Eur J Biochem 268, 6045–6057 (2001) q FEBS 2001 Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN Design of a potent endozepine antagonist ´ ˆ Jerome Leprince1, Hassan Oulyadi2, David Vaudry1, Olfa Masmoudi1, Pierrick Gandolfo1, Christine Patte1, ` Jean Costentin3, Jean-Luc Fauchere4, Daniel Davoust2, Hubert Vaudry1 and Marie-Christine Tonon1 ´ ´ Institut Federatif de Recherches Multidisciplinaires sur les Peptides (IFRMP 23), Laboratoire de Neuroendocrinologie Cellulaire et ´ ´ ´ ´ ´ Moleculaire, Institut National de la Sante et de la Recherche Medicale Unite 413, CNRS, Universite de Rouen, Mont-Saint-Aignan, France; ´ ´ ´ ´ IFRMP 23, Laboratoire de Resonance Magnetique Nucleaire, Institut de Recherche en Chimie Organique Fine, CNRS Unite Mixte de ´ ´ Recherches 6014, Universite de Rouen, Mont-Saint-Aignan, France; 3IFRMP 23, Laboratoire de Neuropsychopharmacologie, CNRS Unite ´ Mixte de Recherches 6036, Universite de Rouen, Rouen, France; 4Institut 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 ([Ca21]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, cyclo128 OP, which was three times more potent and 1.4-times more efficacious than OP to increase [Ca21]i in cultured rat astrocytes Cyclo128 OP also mimicked the effect of both OP and ODN on polyphosphoinositide turnover Conversely, the cyclo128 [D -Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca21]i and phosphoinositide metabolism in astrocytes The structure of these cyclic analogs has been determined by two-dimensional 1H-NMR and molecular dynamics Cyclo128 OP exhibited a single conformation characterized by a g turn comprising residues Pro2 –Leu4 and a type III b turn encompassing residues Leu5–Lys8 Cyclo128 [D -Leu5]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 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 triakontatetraneuropeptide TTN (DBI17250) [2] and the octadecaneuropeptide ODN (DBI33250) [3] which are collectively designated by the term endozepines [4] Intracerebroventricular injection of endozepines provokes anxiogenic effects [5], induces proconflict behavior [1,6], reverses the anticonflict 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 GABAAchloride 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 relationship studies have shown that the C-terminal octapeptide of ODN (OP; ODN11218) 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 Medicale Unite 413, Unite ´ Associee au Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France Fax: 33 235 14 6946, Tel.: 33 235 14 6624, E-mail: hubert.vaudry@univ-rouen.fr Abbreviations: ODN, octadecaneuropeptide; OP, octapeptide; DBI, diazepam-binding inhibitor; BZ, benzodiazepine; TTN, triakontatetraneuropeptide; HMP, 4-hydroxymethyl-phenoxymethylcopolystyrene-1%-divinylbenzene resin; HBTU, O-benzotriazol-1-ylN,N,N ,N -tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; DIEA, N,N-diisopropylethylamine; PEG-PS, poly(ethylene glycol)–polystyrene resin; NMP, N-methylpyrrolidin2-one; DMF, N,N-dimethylformamide; [Ph3P]4Pd, 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 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) Keywords: solid-phase peptide synthesis; cyclic peptides; structure-activity relationship; astrocytes; cytosolic calcium concentration 6046 J Leprince et al (Eur J Biochem 268) 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, cyclization 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 1H-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 M AT E R I A L S A N D M E T H O D S Materials All amino-acid residues, preloaded 4-hydroxymethylphenoxymethyl-copolystyrene-1%-divinylbenzene resin [Fmoc-Lys(Boc)-HMP], O-benzotriazol-1-yl-N,N,N ,N tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), piperidine and N,N-diisopropylethylamine (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-leBretonneux, France) Trifluoroacetic acid, trichloroacetic acid, phenol, thioanisol, ethanedithiol, N-methylpyrrolidin2-one (NMP), N,N-dimethylformamide (DMF), tetrakis (triphenylphosphine)palladium(0) ([Ph3P]4Pd), sodium diethyldithiocarbamate, N-methylmorpholine (NMM), U73122 (1-[6-([(17b)-3-methoxyestra-1,3,5-(10)-trien17-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 ` (Compiegne, France) BSA (fraction V) was from Roche Molecular Biochemicals (Mannheim, Germany) Indo-1acetoxymethyl ester and