Peptides 26 (2005) 1159–1166

Synthesis and biological studies of nociceptin derivatives containing theDTPA chelating group for further labeling with therapeutic radionuclides

Melinda Ligetib,1, Ozge Gunduza,1, Anna Magyarb, Erzsebet Katoc, Andras Z. Ronaic,Claudio Vitad, Imre Vargae, Ferenc Hudeczb,f , Geza Totha, Anna Borsodia, Sandor Benyhea,∗

a Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungaryb Research Group of Peptide Chemistry, E¨otvos University and Hungarian Academy of Sciences, Budapest, Hungary

c Group of Neuropsyhopharmacology, Department of Pharmacology, Semmelweis University, Budapest, Hungaryd Departement d’Ingenierie et d’Etudes des Proteines, CEA, Saclay, France

e Department of Inorganic and Analytical Chemistry, E¨otvos University, Budapest, Hungaryf Department of Organic Chemistry, E¨otvos University, Budapest, Hungary

Received 29 November 2004; received in revised form 25 January 2005; accepted 26 January 2005Available online 2 March 2005

r. Thishysiologi-ttempt too theicaled and pu-lysis.radiola-embrane

sidesient thaner to get a

thecald ofass

theysideptin13he

Abstract

Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the nociceptin (NOP) receptoneuropeptide–receptor system is involved in pain regulation, tolerance to and dependence on opiates as well as many other pcal and pathophysiological events. The role and mechanisms of nociceptin in pathological conditions is not clearly known yet. In an ahave a radiopharmaceutical labeled either with99mTc or111In, we incorporated diethylenetriaminepentaacetic acid (DTPA) as chelator intstructure of [Arg14,Lys15]nociceptin(1–17)-NH2 at the�-amino group of Lys15. Such a radiopeptide may be useful in imaging for diagnostpurposes. Preparation of the peptide ligands was carried out by solid phase synthesis. Two peptides containing DTPA were obtainrified. The products were [Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2 and its cross-linked dimer on the basis of mass spectrometric anaIn 115In3+ binding experiments the conjugates exhibited preserved indium ion chelating properties, indicating the potential use ofbeled DTPA-nociceptin derivatives as radiopharmaceutical. Biological properties of these compounds were studied in rat brain mpreparations by radioligand binding, functional biochemical [35S]GTP�S binding assays and mouse vas deferens (MVD) bioassay. Bethe similar in vitro binding characteristics to nociceptin receptor, both of the DTPA-chelated compounds were more potent and efficnociceptin in functional biochemical and mouse vas deferens bioassays. Our further aim is to radiolabel these compounds in ordradiopharmaceutical which can be used diagnostically.© 2005 Elsevier Inc. All rights reserved.

Keywords:Nociceptin; NOP receptor; Radiopharmaceutical; DTPA chelate; Radioligand binding; GTP�S assay; Mouse vas deferens bioassay

1. Introduction

Nociceptin, also called as orphanin FQ, is the endogenouspeptide agonist of the nociceptin (NOP) receptor[19,28].NOP receptor shares high homology with multiple opioidreceptors, mostly with the�-type, but it does not bind opioidligands. Nociceptin and NOP receptor are widely distributed

∗ Corresponding author. Tel.: +36 62 432 099; fax: +36 62 433 432.E-mail address:benyhe@nucleus.szbk.u-szeged.hu (S. Benyhe).

1 These authors have contributed equally to this work.

in the central and peripheral nervous system, as well asimmune system, and are involved in various physiologiand pathophysiological events. The peptide is compose17 amino acids and derived from its larger molecular mprecursor polypeptide, proorphanin/pronociceptin[24,21].Mature nociceptin heptadecapeptide mostly resembles tonatural�-opioid ligand dynorphin by size, partial homologand the presence of several positively charged amino acidchains. The core sequence and the C-terminus of nocicecontaining the basic Arg-Lys (RK) pairs at the 8–9 and 12–position are crucial for the electrostatic interactions with t

0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.peptides.2005.01.018

1160 M. Ligeti et al. / Peptides 26 (2005) 1159–1166

second extracellular loop of the NOP receptor. In order tobetter understand the receptor–ligand interactions and to findnew agonists and antagonist ligands, a number of structure-activity studies were performed. Okada et al. reported threesynthetic nociceptin analogues containing an additional Arg-Lys (RK) amino acid repeat adjacent to the original RK pairs[25], in order to strengthen the electrostatic interactions at thebinding pocket. This study led to a new nociceptin analogue,[Arg14,Lys15]nociceptin, which is more potent than the nativenociceptin. We applied [Arg14,Lys15]nociceptin(1–17)-NH2as a base molecule and coupled the chelating agent to theside chain epsilon (�) amino-group of Lys15 by amide bond.

