1-yl-
N,N,N
0
,N
0
-tetramethyluronium hexafluorophosphate; HOBt,
1-hydroxybenzotriazole; DIEA, N,N-diisopropylethylamine; PEG-PS,
poly(ethylene glycol)–polystyrene resin; NMP, N-methylpyrrolidin-
2-one; DMF, N,N-dimethylformamide; [Ph
3
P]
4
Pd,
tetrakis(triphenylphosphine)palladium(0); NMM,
N-methylmorpholine; DMEM, Dulbecco’s modified Eagle’s medium;
DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate; IP, inositol
phosphate; PIP, polyphosphoinositide.
Eur. J. Biochem. 268, 6045–6057 (2001) q FEBS 2001
On the basis of these observations, we have undertaken
the design of selective ODN analogs that would exhibit high
affinity for the rat astrocyte endozepine receptor. Backbone
cyclization is an efficient approach which has been widely

used to stabilize the spatial conformation of peptides
without altering the side chain motifs that are often involved
in their biological activity [13,14]. In several cases, cycliz-
ation has been found to enhance the potency of peptides on
their receptors [15 –18]. The aim of the present study was
to prepare head-to-tail cyclic analogs of OP in order to
generate possible agonists and antagonists of ODN. We have
determined the secondary structure of the cyclic OP analogs
by two-dimensional
1
H-NMR and molecular dynamic
simulation, and we have investigated the biological activity
of these analogs by measuring their ability to modify
cytosolic calcium concentrations and polyphosphoinositide
turnover in cultured rat astrocytes.
MATERIALS AND METHODS
Materials
All amino-acid residues, preloaded 4-hydroxymethyl-
phenoxymethyl-copolystyrene-1%-divinylbenzene resin
[Fmoc-Lys(Boc)-HMP], O-benzotriazol-1-yl-N,N,N
0
,N
0
-
tetramethyluronium hexafluorophosphate (HBTU),
1-hydroxybenzotriazole (HOBt), piperidine and N,N-diiso-
propylethylamine (DIEA) were purchased from Applied
Biosystems (St Quentin en Yvelines, France). Preloaded poly
(ethylene glycol)–polystyrene resin [Fmoc-Asp(PEG-PS)-
OAl] was obtained from PerSeptive Biosystems (Voisins-le-

Bretonneux, France). Trifluoroacetic acid, trichloroacetic
acid, phenol, thioanisol, ethanedithiol, N-methylpyrrolidin-
2-one (NMP), N,N-dimethylformamide (DMF), tetrakis
(triphenylphosphine)palladium(0) ([Ph
3
P]
4
Pd), sodium
diethyldithiocarbamate, N-methylmorpholine (NMM),
U73122 (1-[6-([(17b)-3-methoxyestra-1,3,5-(10)-trien-
17-yl]amino)hexyl]1H-pyrrole-2,5-dione), Dulbecco’s
modified Eagle’s medium (DMEM), F12 culture medium,
insulin and
D(1)-glucose were from Sigma-Aldrich Chimie
(St Quentin Fallavier, France). Glutamine, the antibiotic–
antimycotic solution and Hepes were from Bioproducts
(Gagny, France). Fetal bovine serum was from Biosys
(Compie
`
gne, France). BSA (fraction V) was from Roche
Molecular Biochemicals (Mannheim, Germany). Indo-1-
acetoxymethyl ester and fluo-4-acetoxymethyl ester were
from Molecular Probes Europe (Leiden, The Netherlands).
Myo-[
3
H]inositol (100 Ci
:
mmol
21
) was from Amersham

International (Les Ulis, France). Sodium 2,2-dimethyl-
2-silapentane-5-sulfonate (DSS) and D
2
O were from Euriso-
top (CEA, Saclay, France).
Peptide synthesis
ODN (QATVGDVNTDRPGLLDLK) and OP (RPGLLDLK)
were synthesized (0.25 mmol scale for ODN; 0.1 mmol
scales for OP) on a Fmoc-Lys(Boc)-HMP resin using an
Applied Biosystems model 433A peptide synthesizer using
the standard FastMocVMonPrevPKw procedure as pre-
viously described [12]. The synthesis of linear precursors of
cyclo
128
OP and cyclo
128
[D-Leu5]OP (0.25 mmol scale
each) was performed on a Pioneer
TM
PerSeptive Biosystems
peptide synthesizer on a Fmoc-Asp(PEG-PS)-OAl resin
using similar coupling procedure, extended to a capping
cycle of the nonacylated primary amine. After completion
of the chain assembly, deprotection of the allyl ester
was performed manually by Pd(0) under Ar as previously
described [19,20]. The catalyst (3 eq., 0.75 mmol), dis-
solved in 26 mL Ac
2
O/CHCl
3

/NMM mixture (2 : 37 : 1;
v/v/v) was transferred to a sealed tube containing the Fmoc-
peptidyl(resin)-OAl using an Ar flushed gas-tight syringe
and gently agitated for 2.5 h at room temperature. The resin
was then washed sequentially with fresh catalyst dissolving
mixture, 0.5% DIEA in DMF, 0.5% sodium diethyldithio-
carbamate in DMF, 0.5% HOBt in DMF and DCM, and
dried in vacuo. The N-terminal Fmoc group was removed by
treatment with 20% piperidine in NMP. Prior to each manual
step, part of the X-peptidyl(resin)-Y (X ¼ Fmoc or H;
Y ¼ OAl or OH) was completely deprotected by trifluoro-
acetic acid to generate linear peptide sequences for reversed-
phase HPLC (RP-HPLC) analysis. On-resin head-to-tail
cyclization of the peptide was performed twice by addition
of HBTU/DIEA (8 eq., 2 mmol; 1 : 1, mol/mol) in 15 mL
of DMF for 2 Â 3.5 h with occasional gentle agitation.
Cyclization was monitored by the Kaiser’s test. Peptides
were deprotected and cleaved from the resin as previously
described [12].
Peptide purification
All peptides were purified by RP-HPLC on a semiprepara-
tive Vydac C
18
column (1 Â 25 cm; Touzart et Matignon,
Courtaboeuf, France) using a linear gradient (10–50% over
40 min) of acetonitrile/trifluoroacetic acid (99.9 : 0.1, v/v)
at a flow rate of 5 mL
:
min
21