fluo-4-acetoxymethyl ester were from Molecular Probes Europe (Leiden, The Netherlands) Myo-[3H]inositol (100 Ci:mmol21) was from Amersham International (Les Ulis, France) Sodium 2,2-dimethyl2-silapentane-5-sulfonate (DSS) and D2O were from Eurisotop (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 previously described [12] The synthesis of linear precursors of cyclo128 OP and cyclo128 [D -Leu5]OP (0.25 mmol scale each) was performed on a PioneerTM PerSeptive Biosystems peptide synthesizer on a Fmoc-Asp(PEG-PS)-OAl resin q FEBS 2001 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), dissolved in 26 mL Ac2O/CHCl3/NMM mixture (2 : 37 : 1; v/v/v) was transferred to a sealed tube containing the Fmocpeptidyl(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 diethyldithiocarbamate 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 trifluoroacetic acid to generate linear peptide sequences for reversedphase HPLC (RP-HPLC) analysis On-resin head-to-tail cyclization of the peptide was performed twice by addition of HBTU/DIEA (8 eq., mmol; : 1, mol/mol) in 15 mL of DMF for  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 semipreparative Vydac C18 column (1  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 mL:min21 Analytical RP-HPLC was performed on a Vydac C18 column (0.45  25 cm) using a linear gradient (10 –40% over 30 min) of acetonitrile/ trifluoroacetic acid at a flow rate of mL:min21 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 previously described [21] Briefly, cerebral hemispheres from newborn Wistar rats were collected in DMEM/F12 culture medium (2 : 1, v/v) supplemented with mM glutamine, 1% insulin, mM Hepes, 0.4% D (1)-glucose and 1% of the antibiotic/antimicotic solution The tissues were disaggregated 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 106 cells per dish The cells were incubated at 37 8C in a moist atmosphere (5% CO2), and the medium was changed twice a week Confocal imaging Five- to seven-day-old cells were loaded with mM fluo4-acetoxymethyl ester diluted in culture medium, at 37 8C for 30 Thereafter, the calcium-dye-probe was washed off and exchanged with mL of fresh medium The q FEBS 2001 Design of a potent endozepine antagonist (Eur J Biochem 268) 6047 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  480 pixels at one image per 532 ms) and data processing was carried out using the INTERVISION software (Noran Instruments) Cyclo128 OP (1028 M) was ejected for s in the vicinity of the cells by a pressure ejection system Measurement of cytosolic Ca21 concentration Five- to seven-day-old cells were loaded with mM indo1-acetoxymethyl ester diluted in culture medium, at 37 8C for 45 The cells were washed twice with mL of fresh medium The [Ca21]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 [Ca21]i-values were calculated as previously described [22] All secretagogues were ejected for 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:mL21 myo-[3H]inositol (100 Ci:mmol21) at 37 8C in glucose- 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 mL of icecold 10% trichloroacetic acid The cells were homogenized and centrifuged (13 000 g; 15 min; 8C) The supernatant was washed three times with water-saturated diethylether, neutralized with 10 mL of M NaHCO3 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 CHCl3/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 computer Oneand 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 H2O (10% D2O) or D2O 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 1H 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 F2, and 512 experiments in F1 Prior to Fourier transform, data matrices were zero filled in F1 dimension and a phase shifted sine-bell filter function was applied Processing of NMR data was performed on a SGI Indigo 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 3JNH-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 6048 J Leprince et al (Eur J Biochem 268) 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 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 modification, 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] R E S U LT S Synthesis of cyclic peptides The two cyclic peptides cyclo128 OP and cyclo128 [D -Leu5]OP were synthesized directly on solid support and the headto-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 