Changes in the density of receptors during diseases can bemonitored by radioprobes which are labeled with therapeuticradionuclides for the peptide receptor radionuclide therapy(PRRT) [15]. Radioactive metal ions attached to smallmolecules, peptides, or proteins have been used in nuclearmedicine for diagnosis of cancer and for studying variousorgans[4]. In an attempt to prepare a radiolabeled nociceptinderivative that is applicable as an imaging, research, diagnos-tic, or therapeutic agent, we applied diethylenetriaminepen-taacetic acid (DTPA) as a bifunctional chelating moleculeto the Lys15 residue of [Arg14,Lys15]nociceptin(1–17)-NH2.Here we describe two new DTPA-containing peptide ana-logues: [Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2 and itscross-linked peptide dimer. Chelate conjugates were testedb vel-ot lt int ep-t dinga ranef thec iso-l t onlys lsom says.

2

2

( d[ asdo -i scri-b e( ituteL -t ris-h ma–A )-p e

(Boc-Phe-OH), N-Hydroxybenzotriazole (HOBt) andN,N′-dicyclohexylcarbodiimide (DCCI) were from NovaBiochem (Laufelfingen, Switzerland), DTPAA (Diethylen-etriaminepentaacetic acid dianhydride) was obtained fromSigma-Aldrich (Sigma–Aldrich Chimie, Lyon, France).PAL-PEG-PS resin was product of Perseptive Biosystems(PE Biosystems, France). All the solvents and other reagentsused in the synthesis and purification were from SDS(Peypin, France). InCl3 and 8-hydroxyquinoline werepurchased from Sigma–Aldrich (Budapest, Hungary).

2.2. Peptide synthesis

Peptide synthesis was performed by solid phase methodutilizing an Applied Biosystems 433A Peptide Synthe-sizer, using Fmoc-amino acid derivatives (except N-terminal Boc-Phe-OH), polyethylene glycol-polystyreneresin, equipped with a peptide amide linker (PAL-PEG-PSresin) and HOBt/DCCI-mediated coupling (FastMoc pro-tocol on 0.1 mmol scale; with amino acid derivatives sug-gested by the Synthesizer manufacturer). Amino acid sidechain blocking groups were the following:tert-butyl ether(tBu) for Ser, Thr; tert-butyloxycarbonyl (tBoc) for Lys;2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl (Pbf)for Arg; trityl (Trt) for Asn and Gln; orthogonal side chainblocking by 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)3-m

molom tica byt (at3 com-p ainaw hy-d ingD ta mt tingg 5%t ra-t atedae n dis-t fiedb ydac2 h a9 FAi rile( jorp uctsw ana-l lyti-c aphy( ide

y 115In-binding experiments according to the method deped by Nagy et al. with slight modifications[22]. We found

hat the coupling of DTPA to peptide chain did not resuhe loss of chelating ability of the DTPA moiety. These pide derivatives were further investigated in receptor binnd functional biochemical assays using crude memb

ractions from rat brain. Pharmacological properties ofompounds were also studied on electrically stimulated

ated mouse vas deferens. The new ligands displayed noimilar in vitro binding characteristics to nociceptin but aore potent agonistic properties were observed in bioas