. Analytical RP-HPLC was
performed on a Vydac C
18
column (0.45 Â 25 cm) using a
linear gradient (10 –40% over 30 min) of acetonitrile/
trifluoroacetic acid at a flow rate of 1 mL
:
min
21
. The
purified peptides were characterized by FAB-MS on a
conventional EB geometry mass spectrometer JEOL model
AX-500 equipped with a DEC data system (JEOL-Europe
SA, Croissy-sur-Seine, France) or by MALDI-TOF-MS on a
Tofspec E (Micromass, Manchester, UK).
Cell culture
Primary cultures of rat astrocytes were performed as pre-
viously described [21]. Briefly, cerebral hemispheres from
newborn Wistar rats were collected in DMEM/F12 culture
medium (2 : 1, v/v) supplemented with 2 m
M glutamine,
1% insulin, 5 m
M Hepes, 0.4% D(1)-glucose and 1% of the
antibiotic/antimicotic solution. The tissues were disaggre-
gated mechanically using a Pasteur pipette, and filtered
through a 100-mm nylon sieve (Poly Labo, Strasbourg,
France). Dissociated cells were resuspended in culture
medium supplemented with 10% fetal bovine serum and
seeded on coverslips in 35-mm dishes (Dutscher, Brumath,
France) at a density of 10

6
cells per dish. The cells were
incubated at 37 8C in a moist atmosphere (5% CO
2
), and the
medium was changed twice a week.
Confocal imaging
Five- to seven-day-old cells were loaded with 3 m
M fluo-
4-acetoxymethyl ester diluted in culture medium, at 37 8C
for 30 min. Thereafter, the calcium-dye-probe was washed
off and exchanged with 2 mL of fresh medium. The
6046 J. Leprince et al. (Eur. J. Biochem. 268) q FEBS 2001
fluorescence emission of the fluo-4-loaded cells, induced by
excitation at 488 nm (laser Ar/Kr) was recorded with a
500-nm long-pass filter on a Noran OZ confocal microscope
(Noran Instruments, Middleton, WI, USA). Images were
recorded as a time series (512 Â 480 pixels at one image per
532 ms) and data processing was carried out using the
INTERVISION software (Noran Instruments). Cyclo
128
OP
(10
28
M) was ejected for 2 s in the vicinity of the cells by a
pressure ejection system.
Measurement of cytosolic Ca
21
concentration
Five- to seven-day-old cells were loaded with 5 m

M indo-
1-acetoxymethyl ester diluted in culture medium, at 37 8C
for 45 min. The cells were washed twice with 2 mL of fresh
medium. The [Ca
21
]
i
was monitored by a dual-emission
microfluorimeter system constructed from a Nikon Diaphot
inverted microscope, as previously described [12]. The
fluorescence emission of indo-1, induced by excitation at
355 nm, was recorded at two wavelengths (405 nm and
480 nm) by separate photometers (Nikon, Champigny sur
Marne, France). The 405/480 ratio was determined using
an analogic divider (constructed by B. Dufy, University of
Bordeaux, France) after conversion of single photon
currents to voltage signals. All three signals (405 nm,
480 nm and the 405/480 ratio) were continuously recorded
with the
JAD-FLUO 1.2 software (Notocord Systems,
Croissy-sur-Seine, France). The [Ca
21
]
i
-values were calcu-
lated as previously described [22]. All secretagogues were
ejected for 2 s in the vicinity of individual cells by a pressure
ejection system. The indicated doses of peptides correspond
to the concentration contained in the ejection pipette.
Measurement of polyphosphoinositide metabolism

Twelve- to 14-day-old cells were incubated with 10 mCi
:
mL
21
myo-[
3
H]inositol (100 Ci
:
mmol
21
)at378Cinglucose-and
fetal bovine serum-free culture medium, in the absence or
presence of ODN-related peptides. The incubation was
stopped by removing the medium and adding 1 mL of ice-
cold 10% trichloroacetic acid. The cells were homogenized
and centrifuged (13 000 g; 15 min; 4 8C). The supernatant
was washed three times with water-saturated diethylether,
neutralized with 10 mLof1
M NaHCO
3
. Free inositol and
total tritiated inositol phosphates (IPs) were separated by
anion exchange chromatography (AG1-X8 resin; 100–200
mesh; formate form; Bio-Rad Laboratories, Richmond, CA,
USA) using distilled water and 0.8
M ammonium formate in
0.1
M formic acid, respectively, and the radioactivity
contained in each fraction was counted in a b-counter
(LKB 1217 Rack Beta, EG and G Wallace, Evry, France).

Polyphosphoinositides (PIPs) were extracted from the
pellet with 500 mL CHCl
3
/MeOH (2 : 1, v/v) and counted
in a b-counter. The remaining pellets were used for
measurement of protein concentration by the Lowry’s
method.
NMR spectroscopy
NMR experiments were carried out using an AVANCE
DMX 600 MHz spectrometer (Bruker S.A., Wissenbourg,
France) equipped with a SGI indigo 2 computer. One-
and two-dimensional NMR spectra were obtained at
temperatures of 275, 280, 285, 293 and 298 K. NMR
samples were prepared by dissolving the peptides in 550 mL
of H
2
O (10% D
2
O) or D
2
O. One- and two-dimensional
spectra were recorded with carrier frequency in the middle
of the spectrum coinciding with the water resonance which
was suppressed either by a presaturation using continuous
irradiation during relaxation delay or by using the gradient
pulse
WATERGATE [23]. One- and two-dimensional
1
H NMR
spectra were calibrated using DSS as an external reference.