cyclo128 OP are shown in Fig The HPLC profiles revealed the existence of two peaks which eluted at 17.0 and 26.2 (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 q FEBS 2001 peptide from the resin, RP-HPLC analysis revealed the occurrence of a new major peak eluting at 18.6 and the disappearance of the linear precursor form (Fig 2C) Similar chromatograms were obtained during the synthesis of cyclo128 [D -Leu5]OP (data not shown) Effect of ODN analogs on [Ca21]i The spatial-temporal [Ca21]i changes in rat astrocytes were visualized by means of a confocal laser scanning microscope Ejection of 1028 M cyclo128 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 1028 M , cyclo128 OP was more efficient than ODN and OP (Fig 3C) Administration of graded concentrations of OP and cyclo128 OP induced a bell-shaped [Ca21]i response (Fig 4) For concentrations ranging from 10211 to 1028 M , OP and cyclo128 OP provoked a dose-related increase in [Ca21]i with maximum responses at concentrations of 1028 M and 3.16  1029 M , respectively At higher concentrations (102721025 M ), the effect of OP and cyclo128 OP gradually declined The efficacy of cyclo128 OP in raising [Ca21]i was 1.4-fold higher than that of OP Repeated pulses of cyclo128 OP (3.16  1029 M ) resulted in sequential increases in [Ca21]i with gradual attenuation of the response (Fig 4) Administration of cyclo128 [D -Leu5]OP, for concentrations ranging from 10210 to 1025 M , did not affect [Ca21]i in rat astrocytes (Fig 5) A 10-min preincubation of astrocytes with graded concentrations of cyclo128 [D -Leu5]OP (1021021026 M ) provoked a dose-dependent inhibition of the ODN-induced [Ca21]i increase with a pIC50 value of 7.17 ^ 0.29 (Fig 6) At a concentration of 1026 M , cyclo128 [D -Leu5]OP totally abolished the [Ca21]i response to ODN Cyclo128 [D -Leu5]OP also suppressed the [Ca21]i increase evoked by 1028 M OP (Fig 7A) On the other hand, cyclo128 [D -Leu5]OP, at concentrations of 1026 and 1025 M , significantly reduced (P , 0.001) but did not abolish the [Ca21]i increase induced by cyclo128 OP (Fig 7B) Fig 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) Ac2O/CHCl3/NMM (B) (1) [Ph3P]4Pd/ AcOH/CHCl3/NMM, (2) AcOH/CHCl3/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/H2O Xaa ¼ Leu (cyclo128 OP) or D -Leu (cyclo128 [D -Leu5]OP) q FEBS 2001 Design of a potent endozepine antagonist (Eur J Biochem 268) 6049 Fig RP-HPLC monitoring of N- and C-terminal deprotections and on-resin cyclization of the linear precursor of cyclo128 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 C18 analytical column The dotted lines represent the profile of the elution gradient (% acetonitrile) Effect of ODN analogs on polyphosphoinositide metabolism Exposure of cultured astrocytes to ODN, OP, or cyclo128 OP (1028 M each) caused a significant increase in the formation Fig Effect of cyclo128 OP on [Ca21]i in cultured rat astrocytes Time series of pseudocolor images illustrating [Ca21]i changes in astrocytes loaded with fluo-4-acetoxymethylester (A) Intracellular Ca21-wave following ejection of cyclo128 OP (1028 M ) (B) Intracellular calcium in cells from the same field after ejection of medium alone Sampling rate, image per 532 ms The pseudocolor scale indicates the corresponding [Ca21]i changes expressed in arbitrary units (C) Effects of ODN, OP and cyclo128 OP (1028 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 of [3H]IPs (Fig 8A) and a concomitant decrease in the levels of [3H]PIPs (Fig 8B) In contrast, cyclo 128 [D -Leu5]OP, even at a high concentration (1026 M ), did not affect the basal level of [3H]IPs and [3H]PIPs (Fig 8A,B) In the presence of 1026 M cyclo 128 [D -Leu5]OP, the effect of ODN, OP and cyclo128 OP (1028 M each) on [3H]IPs and [3H]PIPs was totally abolished (Fig 8C,D) Similarly, incubation of astrocytes 6050 J Leprince et al (Eur J Biochem 268) Fig Effect of graded concentrations of OP and cyclo128 OP on [Ca21]i in cultured rat astrocytes A 2-s pulse of OP (1021121025 M ) and cyclo128 OP (1021121026 M ) was administred in the vicinity of the cells and [Ca21]i was measured by microfluorimetry Each value represents the mean amplitude (^ SEM) of the calcium response induced by OP (B) and cyclo128 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  1029 M cyclo128 OP (arrows) for 10 with the phospholipase C inhibitor