. Materials and methods

.1. Chemicals

[3H]Nociceptin-NH2 (24.5 Ci/mmol), [3H]Naloxone28 Ci/mmol), [3H]Dynorphin-1–17 (40 Ci/mmol) an3H]Ile5,6deltorphin-II (28 Ci/mmol) were preparedescribed [3,23,34]. [3H]DAMGO (54.5 Ci/mmol) wasbtained from Amersham. Ac-RYYRIK-NH2 was synthes

zed and generously provided by Kocsis et al., as deed previously [16]. Guanosine-5′-[�-35S]-triphosphat1204 Ci/mmol) was purchased from the Isotope Insttd. (Budapest, Hungary). MgCl2, KCl, ethylenediamine

etraacetic acid (EDTA), polyethyleneimine (PEI), Tydroxymethyl-aminomethane were products of Sigldrich. All Fluorenylmethyl-oxycarbonyl (Fmocrotected amino acids,tert-butyloxycarbonyl-phenylalanin

ethyl-butyl (ivDde) for Lys15.The polypeptide chain was assembled by coupling 1 m

f Fmoc-amino acid derivatives (10-fold molar excess) inN-ethylpyrrolidone (NMP), followed by capping with acenhydride. Temporary Fmoc-protection was removed

reatments with 20% piperidine in NMP. UV monitoring01 nm) was used to add deprotection cycles until it waslete in case of difficult cleavage. After completion of chssembly, the ivDde protecting group of Lys15 side chainas specifically removed by three treatments with 2%razine in NMP for 3 min; DTPA was then incorporated usTPAA, N,N′-diisopropylcarbodiimide (DIPCI) and HOBs described previously[22]. The peptide was cleaved fro

he resin with simultaneous removal of side chain protecroups by treatment with 90% trifluoroacetic acid (TFA):

riisopropylsilane (TIS):5% water for 2 h at room tempeure. The resin was then filtered off, TFA was evapornd the residue was extracted with water/methyl-tert-butylther. The inorganic phase was evaporated, dissolved i

illed water and lyophilized. The crude product was puriy reverse-phase HPLC on a semi preparative C18 V18TP 1022 column, eluted at 10 ml/min flow rate wit0 min 0–40% linear gradient of 5% acetonitrile–0.1% T

n water (solvent A)/10% water–0.09% TFA in acetonitsolvent B) with detection at 214 nm. Fractions of maeaks were collected and lyophilized. Two major prodere found. Purified derivatives were characterized by

ytical HPLC and electrospray mass spectrometry. Anaal reverse-phase high performance liquid chromatogrRP-HPLC) separations were performed on Supelco W

M. Ligeti et al. / Peptides 26 (2005) 1159–1166 1161

Pore C18 column, eluted at 1 ml/min flow rate with a 30 min5–40% linear gradient, solvents and detection the same asdescribed above.

2.3. 115In3+ chelating of DTPA-peptide conjugates

We studied the115In3+ chelating properties of conjugatesby total-reflexion X-ray fluorescence (TXRF) spectroscopy.For preparation of115In3+-DTPA-peptide we applied the pro-cedure described by Nagy et al.[22]. Stock solution of conju-gate (3.5× 10−4 M) in 0.1 M sodium–acetate buffer, pH 5.6was prepared. InCl3 was dissolved in a minimal amount ofAcOH and diluted with 0.1 M sodium–acetate buffer, pH 5.6to achieve a final concentration of 7.0× 10−4 M. The solu-tion of DTPA-peptide conjugate was mixed with the stocksolution of InCl3 at various molar ratios (0.5–1.1) and stirredfor 2 h at RT. Uncoordinated metal ions were removed from115In3+-DTPA-peptide samples by extraction with equal vol-ume of 0.1 M 8-hydroxyquinoline dissolved in chloroform.After 1 h the aqueous phase was tested for chelated115In3+

content by TXRF.

2.4. Total-reflection X-ray fluorescence spectroscopy

TXRF was performed on an Extra II A (AtomikaGmbH, Munich, Germany) instrument using 2.0× 10−2 MC s ofs tan-d leanb t byc kV,3 d bya r at2 .T

2

v malH gary)f t thiss our,r andm ofs peanC theH rch(

2

istarrB ere-b e in

50 mM Tris–HCl buffer (pH 7.4) using a teflon–glass ho-mogenizer. The homogenate was centrifuged at 40,000×gfor 20 min at 4◦C and the resulting pellet was resuspended infresh buffer and incubated for 30 min at 37◦C. The centrifu-gation step was repeated, and the final pellet was resuspendedin 50 mM Tris–HCl buffer (pH 7.4) containing 0.32 M su-crose and stored at−70◦C until use. Before use membraneswere thawed, washed by centrifugation to remove sucroseand used immediately in the binding assays.