Spin systems identification and sequential assignment were
achieved by TOCSY, COSY and NOESY experiments. The
TOCSY spectra were recorded with spin-lock time of 80 ms
by using Mlev17 sequence for the isotropic mixing. Four
mixing times (80, 150, 200 and 300 ms) were used for
NOESY spectra in order to identify diffusion effects. All
two-dimensional NMR experiments were acquired with
a total of 2048 complex data points in F
2
, and 512
experiments in F
1
. Prior to Fourier transform, data
matrices were zero filled in F
1
dimension and a phase
shifted sine-bell filter function was applied. Processing of
NMR data was performed on a SGI Indigo 2 workstation
using the manufacturer’s programs
XWINNMR 2.1 and
AURELIA 2.0.
Structure calculations
The volumes of the cross-peaks of the NOESY spectra
acquired with a mixing time of 200 ms were integrated
using the
AURELIA 2.0 software from Bruker. Interproton
distances were calculated using the isolated spin pair
approximation and setting the average methylene cross peak
volume at 0.18 nm. A range of 20% of the distance values
was used for defining the upper and lower bonds of the

constraints. For the methyl protons and protons that could
not be stereospecifically assigned, pseudoatoms were
generated during primary structure construction. For these
protons the lower bond of the distance constraints was set
to the sum of the Van der Waals radii. Backbone dihedral
restraint residues were deduced from
3
J
NH-Ha
coupling
constant by using the empirically Karplus-type relations
[24]. For coupling constant where more than a single
F-value is possible, additional secondary information from
NOE data was used to reduce the number of solutions. A
range of ^ 308 was used for defining the upper and lower
angle of the constraints. The structures were generated from
the experimental data with a standard dynamical simulated
annealing protocol using the
X-PLOR 3.1 program. A force
field adapted for NMR structure determination (file
parallhdg.pro and topallhdg.pro) was used, and an initial
structure was built by randomly generating F and C angles.
In the calculations starting from random structure, we
used higher force constants for the bond lengths, bond
angles, improper and planarity angles. After a short energy
minimization, the first stage started with scaling of the
weights of the NOE, constrained improper dihedrals, and
nonbonded terms initially small values to more realistic ones
using in total 30 000 time steps each of 0.002 ps at 1000 K.
The second stage performed a slow cooling of the system to

100 K in 50 stages each with 5000 time steps of 0.002 ps.
The final stage involved 2000 cycles of constrained Powell
energy minimization. Structures with the lowest distance
and dihedral constraint energy were selected and refined by
restrained Powell energy minimization using a more
q FEBS 2001 Design of a potent endozepine antagonist (Eur. J. Biochem. 268) 6047
realistic force field based on the CHARMM 19 program. The
Van der Waals energy was calculated with switched
Lennard–Jones potential, and the electric energy was
calculated with a shifted Coulomb potential with a dielectric
constant 1 ¼ 80.7. Structures were displayed using the
SYBYL software package (Tripos Associates, Inc., St Louis,
MO, USA). The whole structural analysis described in the
text was performed using structures calculated without
explicit H-bond restraints. The final structures were
examined to obtain pairwise root mean square differences
(rmsd) over the backbone heavy atoms (N, Ca and C).
Structure calculations without dihedral constraints were also
carried out. This did not produce any structural modifi-
cation, and only a slight change of the rmsd values was
observed for the backbone atoms.
Calculations and statistics
Data are expressed as mean ^ SEM. Student’s t-test
was used to determine statistical differences between
control and experimental values within the same set of
experiments [25].
RESULTS
Synthesis of cyclic peptides
The two cyclic peptides cyclo
128

OPandcyclo
128
[D-Leu5]OP
were synthesized directly on solid support and the head-
to-tail cyclization was achieved using orthogonal allyl
protection for the a-carboxylic function of aspartic acid
(Fig. 1). After the last coupling cycle and removal of the
allyl protecting group with Pd(0) and N-methylmorpholine,
an aliquot of Fmoc-peptidyl(PEG-PS)-OH was treated with
the cleavage mixture (reagent K) [26] and submitted to
RP-HPLC analysis. The chromatograms corresponding to
the different steps of the synthesis of cyclo
128
OP are shown
in Fig. 2. The HPLC profiles revealed the existence of two
peaks which eluted at 17.0 and 26.2 min (Fig. 2A). The
major one (retention time 26.2 min), which was assumed to
be the Fmoc-peptidyl-OH derivative, completely vanished
after treatment with piperidine (Fig. 2B). After two periods
of 3.5 h of lactamization with intermediate reactivation,
followed by side-chain deprotection and cleavage of the
peptide from the resin, RP-HPLC analysis revealed the
occurrence of a new major peak eluting at 18.6 min and
the disappearance of the linear precursor form (Fig. 2C).
Similar chromatograms were obtained during the synthesis
of cyclo
128
[D-Leu5]OP (data not shown).
Effect of ODN analogs on [Ca
21

]
i
The spatial-temporal [Ca
21
]
i
changes in rat astrocytes were
visualized by means of a confocal laser scanning
microscope. Ejection of 10
28
M cyclo
128
OP in the vicinity
of cultured cells provoked a wave of calcium in the
cytoplasm of astrocytes (Fig. 3A) while ejection of culture
medium alone had no effect (Fig. 3B). Comparison of the
amplitude of the response with that of ODN and OP revealed
that, at a concentration of 10
28
M, cyclo
128
OP was more
efficient than ODN and OP (Fig. 3C). Administration of
graded concentrations of OP and cyclo
128
OP induced a
bell-shaped [Ca
21
]
i

response (Fig. 4). For concentrations
ranging from 10
211
to 10
28
M, OP and cyclo
128
OP pro-
voked a dose-related increase in [Ca
21
]
i
with maximum
responses at concentrations of 10
28
M and 3.16 Â 10
29
M,
respectively. At higher concentrations (10
27
210
25
M), the
effect of OP and cyclo
128
OP gradually declined. The
efficacy of cyclo
128
OP in raising [Ca
21