U73122 (1025 M ) abrogated the effects of ODN, OP and cyclo128 OP (1028 M each) on [3H]IPs and [3H]PIPs (Fig 8E,F) NMR solution structure of cyclo128 OP Amino-acid spin systems of cyclo128 OP were readily identified from two-dimensional COSY and two-dimensional TOCSY spectra starting from amide protons in the region of Fig Effect of graded concentrations of cyclo128 [D -Leu5]OP on [Ca21]i in cultured rat astrocytes A 2-s pulse (arrow) of cyclo128 [D -Leu5]OP (1021021025 M ) was administered in the vicinity of different cells The number of cells studied is indicated in parentheses q FEBS 2001 Fig Effect of graded concentrations of cyclo128 [D -Leu5]OP on the ODN-evoked [Ca21]i increase in cultured rat astrocytes The cells were incubated for 15 in the absence (W) or presence (X) of cyclo128 [D -Leu5]OP (1021021026 M ) before administration of a 2-s pulse of ODN (1028 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 9.5 –7 p.p.m and were confirmed by inspection of crosspeaks in the high field region corresponding to side-chain through-bond connectivities NOESY data as those presented in Fig were then used to determine the sequential assignments and the chemical shifts reported in Table Amide protons which were relatively slow to exchange with solvent were identified by dissolving cyclo128 OP in deuterated solvent D2O Then, one-dimensional 1H-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 3JNH-Ha coupling constants for the amide protons of cyclo128 OP were measured from the 1D 1H-NMR spectrum (Table 1) The 3JNH-Ha coupling constants of the three leucine residues (Leu4, Leu5 and Leu7) differed from the averaged value usually observed for small peptides (< Hz) The examination of short and medium range NOEs (Fig 11), in particular NOE observed between HaPro2 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 cyclo128 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 q FEBS 2001 Design of a potent endozepine antagonist (Eur J Biochem 268) 6051 Fig Effect of cyclo128 [D -Leu5]OP on the OP- and cyclo128 OP-evoked [Ca21]i increase in cultured rat astrocytes The cells were incubated for 15 in the absence or presence of cyclo128 [D -Leu5]OP (1026 or 1025 M ) before administration of a 2-s pulse (arrow) of OP (A) or cyclo128 OP (B) (1028 M , each) Each value represents the mean amplitude (^ SEM) of the ODN-evoked response calculated from at least different dishes from two independent cultures The number of cell studied is indicated in parentheses 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 peptidylprolyl 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 dadd0 (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 Amide protons that exchange with solvent were identified by dissolving cyclo128 [D -Leu5]OP in D2O Due to fast H/D exchange all Ramachandran diagramme Ten structures with the lowest distance and dihedral constraint energies were selected, providing a well-defined shape of the backbone foldings of cyclo128 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 cyclo128 [D -Leu5]OP The 1H-NMR spectrum of cyclo128 [D -Leu5]OP in H2O at 280 K was assigned by using the same strategy as for cyclo128 OP In a first step, two-dimensional spectra COSY and TOCSY were used for the identification of the aminoacid 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, Table 1H-NMR assignments, 3JHN-Ha coupling constant and backbone angles F, C for cyclo128 OP at 280 K Chemical shifts are relative to DSS Chemical shift (p.p.m.) Residue HN Ha Hb Hg Hd H1 JHN-Ha (Hz) Arg1 Pro2 Gly3 Leu4 Leu5 Asp6 Leu7 Lys8 7.56 4.74 4.27 4.20/3.70 4.68 4.14 4.49 4.24 3.99 1.91 2.31/1.88 1.64/1.50 2.14/1.95 3.22 3.82/3.57 7.26 7.9 1.68/1.58 1.71/1.60 3.10/2.88 1.84/1.67 1.88 1.58 1.71 0.88 0.94/0.89 1.67 1.35 0.90 1.65 2.98 NH3 : 7.59 6.3 8.9 3.4 – 7.9 6.1 8.84 7.87 8.79 8.52 7.85 8.07 Backbone angles (8) F C 2164 290 86 2114 78 244 272 2129 166 128 254 105 62 227 230 6052 J Leprince et al (Eur J Biochem 268) q FEBS 2001 Fig Region of 600- MHz NOESY spectrum of cyclo128 OP obtained in H2O at 280 K The spectrum was recorded with a mixing time of 200 ms 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 Effect of cyclo128 [D -Leu5]OP and U73122 on inositol phosphate formation and polyphosphoinositide breakdown induced by ODN, OP and cyclo128 OP in cultured rat astrocytes The cells were