2.7. Receptor binding assay

All binding assays were performed in 50 mM Tris–HClbuffer (pH 7.4) in a final volume of 1 ml, containing0.2–0.4 mg/ml membrane protein. Rat brain membranes wereincubated with [3H]nociceptin(1–17)-NH2 in the presence ofunlabeled compounds (10−5 to 10−11 M). Up to 1 nM of radi-oligand was prepared in 1 mg/ml protease-free bovine serumalbumin solution (Protease-free BSA, Fraction V, Sigma).Reactions were terminated by rapid filtration under vacuum,and washed three times with 5 ml ice-cold 50 mM Tris–HCl(pH 7.4) buffer through Whatman GF/C glass fibers by us-ing Brandel M24R Cell Harvester. The filter fibers were pre-soaked 30 min in 0.3% polyethyleneimine (PEI) solution (pH10). After filtration filter fibers were dried and bound radioac-tivity was measured in UltimaGoldTM scintillation cocktailu er.P thodw -i ateda (ver-s erefi titionfiw n

2

ureda tb eiT ,p gc di l.T stedc thep mt tionw db lassfi old5 RC wasd ).

d(NO3)2 as an internal standard (Merck). Ten microlitreample solution mixed with the same volume of internal sard solution was dried directly on quartz plates in a cench at RT for 30 min. The excitation was carried ouontinuum radiation of a line focused tungsten tube (500 mA). The emitted fluorescence intensity was detecteSi(Li) detector coupled with a multichannel analyze

3.17 keV for Cd K� and 24.21 keV for In K�, respectivelyhe overall measuring period was 300 s.

.5. Animals

NMRI mice (from LATI, Godollo, Hungary) for the initro pharmacological tests and inbred Wistar rats (Aniouse of the Biological Research Center, Szeged, Hun

or the biochemical experiments were used throughoutudy. Mice and rats were kept in groups of eight and fespectively, allowed free access to food and wateraintained on a 12 h/12 h light/dark cycle until the time

acrifice. Animals were treated according to the Euroommunities Council Directives (86/609/ECC) andungarian Act for the Protection of Animals in Resea

XXVIII.tv. Section 32).

.6. Rat brain membrane preparations

Crude membrane fractions were prepared from Wat brains according to the method previously described[2].riefly, rats were decapitated and the brains without cellum were quickly removed and homogenized on ic

sing Packard Tricarb 2300TR Liquid Scintillation Analyzrotein concentration was measured by the Bradford meith bovine serum albumine as standard[6]. Receptor bind

ng experiments were performed in duplicates and repet least 3 times. Data were analyzed by GraphPad Prismion 3.0, San Diego, CA, USA). Displacement curves wtted by non-linear regression using the one-site competting option. The equilibrium inhibition constant (Ki value)as calculated according to the Cheng–Prusoff equatio[9].

.8. [35S]GTPγS binding assays

Receptor-mediated G-protein activation was meass described previously[32], with slight modifications. Rarain membrane fractions (≈10�g of protein/sample) wer

ncubated at 30◦C for 60 min in Tris–EGTA buffer (50 mMris–HCl, 1 mM EGTA, 3 mM MgCl2, 100 mM NaClH 7.4) containing [35S]GTP�S (0.05 nM) and increasinoncentrations (10−9 to 10−5 M) of the compounds testen the presence of 30�M GDP in a final volume of 1 motal binding was measured in the absence of the teompound, non-specific binding was determined inresence of 10�M unlabeled GTP�S and subtracted fro

otal binding to calculate the specific binding. The reacas started by addition of [35S]GTP�S and terminatey filtrating the samples through Whatman GF/B gber filters. Filters were washed three times with ice-c0 mM Tris–HCl buffer (pH 7.4) using Brandel M24ell Harvester, then dried, and bound radioactivityetected in UltimaGoldTM scintillation cocktail (Packard

1162 M. Ligeti et al. / Peptides 26 (2005) 1159–1166

Stimulation is given as percent of the specific [35S]GTP�Sbinding observed in the absence of receptor ligands (basalactivity). Data from triplicate experiments were analyzed bysigmoid dose–response curve fit option of Prism 3.0.