]
i
was 1.4-fold
higher than that of OP. Repeated pulses of cyclo
128
OP
(3.16 Â 10
29
M) resulted in sequential increases in [Ca
21
]
i
with gradual attenuation of the response (Fig. 4).
Administration of cyclo
128
[D-Leu5]OP, for concentra-
tions ranging from 10
210
to 10
25
M, did not affect [Ca
21
]
i
in
rat astrocytes (Fig. 5). A 10-min preincubation of astrocytes
with graded concentrations of cyclo
128
[D-Leu5]OP
(10

210
210
26
M) provoked a dose-dependent inhibition of
the ODN-induced [Ca
21
]
i
increase with a pIC
50
value of
7.17 ^ 0.29 (Fig. 6). At a concentration of 10
26
M,
cyclo
128
[D-Leu5]OP totally abolished the [Ca
21
]
i
response
to ODN. Cyclo
128
[D-Leu5]OP also suppressed the [Ca
21
]
i
increase evoked by 10
28
M OP (Fig. 7A). On the other

hand, cyclo
128
[D-Leu5]OP, at concentrations of 10
26
and
10
25
M, significantly reduced (P , 0.001) but did not
abolish the [Ca
21
]
i
increase induced by cyclo
128
OP
(Fig. 7B).
Fig. 1. Scheme for the stepwise approach in the
synthesis of the cyclic peptides. (A) (1) piperidine
20% NMP, (2) Fmoc-AA-OH/HBTU/HOBt/
DIEA, (3) Ac
2
O/CHCl
3
/NMM. (B) (1) [Ph
3
P]
4
Pd/
AcOH/CHCl
3

/NMM, (2) AcOH/CHCl
3
/NMM, (3)
DIEA 0.5% DMF, (4) sodium
diethyldithiocarbamate 0.5% DMF, (5) HOBt
0.5% DMF, (6) DCM; (C) (1) piperidine 20%
NMP, (2) HBTU/DIEA; (D) trifluoroacetic acid/
phenol/thioanisol/ethanedithiol/H
2
O. Xaa ¼ Leu
(cyclo
128
OP) or D-Leu (cyclo
128
[D-Leu5]OP).
6048 J. Leprince et al. (Eur. J. Biochem. 268) q FEBS 2001
Effect of ODN analogs on polyphosphoinositide
metabolism
Exposure of cultured astrocytes to ODN, OP, or cyclo
128
OP
(10
28
M each) caused a significant increase in the formation
of [
3
H]IPs (Fig. 8A) and a concomitant decrease in the
levels of [
3
H]PIPs (Fig. 8B). In contrast, cyclo

128
[D-Leu5]OP, even at a high concentration (10
26
M), did
not affect the basal level of [
3
H]IPs and [
3
H]PIPs
(Fig. 8A,B). In the presence of 10
26
M cyclo
128
[D-Leu5]OP, the effect of ODN, OP and cyclo
128
OP
(10
28
M each) on [
3
H]IPs and [
3
H]PIPs was totally
abolished (Fig. 8C,D). Similarly, incubation of astrocytes
Fig. 2. RP-HPLC monitoring of N- and C-terminal deprotections
and on-resin cyclization of the linear precursor of cyclo
128
OP.
Aliquots of the reaction media after allyl ester deprotection (A), Fmoc
removal (B) and cyclization of the linear precursor (C) were cleaved and

injected onto a Vydac C
18
analytical column. The dotted lines represent
the profile of the elution gradient (% acetonitrile).
Fig. 3. Effect of cyclo
128
OP on [Ca
21
]
i
in cultured rat astrocytes.
Time series of pseudocolor images illustrating [Ca
21
]
i
changes in
astrocytes loaded with fluo-4-acetoxymethylester. (A) Intracellular
Ca
21
-wave following ejection of cyclo
128
OP (10
28
M). (B) Intra-
cellular calcium in cells from the same field after ejection of medium
alone. Sampling rate, 1 image per 532 ms. The pseudocolor scale
indicates the corresponding [Ca
21
]
i

changes expressed in arbitrary
units. (C) Effects of ODN, OP and cyclo
128
OP (10
28
M each) on the
amplitude of the calcium response measured by microfluorimetry. Each
value represents the mean amplitude (^ SEM) of the calcium response
calculated from at least 10 different dishes from five independent
cultures. The number of cells studied is indicated in parentheses. NS not
statistically significant, *P , 0.05, **P , 0.01.
q FEBS 2001 Design of a potent endozepine antagonist (Eur. J. Biochem. 268) 6049
for 10 min with the phospholipase C inhibitor U73122
(10
25
M) abrogated the effects of ODN, OP and cyclo
128
OP (10
28
M each) on [
3
H]IPs and [
3
H]PIPs (Fig. 8E,F).
NMR solution structure of cyclo
128
OP
Amino-acid spin systems of cyclo
128
OP were readily

identified from two-dimensional COSY and two-dimensional
TOCSY spectra starting from amide protons in the region of
9.5–7 p.p.m. and were confirmed by inspection of cross-
peaks in the high field region corresponding to side-chain
through-bond connectivities. NOESY data as those pre-
sented in Fig. 9 were then used to determine the sequential
assignments and the chemical shifts reported in Table 1.
Amide protons which were relatively slow to exchange
with solvent were identified by dissolving cyclo
128
OP in
deuterated solvent D
2
O. Then, one-dimensional
1
H-NMR
spectra were recorded at regular interval (Fig. 10). Due to
fast H/D exchange, the amide NH of residues Gly3, Leu5
and Asp6 disappeared rapidly (within one hour or less)
compared to the other amide protons.
The
3
J
NH-Ha
coupling constants for the amide protons of
cyclo
128
OP were measured from the 1D
1
H-NMR spectrum