incubated for with ODN, OP or cyclo128 OP (1028 M each) in the absence (A, B) or presence (C, D) of cyclo128 [D -Leu5]OP (1026 M ) In another set of experiments (E, F), the cells were preincubated with U73122 (1025 M ) for 10 and then incubated for with ODN, OP or cyclo128 OP (1028 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 amide protons of cyclo128 [D -Leu5]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 cyclo128 [D -Leu5]OP using the same protocol as for cyclo128 OP Ten final best structures obtained for the trans and cis conformers were selected providing a welldefined shape of the backbone foldings (Fig 15) The rmsd values calculated relatively to the mean structures over all Fig 10 Region of 600-MHz amide proton NMR spectra (A) Spectrum of cyclo128 OP in H2O (B–D) Spectra of cyclo128 OP in D2O recorded after 10, 75 and 255 min, respectively q FEBS 2001 Design of a potent endozepine antagonist (Eur J Biochem 268) 6053 Fig 11 Summary of NOEs observed in a 600-MHz NOESY spectrum of cyclo128 OP obtained in H2O 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 h are indicated by black squares above the sequence Fig 14 Summary of NOEs observed in a 600-MHz NOESY spectrum of cyclo128 [D -Leu5]OP obtained in H2O 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 12 Lowest energy conformer of cyclo128 OP from simulated annealing The dotted lines indicate hydrogen bonds consistent with NMR data Fig 13 Regions of a 600-MHz NOESY spectra of cyclo128 [D -Leu5]OP recorded with a mixing time of 200 ms at 280 K (A) NH-aCH cross peaks in H2O: cis isomer, bold letters; trans isomer, italic letters (B) aCH-aCH region in D2O solution on [Ca21]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 cyclo128 OP and cyclo128 [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 1H-NMR and molecular dynamics Introduction of conformational restraint through cyclization 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 6054 J Leprince et al (Eur J Biochem 268) q FEBS 2001 Table 1H-NMR assignments, 3JHN-Ha coupling constant and backbone angles F, C for cis and trans conformers of cyclo128 [D -Leu5]OP at 280 K Chemical shift is relative to DSS Chemical shift (p.p.m.) Residue HN Ha Hb Hg Hd Arg1a Arg1b Pro2a Pro2b Gly3a Gly3b Leu4a Leu4b Leu5a Leu5b Asp6a Asp6b Leu7a Leu7b Lys8a Lys8b 8.88 7.6 4.67 4.79 4.49 4.46 4.11/3.60 4.09/3.88 4.47 4.57 4.17 4.35 4.8 4.85 4.31 4.22 4.14 4.18 1.83 1.8 2.39/1.95 2.38/2.25 1.83 1.62 2.14/2.03 1.95/1.77 3.45/3.21 3.25 3.68 3.56 JHN – Ha (Hz) H1 7.27 7.3 a 9.11 8.89 8.24 8.19 8.66 8.27 9.3 8.02 7.55 8.49 8.21 8.36 Backbone angles (8) 1.72 1.7 1.64 1.83/1.64 3.04/2.83 2.92 1.75/1.51 1.80/1.65 1.74 1.89 1.72 1.57 1.64 1.64 0.96/0.89 0.92 0.94 0.94/0.89 1.75 1.8 1.56 1.50/1.42 1.03/0.93 0.96/0.91 1.74 1.7 – – 3.06 NH3 : 7.64 3.02 NH3 : 7.65 11.3 11.7 7.3 8.7 4.4 – 8.3 – 7.1 4.9 4.4 6.8 F C 246 2120 256 272 90 2170 2129 2118 104 124 298 2132 2140 43 2142 2178 135 96 140 68 153 237 179 251 2117 75 33 2163 2154 234 137 54 Cis conformer b Trans conformer 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 Na-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 hydrogenolysis 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 cyclo128 OP induced calcium waves in cultured rat astrocytes, suggesting that cyclization did not impair the agonistic activity of OP Quantitative measurement of [Ca21]i by microfluorimetry showed that cyclo128 OP induced a bellshaped increase in [Ca21]i which was reminiscent of those previously observed with ODN and OP [12] However, we found that the potency of cyclo128 OP was increased by a factor of three and its efficacy by a factor of 1.4 compared to its linear counterpart in eliciting [Ca21]i rise in rat astrocytes As the dose–response curves obtained with ODN and OP are Fig 15 Lowest energy conformers of cyclo128 [D -Leu5]OP from simulated annealing (A) Cis conformer (B) Trans conformer q FEBS 2001 Design of a potent endozepine antagonist (Eur J Biochem 268) 6055 strictly superimposable, these data indicate that cyclo128 OP is a potent agonist of both ODN and OP Cyclo128 [D -Leu5]OP did not modify [Ca21]i in cultured rat astrocytes but totally blocked both the OP- and the ODNevoked [Ca21]i increase The fact that the linear counterpart, [D -Leu5]OP, only partially reduced the stimulatory effect of ODN on [Ca21]i [12] indicates that cyclization of the peptide strongly enhances its antagonistic activity Concurrently, cyclo128 [D -Leu5]OP did not completely abolish the cyclo128 OP-induced [Ca21]i increase, confirming that cyclo128 OP is a more potent agonist than the linear peptide We have previously demonstrated that the effect of ODN on [Ca21]i is mediated through activation of a membrane receptor coupled to a phopholipase C [10] The present study shows that cyclo128 OP stimulates inositol phosphate formation and polyphosphoinositide breakdown in cultured rat astrocytes and that cyclo128 [D -Leu5]OP totally abolishes the effects of ODN, OP and cyclo128 OP on inositol phosphate turnover In addition, the effects of cyclo128 OP on polyphosphoinositide metabolism were totally blocked by U73122, a specific inhibitor of phospholipase C activity [34] These results confirm that cyclo128 OP and cyclo128 [D -Leu5]OP act, respectively, as an agonist and an antagonist of the metabotropic receptor which mediates the ODN-induced [Ca21]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 [Ca21]i, indicating that cyclo128 OP and cyclo128 [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 cyclo128 OP and cyclo128 [D -Leu5]OP in solution by 1H-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 3JNH-Na coupling constant and NOE cross peak, respectively Analysis of the (F, C) angles, and the potential hydrogen bonding in calculated structures of cyclo128 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 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 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 C¼O of Leu5 (< 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 cyclo128 [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 dadd0 (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 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 cyclo128 [D -Leu5]OP showed that the residues of the cis and trans conformers of cyclo128 [D -Leu5]OP not fall into any classical turn Cis and trans conformers generally cannot be isolated as stable compounds 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 cyclo128 OP differs from that determined for either its cis and trans diastereoisomer cyclo128 [D -Leu5]OP and also from that of the linear ODN peptide (data not shown) in that the structure of cyclo128 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 gonadotropinreleasing hormone [44] while a g turn is present in bradykinin [45] The fact that cyclo128 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, cyclo128 OP adopts a single conformation in solution encompassing a g turn and a type III b turn whereas cyclo128 [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 Gerard Cauchois for skillful technical assistance This study was 6056 J Leprince et al (Eur J Biochem 268) supported by INSERM (U413), the LARC-Neuroscience network and ´ the Conseil Regional de Haute-Normandie J L was the recipient of a ´ scholarship from Oril-Servier Laboratories and the Conseil Regional de Haute-Normandie O M was the recipient of a scholarship from the French Ministry of Research REFERENCES Guidotti, A., Forchetti, C.M., Corda, M.G., Konkel, D., Bennett, C.D & Costa, E (1983) Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors Proc Natl Acad Sci USA 80, 3531–3535 Slobodyansky, E., Guidotti, A., Wambebe, C., Berkovich, A & Costa, E (1989) Isolation and characterization of a rat brain triakontatetraneuropeptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro 5–4864 recognition site J Neurochem 53, 1276–1284 Ferrero, P., Santi, 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constrained analog Science 210, 656 –658 Cann, J.R., London, R.E., Matwiyoff, N.A & Stewart, J.M (1983) A combined spectroscopic study of the solution conformation of bradykinin Adv Exp Med Biol 156, 495– 500 Berkovich, A., McPhie, P., Campagnone, M., Guidotti, A & Hensley, P (1990) A natural processing product of rat diazepam binding inhibitor, triakontatetraneuropeptide (diazepam binding inhibitor 17–50) contains an a-helix, which allows discrimination between benzodiazepine binding site subtypes Mol Pharmacol 37, 164 –172  ... 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 1H-NMR and molecular dynamic... 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... 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