2.9. Mouse vas deferens (MVD) bioassay

Vasa deferentia taken from NMRI mice weighing35–40 g were prepared, mounted and field-stimulated asdescribed previously[16,25]. Nociceptin agonists weretested in non-cumulative manner at 4–6 concentrationlevels. Agonist potencies were characterized by the 50%inhibitory concentrations (IC50, i.e., x at y= 50%) cal-culated from the logarithmic regressions of individualconcentration–response curves. To the pooled data pointsnon-linear curve fitting was used according to the Hillequation (Sigmaplot 8.0). A synthetic hexapeptide NOPreceptor antagonist, Ac-RYYRIK-NH2 [10], was usedto inhibit effects exerted by the agonist peptides; 30 minpre-equilibration period was applied for the antagonist.

3. Results

3.1. Peptide synthesis and chelation with115In3+

Two novel nociceptin derivatives conjugated with thec acid

(DTPA) were prepared by solid phase peptide synthesis. Oneof the conjugates (Fig. 1B) contains one peptide (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln-NH2) and one DTPA moiety at side chain of Lys15.The other conjugate (Fig. 1C) possesses two peptide chainsand one DTPA moiety connected via -Lys15-DTPA-Lys15-bridge between�-amino groups. Structures were confirmedby electrospray mass spectrometry: calculated/found masses[M+ H]+ are 2283.2/2283.7m/z; 4172.3/4173.0m/z, respec-tively. The complex of DTPA-peptide conjugates with115In3+

were prepared according to Nagy et al. with minor modi-fications[22]. Complexed115In3+ content was determinedby TXRF spectroscopy. The115In-binding experimentsrevealed that the coupling of DTPA to peptide chain did notresult in the loss of chelating ability of the DTPA moiety. Wefound that the chelating efficiencies were more than 95%, inboth cases.

3.2. Receptor binding and affinity of nociceptin peptides

Equilibrium competition experiments were conductedon rat brain membranes with [3H]nociceptin-NH2. Thenewly synthesized DTPA-nociceptin analogues were ef-fective in displacing [3H]nociceptin-NH2 binding (Fig. 2,Table 1). Heterologous competition curves for each com-p -site

helating molecule diethylene-triaminepentaacetic

Fig. 1. Schematic structure of diethylenetriaminepentaa

ound were satisfactorily fitted according to the single

cetic acid (DTPA) and DTPA-fused nociceptin derivatives.

M. Ligeti et al. / Peptides 26 (2005) 1159–1166 1163

Table 1Equilibrium competition binding data of nociceptin and [Arg14,Lys15]nociceptin-DTPA conjugates in rat brain membranes

Peptide (no. of experiments) IC50 (nM) log IC50 ± SEM Ki (nM)

Nociceptin-OH (n= 7) FGGFTGARKSARKLANQ 2.8 −8.55± 0.09 2.2[Arg14,Lys(DTPA)15]Noc(1–17)-NH2 (n= 3) FGGFTGARKSARKRK(DTPA)NQ-NH2 1.3 −8.884± 0.09 1.0Cross-linked dimer (n= 3) (seeFig. 1for structure) 8.2 −8.088± 0.11 6.5

binding model. In the case of the dimer, experimen-tal data could also be analyzed by the two-site model,indicating that another binding component or differentaffinity states of the same receptor population might bepresent. Two-site fitting was not statistically better, there-fore only the one-site curve is drawn on the figure. Both[Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2 and the cross-linked dimer displayed high affinities withKi values of 1 nMfor [Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2 and 6.5 nMfor the cross-bridged derivative. DTPA-conjugated peptidesshowed very weak affinity toward opioid binding siteslabeled with the�-receptor selective [3H]Ile5,6deltorphin-II[23] or the �-specific [3H]dynorphin(1–17).�-Opioid re-ceptor affinities determined by [3H]DAMGO (0.4 nM)were considerably higher, exhibiting IC50 valuesof 90± 28 nM for the dimer, and 358± 73 nM for[Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2.