(Table 1). The
3
J
NH-Ha
coupling constants of the three
leucine residues (Leu4, Leu5 and Leu7) differed from the
averaged value usually observed for small peptides
(< 7 Hz). The examination of short and medium range
NOEs (Fig. 11), in particular NOE observed between Ha-
Pro2 and Ha-Leu4, Ha-Leu5 and NH-Leu7, Ha-Leu6 and
NH-Lys8, in combination with the coupling constants and
slow H/D exchange of NH-Leu4, NH-Lys8, suggested a first
turn centered on the Gly3 and Leu4 residues, and a second
one in the region encompassing the Leu5, Asp6 and Leu7
residues. In order to better localize the different turns and to
identify each type of turn, molecular modeling under
experimental NMR restraints was performed.
NOEs data and coupling constants detected for cyclo
128
OP were used to drive a set of 47 distance and six dihedral
angle restraints. These restraints were used to generate a set
of 30 structures by simulated annealing as described in
Materials and methods. All the calculated structures fitted
the experimental data quite well and converged with high
precision. Analysis of the F and C angles showed that all
the residues were in the energetically favorable region of the
Fig. 5. Effect of graded concentrations of cyclo
128
[D-Leu
5

]OP on
[Ca
21
]
i
in cultured rat astrocytes. A 2-s pulse (arrow) of cyclo
128
[D-Leu5]OP (10
210
210
25
M) was administered in the vicinity of
different cells. The number of cells studied is indicated in parentheses.
Fig. 6. Effect of graded concentrations of cyclo
128
[D-Leu
5
]OP on
the ODN-evoked [Ca
21
]
i
increase in cultured rat astrocytes. The
cells were incubated for 15 min in the absence (W) or presence (X)of
cyclo
128
[D-Leu5]OP (10
210
210
26

M) before administration of a 2-s
pulse of ODN (10
28
M). Each value represents the mean amplitude
(^ SEM) of the ODN-evoked response calculated from at least four
different dishes from two independent cultures. The number of cells
studied is indicated in parentheses.
Fig. 4. Effect of graded concentrations of OP and cyclo
128
OP on
[Ca
21
]
i
in cultured rat astrocytes. A 2-s pulse of OP (10
211
210
25
M)
and cyclo
128
OP (10
211
210
26
M) was administred in the vicinity of
the cells and [Ca
21
]
i

was measured by microfluorimetry. Each value
represents the mean amplitude (^ SEM) of the calcium response
induced by OP (B) and cyclo
128
OP (X) calculated from at least four
different dishes from two independent cultures. The number of cells
studied is indicated in parentheses. The inset shows a typical profile of
the calcium response to 2-s pulses of 3.16 Â 10
29
M cyclo
128
OP
(arrows).
6050 J. Leprince et al. (Eur. J. Biochem. 268) q FEBS 2001
Ramachandran diagramme. Ten structures with the lowest
distance and dihedral constraint energies were selected,
providing a well-defined shape of the backbone foldings of
cyclo
128
OP (Fig. 12). The rmsd value calculated relatively
to the mean structures over all backbone atoms of the cycle
was 0.009 nm.
NMR solution structure of cyclo
128
[D-Leu5]OP
The
1
H-NMR spectrum of cyclo
128
[D-Leu5]OP in H

2
Oat
280 K was assigned by using the same strategy as for
cyclo
128
OP. In a first step, two-dimensional spectra COSY
and TOCSY were used for the identification of the amino-
acid spin system. In these spectra the glycine residue was
easily identified from its characteristic remote peaks (Ha1,
Ha2) at the amide proton frequency. Two glycine-type
remote peaks were clearly observed instead of one expected,
suggesting the existence of two spectroscopically distinct
molecular conformers. Analysis of other spectra confirmed
the occurrence in the solution of two distinct species
corresponding to the same primary structure. In a second
step, sequential assignment was simultaneously conducted
for the two conformers by using daN, dbN, and dNN in the
NOESY experiments (Fig. 13A). At this stage, the origin of
the structural heterogeneity was identified as a peptidyl-
prolyl cis-trans isomerism of the peptide bond Arg1-Pro2.
Some of the spectral data are illustrated in Fig. 13B, that
show a region of the NOESY spectrum containing the
sequential daa(Arg1, Pro2) and dadd
0
(Arg1, Pro2)
connectivities, characteristic of the two isomeric forms.
The proton chemical shift values of the cis and trans
conformers at 280 K are given in Table 2. Amide protons
that exchange with solvent were identified by dissolving
cyclo

128
[D-Leu
5
]OP in D
2
O. Due to fast H/D exchange all
Table 1.
1
H-NMR assignments,
3
J
HN-Ha
coupling constant and backbone angles F, C for cyclo
128
OP at 280 K. Chemical shifts are relative to
DSS.
Residue
Chemical shift (p.p.m.)
3
J
HN-Ha
(Hz)
Backbone angles (8)
HN Ha Hb Hg Hd H1FC
Arg1 7.56 4.74 1.91 1.64/1.50 3.22 7.26 7.9 2164 166
Pro2 4.27 2.31/1.88 2.14/1.95 3.82/3.57 290 128
Gly3 8.84 4.20/3.70 6.3 86 254
Leu4 7.87 4.68 1.68/1.58 1.58 0.88 8.9 2114 105
Leu5 8.79 4.14 1.71/1.60 1.71 0.94/0.89 3.4 78 62
Asp6 8.52 4.49 3.10/2.88 – 244 227

