Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN Design of a potent endozepine antagonist Je´roˆme Leprince1, Hassan Oulyadi2,
Trang 1Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN
Design of a potent endozepine antagonist
Je´roˆme Leprince1, Hassan Oulyadi2, David Vaudry1, Olfa Masmoudi1, Pierrick Gandolfo1, Christine Patte1, Jean Costentin3, Jean-Luc Fauche`re4, Daniel Davoust2, Hubert Vaudry1and Marie-Christine Tonon1
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;3IFRMP 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 ([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, cyclo128OP, which was three times
more potent and 1.4-times more efficacious than OP to
increase [Ca21]iin cultured rat astrocytes Cyclo128OP also
mimicked the effect of both OP and ODN on
polyphos-phoinositide 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-dimen-sional 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
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 (DBI17250) [2] and the octadecaneuro-peptide ODN (DBI33250) [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 GABAA -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; 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 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: hubert.vaudry@univ-rouen.fr
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.
Trang 2On 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 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 A T E R I A L S A N D M E T H O D S
Materials
All amino-acid residues, preloaded
4-hydroxymethyl-phenoxymethyl-copolystyrene-1%-divinylbenzene resin
[Fmoc-Lys(Boc)-HMP], O-benzotriazol-1-yl-N,N,N0,N0
-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) ([Ph3P]4Pd), 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 andD(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-Indo-1-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-dimethyl-2-silapentane-5-sulfonate (DSS) and D2O 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
cyclo128OP and cyclo128[D-Leu5]OP (0.25 mmol scale
each) was performed on a PioneerTMPerSeptive 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 Ac2O/CHCl3/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 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 C18column (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 5 mL:min21 Analytical RP-HPLC was performed on a Vydac C18column (0.45 25 cm) using a linear gradient (10 – 40% over 30 min) of acetonitrile/ trifluoroacetic acid at a flow rate of 1 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 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 mM glutamine, 1% insulin, 5 mMHepes, 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 106cells 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 3 mM 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
Trang 3fluorescence 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
(1028M) was ejected for 2 s in the vicinity of the cells by a
pressure ejection system
Measurement of cytosolic Ca21concentration
Five- to seven-day-old cells were loaded with 5 mM
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 [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
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: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 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 mL of 1M 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 0.8Mammonium formate in
0.1M 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 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 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 pulseWATERGATE[23] One- and two-dimensional1H 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 2 workstation using the manufacturer’s programs XWINNMR 2.1 and
AURELIA2.0
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 theX-PLOR3.1 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
Trang 4realistic force field based on theCHARMM19 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
SYBYLsoftware 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]
R E S U L T S
Synthesis of cyclic peptides
The two cyclic peptides cyclo128OP and cyclo128[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 cyclo128OP 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 cyclo128[D-Leu5]OP (data not shown)
Effect of ODN analogs on [Ca21]i The spatial-temporal [Ca21]ichanges in rat astrocytes were visualized by means of a confocal laser scanning microscope Ejection of 1028Mcyclo128OP 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 1028M, cyclo128OP 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 10211to 1028M, OP and cyclo128OP pro-voked a dose-related increase in [Ca21]i with maximum responses at concentrations of 1028M and 3.16 1029M, respectively At higher concentrations (102721025M), 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 1029M) resulted in sequential increases in [Ca21]i with gradual attenuation of the response (Fig 4)
Administration of cyclo128 [D-Leu5]OP, for concentra-tions ranging from 10210to 1025M, did not affect [Ca21]iin rat astrocytes (Fig 5) A 10-min preincubation of astrocytes with graded concentrations of cyclo128 [D-Leu5]OP (1021021026M) 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 1026M, cyclo128[D-Leu5]OP totally abolished the [Ca21]iresponse
to ODN Cyclo128[D-Leu5]OP also suppressed the [Ca21]i increase evoked by 1028M OP (Fig 7A) On the other hand, cyclo128[D-Leu5]OP, at concentrations of 1026and
1025M, significantly reduced (P , 0.001) but did not abolish the [Ca21]i increase induced by cyclo128 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 OP) or -Leu (cyclo [ -Leu5]OP).
Trang 5Effect of ODN analogs on polyphosphoinositide
metabolism
Exposure of cultured astrocytes to ODN, OP, or cyclo128OP
(1028 each) caused a significant increase in the formation
of [3H]IPs (Fig 8A) and a concomitant decrease in the levels of [3H]PIPs (Fig 8B) In contrast, cyclo128 [D-Leu5]OP, even at a high concentration (1026M), did not affect the basal level of [3H]IPs and [3H]PIPs (Fig 8A,B) In the presence of 1026 M cyclo128 [D-Leu5]OP, the effect of ODN, OP and cyclo128 OP (1028M each) on [3H]IPs and [3H]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 [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 cyclo 128 OP (1028M ) (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 [Ca21] i changes expressed in arbitrary units (C) Effects of ODN, OP and cyclo 128 OP (1028M 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.
Trang 6for 10 min with the phospholipase C inhibitor U73122
(1025M) abrogated the effects of ODN, OP and cyclo128
OP (1028Meach) on [3H]IPs and [3H]PIPs (Fig 8E,F)
NMR solution structure of cyclo128OP
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
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 cyclo128OP 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
The3JNH-Hacoupling constants for the amide protons of cyclo128OP were measured from the 1D1H-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 (< 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 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
Fig 5 Effect of graded concentrations of cyclo 128 [ D -Leu5]OP on
[Ca21] i in cultured rat astrocytes A 2-s pulse (arrow) of cyclo 128
[ D -Leu5]OP (1021021025M ) 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 [Ca21] 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 (1028M ) 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
[Ca21] i in cultured rat astrocytes A 2-s pulse of OP (1021121025M )
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 1029M cyclo 128 OP
(arrows).
Trang 7Ramachandran diagramme Ten structures with the lowest
distance and dihedral constraint energies were selected,
providing a well-defined shape of the backbone foldings of
cyclo128OP (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
The1H-NMR spectrum of cyclo128[D-Leu5]OP in H2O at
280 K was assigned by using the same strategy as for
cyclo128OP 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 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 2 Amide protons that exchange with solvent were identified by dissolving cyclo128[D-Leu5]OP in D2O Due to fast H/D exchange all
Table 1.1H-NMR assignments,3J 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)
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) (1028M , 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.
Trang 8amide 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 cyclo128OP 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
D I S C U S S I O N
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 (1028M each)
in the absence (A, B) or presence (C, D) of cyclo 128 [ D -Leu5]OP
(1026 M ) In another set of experiments (E, F), the cells were
preincubated with U73122 (1025M ) 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 O recorded after 10, 75 and 255 min, respectively.
Trang 9on [Ca21]iin 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 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.
Trang 10head-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
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 cyclo128OP 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 cyclo128OP induced a bell-shaped 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]irise in rat astrocytes
As the dose–response curves obtained with ODN and OP are
Table 2.1H-NMR assignments,3J 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)
a
Cis conformer.bTrans conformer.
Fig 15 Lowest energy conformers of cyclo 128
[ D -Leu5]OP from simulated annealing (A) Cis conformer (B) Trans conformer.