Leu7 7.85 4.24 1.84/1.67 1.67 0.90 7.9 272 230
Lys8 8.07 3.99 1.88 1.35 1.65 2.98 NH
3
: 7.59 6.1 2129 3
Fig. 7. Effect of cyclo
128
[D-Leu5]OP on the
OP- and cyclo
128
OP-evoked [Ca
21
]
i
increase
in cultured rat astrocytes. The cells were
incubated for 15 min in the absence or presence of
cyclo
128
[D-Leu5]OP (10
26
or 10
25
M) before
administration of a 2-s pulse (arrow) of OP (A) or
cyclo
128
OP (B) (10
28
M, each). Each value
represents the mean amplitude (^ SEM) of the

ODN-evoked response calculated from at least 4
different dishes from two independent cultures.
The number of cell studied is indicated in
parentheses.
q FEBS 2001 Design of a potent endozepine antagonist (Eur. J. Biochem. 268) 6051
amide protons of cyclo
128
[D-Leu
5
]OP disappeared rapidly
(within one hour or less) (Fig. 14).
In the NOESY spectrum, the cross-peaks of the cis and
trans conformers could be clearly separated and analyzed
for structure determination. In addition, no exchange of
cross-peak could be observed between the resonances of the
two conformers, indicating that the interconversion is very
slow on the NMR time-scale. NOEs detected for trans and
the cis isomers in combination with the coupling constant,
measured from the one-dimensional spectrum, supported
the existence of a well-defined structure and were used to
drive a set of 27 distance and four dihedral angle constraints
for the trans conformer and 31 distance and six dihedral
angle constraints for the cis conformer. These restraints
were used to generate a set of 30 structures for each
conformer of cyclo
128
[D-Leu5]OP using the same protocol
as for cyclo
128
OP. Ten final best structures obtained for the

trans and cis conformers were selected providing a well-
defined shape of the backbone foldings (Fig. 15). The rmsd
values calculated relatively to the mean structures over all
backbone atoms of the cycle was 0.009 nm for the trans
conformer and 0.007 nm for the cis conformer.
DISCUSSION
It has been previously reported that OP is the minimum
active sequence of ODN that possesses full agonistic activity
Fig. 9. Region of 600- MHz NOESY spectrum of cyclo
128
OP
obtained in H
2
O at 280 K. The spectrum was recorded with a mixing
time of 200 ms.
Fig. 8. Effect of cyclo
128
[D-Leu5]OP and U73122 on inositol
phosphate formation and polyphosphoinositide breakdown induced
by ODN, OP and cyclo
128
OP in cultured rat astrocytes. The cells
were incubated for 5 min with ODN, OP or cyclo
128
OP (10
28
M each)
in the absence (A, B) or presence (C, D) of cyclo
128
[D-Leu5]OP

(10
26
M). In another set of experiments (E, F), the cells were
preincubated with U73122 (10
25
M) for 10 min and then incubated for
5 min with ODN, OP or cyclo
128
OP (10
28
M each). Each bar
represents the mean (^ SEM) value from at least three independent
experiments. The number of determinations is indicated in parentheses.
**P , 0.01, ***P , 0.001.
Fig. 10. Region of 600-MHz amide proton NMR spectra. (A)
Spectrum of cyclo
128
OP in H
2
O. (B–D) Spectra of cyclo
128
OP in
D
2
O recorded after 10, 75 and 255 min, respectively.
6052 J. Leprince et al. (Eur. J. Biochem. 268) q FEBS 2001
on [Ca
21
]
i

in cultured rat astrocytes [12]. It has also been
shown that the [
D-Leu5]OP analog behaves as a weak
antagonist in the same in vitro model [12]. The present study
demonstrates that the cyclic analogs cyclo
128
OP and
cyclo
128
[D-Leu5]OP exhibit, respectively, potent agonistic
and antagonistic activities both on calcium mobilization and
on polyphosphoinositide metabolism in rat astroglial cells.
The secondary structure of these two ODN analogs has been
determined by combining two-dimensional
1
H-NMR and
molecular dynamics.
Introduction of conformational restraint through cycliza-
tion has become a standard strategy in medicinal chemistry
for increasing the receptor affinity and selectivity of peptide
ligands [13,14,27]. The Ala-scan of OP has revealed that
the side chain of each residue is required for the full activity
of the peptide [12]. These data led us to synthesize cyclic
analogs of OP and to use the N- and C-terminus for
cyclization in order to keep the side chains unmodified. We
have taken advantage of the presence of an aspartic acid
residue in the core sequence of OP and its analog to carry out
Fig. 11. Summary of NOEs observed in a 600-MHz NOESY
spectrum of cyclo
128

OP obtained in H
2
O at 280 K. The sequence is
displayed with the one-letter code. The heights of the bars indicate the
intensities of the NOEs. Residues with exchanging times of amide
protons larger than 1 h are indicated by black squares above the
sequence.
Fig. 12. Lowest energy conformer of cyclo
128
OP from simulated
annealing. The dotted lines indicate hydrogen bonds consistent with
NMR data.
Fig. 14. Summary of NOEs observed in a 600-MHz NOESY
spectrum of cyclo
128
[D-Leu
5
]OP obtained in H
2
O at 280 K. (A) Cis
isomer. (B) Trans isomer. The sequence is displayed with the one-letter
code. The heights of the bars indicate the intensities of the NOEs.
Fig. 13. Regions of a 600-MHz NOESY spectra
of cyclo
128
[D-Leu5]OP recorded with a mixing
time of 200 ms at 280 K. (A) NH-aCH cross
peaks in H
2
O: cis isomer, bold letters; trans

isomer, italic letters. (B) aCH-aCH region in D
2
O
solution.
q FEBS 2001 Design of a potent endozepine antagonist (Eur. J. Biochem. 268) 6053
head-to-tail cyclization on peptides bounded to the resin
according to the strategy of Trzeciak & Bannwarth [19],
rather than cyclization of a protected peptide in solution.
With this procedure, aspartic acid was attached to the solid
support via the b-carboxyl group whereas the a-carboxylic
group was protected as allyl ester. Monitoring of peptide
deprotection and lactamization processes by analytical
RP-HPLC revealed that the N
a
-Fmoc group was not totally
stable in reductive media. This observation was at variance
with the data reported by Carpino & Han [28], who found
that Fmoc-derivatives are not sensitive to catalytic hydro-
genolysis. Such a phenomenon, which has been already
reported by others [29,30], can be ascribed to the occurrence
of traces of dimethylamine in DMF or to the resonance
stabilization properties of the fluorenyl system. HPLC
analysis revealed that the peptides were entirely end-to-end
cyclized indicating that the experimental conditions favored
intramolecular rather than intermolecular cyclization. The
low reticulation grade of the solid support and the PEG
spacer used in this study produced a pseudodilution
phenomenon [31] and complete solvatation of the reactive
sites [32,33] which contributed to the efficacy of the
intramolecular cyclization.

Using a video imaging confocal microscopy technique,
we found that cyclo
128
OP induced calcium waves in cultured
rat astrocytes, suggesting that cyclization did not impair the
agonistic activity of OP. Quantitative measurement of [Ca
21
]
i
by microfluorimetry showed that cyclo
128
OP induced a bell-
shaped increase in [Ca
21
]
i
which was reminiscent of those
previously observed with ODN and OP [12]. However, we
found that the potency of cyclo
128
OP was increased by a
factor of three and its efficacy by a factor of 1.4 compared to
its linear counterpart in eliciting [Ca
21
]
i
rise in rat astrocytes.
As the dose–response curves obtained with ODN and OP are
Table 2.
1

H-NMR assignments,
3
J
HN-Ha
coupling constant and backbone angles F, C for cis and trans conformers of cyclo
128
[D-Leu5]OP at
280 K. Chemical shift is relative to DSS.
Residue
Chemical shift (p.p.m.)
3
J
HN –Ha
(Hz)
Backbone angles (8)
HN Ha Hb Hg Hd H1FC
Arg1
a
8.88 4.67 1.83 1.83 3.45/3.21 7.27 – 246 135
Arg1
b
7.6 4.79 1.8 1.62 3.25 7.3 – 2120 96
Pro2
a
4.49 2.39/1.95 2.14/2.03 3.68 256 140
Pro2
b
4.46 2.38/2.25 1.95/1.77 3.56 272 68
Gly3
a

9.11 4.11/3.60 11.3 90 153
Gly3
b
8.89 4.09/3.88 11.7 2170 237
Leu4
a
8.24 4.47 1.72 1.72 0.96/0.89 7.3 2129 179
Leu4
b
8.19 4.57 1.7 1.57 0.92 8.7 2118 251
Leu5
a
8.66 4.17 1.64 1.64 0.94 4.4 104 2117
Leu5
b
8.27 4.35 1.83/1.64 1.64 0.94/0.89 – 124 75
Asp6
a
9.3 4.8 3.04/2.83 8.3 298 33
Asp6
b
8.02 4.85 2.92 – 2132 2163
Leu7
a
7.55 4.31 1.75/1.51 1.75 1.03/0.93 7.1 2140 2154
Leu7
b
8.49 4.22 1.80/1.65 1.8 0.96/0.91 4.9 43 234
Lys8
a

8.21 4.14 1.74 1.56 1.74 3.06 NH3 : 7.64 4.4 2142 137
Lys8
b
8.36 4.18 1.89 1.50/1.42 1.7 3.02 NH3 : 7.65 6.8 2178 54
a
Cis conformer.
b
Trans conformer.
Fig. 15. Lowest energy conformers of cyclo
128
[D-Leu5]OP from simulated annealing. (A) Cis
conformer. (B) Trans conformer.
6054 J. Leprince et al. (Eur. J. Biochem. 268) q FEBS 2001
strictly superimposable, these data indicate that cyclo
128
OP
is a potent agonist of both ODN and OP.
Cyclo
128
[D-Leu5]OP did not modify [Ca
21
]
i
in cultured
rat astrocytes but totally blocked both the OP- and the ODN-
evoked [Ca
21
]
i
increase. The fact that the linear counterpart,

[
D-Leu5]OP, only partially reduced the stimulatory effect of
ODN on [Ca
21
]
i
[12] indicates that cyclization of the
peptide strongly enhances its antagonistic activity. Con-
currently, cyclo
128
[D-Leu5]OP did not completely abolish
the cyclo
128
OP-induced [Ca
21
]
i
increase, confirming that
cyclo
128
OP is a more potent agonist than the linear peptide.
We have previously demonstrated that the effect of ODN
on [Ca
21
]
i
is mediated through activation of a membrane
receptor coupled to a phopholipase C [10]. The present
study shows that cyclo
128

OP stimulates inositol phosphate
formation and polyphosphoinositide breakdown in cultured
rat astrocytes and that cyclo
128
[D-Leu5]OP totally abolishes
the effects of ODN, OP and cyclo
128
OP on inositol
phosphate turnover. In addition, the effects of cyclo
128
OP
on polyphosphoinositide metabolism were totally blocked
by U73122, a specific inhibitor of phospholipase C activity
[34]. These results confirm that cyclo
128
OP and cyclo
128
[D-Leu5]OP act, respectively, as an agonist and an
antagonist of the metabotropic receptor which mediates
the ODN-induced [Ca
21
]
i
increase in rat astrocytes.
It has been previously shown that a vacant C-terminal
carboxylic group is essential for ODN and OP to displace
benzodiazepines from their binding site [3,35]. In contrast,
the present data indicate that free N- and C-terminal
extremities are not required for the agonistic and
antagonistic activities of the two OP analogs on [Ca

21
]
i
,
indicating that cyclo
128
OP and cyclo
128
[D-Leu5]OP are
specific ligands for the metabotropic receptor that should not
recognize central-type benzodiazepine receptors. This
observation is of particular interest as the anxiogenic effects
of ODN are mediated through central-type benzodiazepine
receptors [5] while the anorexic action of ODN is likely to
be mediated through the metabotropic receptor [7].
We have next investigated the conformation of cyclo
128
OP and cyclo
128
[D-Leu5]OP in solution by
1
H-NMR and
restrained molecular dynamics. The structure calculations
were obtained without application of hydrogen bonds that
could be derived from amide proton exchange rate, using
only the angle and distance restraints deduced from
3
J
NH-Na
coupling constant and NOE cross peak, respectively.

Analysis of the (F, C) angles, and the potential hydrogen
bonding in calculated structures of cyclo
128
OP revealed
that the sequence consists of a g turn centered on
Pro2-Gly3-Leu4 and a type III b turn centered on
Leu5-Asp6-Leu7-Lys8 as indicated by the following
structural parameters: (a) the (F, C) dihedral angles of
residue Gly3 (86, 254); (b) the formation of intramolecular
hydrogen bonds (i, i 1 2) between the C ¼ O of Pro2 and
the NH of Leu4 to form a ring associated with a standard
g turn, and (c) a hydrogen bond (i, i 1 3) between the
C ¼ O of Leu5 and NH of Lys8 associated with the average
(F, C) dihedral angles of residues Asp6 (244, 227) and
Leu7 (272, 230) which correspond to almost canonical
values for a type III b turn [36,37]. Furthermore, the analysis
of the calculated structures revealed a short distances
between NH of Leu7 and the side chain carboxyl group of
the Asp6 residue (< 0.28 nm), and NH of Leu7 residue and
C¼OofLeu5(< 0.24 nm). The proximity of these
two C¼O acceptors for one NH donor could explain the slow
exchange H/D observed for NH of the Leu7 residue.
Analysis of NMR data of cyclo
128
[D-Leu5]OP, showed
that two equimolar conformer populations corresponding to
the same primary structure are present in solution. NOE
cross-peaks daa(Arg1, Pro2) and dadd
0
(Arg1, Pro2) clearly

indicated a peptidyl-prolyl cis-trans isomerism of the
peptide bond Arg1-Pro2. This observation suggests that the
presence of a
D-Leu residue in position 5 destabilizes the b,
g turn conformer and gives rise to a population containing a
cis X-Pro bond. The occurrence of cis bonds has been
reported in a number of small peptides, particularly in
peptides containing amide bonds involving a proline residue
[38,39]. Analysis of hydrogen bonds and dihedral angles
(F, C) for the average structures generated from members
of cis and trans family of cyclo
128
[D-Leu5]OP showed that
the residues of the cis and trans conformers of cyclo
128
[D-Leu5]OP do not fall into any classical turn. Cis and trans
conformers generally cannot be isolated as stable com-
pounds and therefore cannot be tested separately for
biological activity. The cyclic tetrapeptide [Sar1]-tentoxin
appears to be the only molecule known so far that has been
isolated as two different conformers [40].
The solution structure of cyclo
128
OP differs from that
determined for either its cis and trans diastereoisomer
cyclo
128
[D-Leu5]OP and also from that of the linear ODN
peptide (data not shown) in that the structure of cyclo
128

OP
consists of a g turn centered on Pro2-Gly3-Leu4 residues
and a type III b turn involving Leu5-Asp6-Leu7-Lys8
residues. g and b turns are typical elements of peptide and
protein conformation that are involved in peptide folding
and thus are intimately associated with biological activity
[41]. For instance, a b turn is found in the core sequence
of somatostatin [42], neurotensin [43] and gonadotropin-
releasing hormone [44] while a g turn is present in brady-
kinin [45]. The fact that cyclo
128
OP exhibits a defined
solution structure while ODN has a very low tendency to
form secondary structures [46] suggests that the g turn and/
or the type III b turn may play a pivotal role in enhancing the
calcium-mobilizing activity of the peptide on rat astrocytes.
In conclusion, we have shown that head-to-tail cyclization
of the ODN agonist OP and the weak antagonist [
D-Leu5]OP
enhances their biological activity on rat astrocytes leading to
a super-agonist and to the first efficacious ODN antagonist
described so far. Furthermore, cyclo
128
OP adopts a single
conformation in solution encompassing a g turn and a type
III b turn whereas cyclo
128
[D-Leu5]OP is present as two
equimolar conformers resulting from cis/trans isomerism.
Characterization of the secondary structure of these two

ligands of ODN receptors should provide useful information
for the design of potent, stable and selective agonists and
antagonists of these receptors. As ODN exerts anxiogenic
effects and induces proconflict behavior [5,6], specific
antagonists may have a therapeutic value for treatment of
psychiatric disorders. In addition, the development of
specific agonists that would selectively mimic the
anorexigenic effect of ODN [7] might be of interest for
the treatment of obesity and feeding disorders.
ACKNOWLEDGEMENTS
The authors wish to thank Mrs Catherine Buquet, Huguette Lemonnier
and Ge
´
rard Cauchois for skillful technical assistance. This study was
q FEBS 2001 Design of a potent endozepine antagonist (Eur. J. Biochem. 268) 6055
supported by INSERM (U413), the LARC-Neuroscience network and
the Conseil Re
´
gional de Haute-Normandie. J. L. was the recipient of a
scholarship from Oril-Servier Laboratories and the Conseil Re
´
gional de
Haute-Normandie. O. M. was the recipient of a scholarship from the
French Ministry of Research.
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