3.3. GTPγS binding

G-protein activation following the agonist occupation ofthe G-protein coupled receptor (GPCR) can be measuredby in vitro [35S]GTP�S binding assays[12]. In order toevaluate the functional activity of the peptides, [35S]GTP�Sbinding assays were performed in crude brain homogenates.P n)o se ofn lated

F -b d with0u ;( ntm n du-p

Fig. 3. Stimulation of [35S]GTP�S binding by various concentrations ofthe peptides. Stimulation is given as a percentage of the non-stimulated(basal) level. Basal activity (Basal = ‘Total binding’− ‘Non-specific bind-ing’) is taken as 100%. Non-specific binding is determined in the presenceof 10�M unlabeled GTP�S. Incubations were carried out for 60 min at30◦C. Points represent the means± SEM of at least three independent de-terminations each performed in triplicates. Symbols are: (©) nociceptin;(�) [Arg14,Lys(DTPA)15]Noc(1–17)-NH2; (�) dimer.

the [35S]GTP�S binding to rat brain membranes (Fig. 3).Experimental data of nociceptin and the monomer formof the conjugate were clearly fitted with monophasicsigmoid activation curves. However, results for the dimerpeptide was acceptably described only by fitting a sigmoidaldose–response curve with variable slope to the data points.The slope-factor or Hill-coefficient value was calculatedto be 0.45 in this case, and suggests again that morecomponents may be involved in the binding process andG-protein activation of the dimer. The maximal stimulationand the ED50 values are shown inTable 2. The efficacy of[Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2 was very simi-lar to the efficacy of nociceptin while the cross-linked dimerwas more efficient than nociceptin. [35S]GTP�S binding ex-periments with the dimer were also performed in membranesprepared from Chinese hamster ovary (CHO) cells stablyexpressing�-opioid receptors (not shown). However, thepeptide showed no stimulation up to 1�M concentration in

Table 2Stimulation of [35S]GTP�S binding by nociceptin and nociceptin-DTPAconjugates in rat brain membranes

Peptide Bindingstimulation(% of basal)

ED50 (nM)

Nociceptin-OH 173± 5 252± 59[C

otency (ED50 values) and efficacy (% maximal stimulatiof the analogues were measured and compared to thoociceptin. All three peptides dose-dependently stimu

ig. 2. Inhibition of [3H]nociceptin(1–17)-NH2 binding to rat brain memranes by nociceptin derivatives. Rat brain membranes were incubate.5 nM [3H]nociceptin(1–17)-NH2 in the presence of (10−12 to 10−5 M)nlabeled compounds for 30 min at 24◦C. Symbols are: (©) nociceptin�) [Arg14,Lys(DTPA)15]Noc(1–17)-NH2; (�) dimer. Points represeean± SEM of at least three different experiments each performed ilicate.

Arg14,Lys(DTPA)15]Noc(1–17)-NH2 177± 6 220± 129ross-linked dimer 253± 16 92± 41

1164 M. Ligeti et al. / Peptides 26 (2005) 1159–1166

Fig. 4. Effects of nociceptin derivatives on mouse vas deferens bioas-say. (A) The inhibitory effect of peptides in field-stimulated mousevas deferens. Points represent the mean, vertical lines the SEM of4–16 independent determinations. Non-linear curve fitting accordingto the Hill-equation was used. Symbols are: (�) nociceptin; (©)[Arg14,Lys(DTPA)15]Noc(1–17)-NH2; (�) cross-linked dimer. (B) The an-tagonism of agonist action by 10−6 M Ac-RYYRIK-NH 2 on mouse vas def-erens. The inhibitory concentration–response curves of nociceptin (©) and[Arg14,Lys(DTPA)15]Noc(1–17)-NH2 () before (open symbols) and after(dark symbols: (�) (�)) 30 min incubation with 10−6 M Ac-RYYRIK-NH 2.Non-linear curve fitting (Hill-equation) was applied to the pooled points of2–2 independent experiments.

�CHO-cell membranes. In light of the biochemical resultsboth [Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2 and thecross-linked dimer appear to be high affinity agonists for theNOP receptor.

3.4. Mouse vas deferens (MVD) bioassay

Nociceptin(1–17)-OH, [Arg14,Lys(DTPA)15]nociceptin-NH2 and the cross-linked dimer inhibited the electri-cally induced contractions of MVD in a concentration-dependent manner (Fig. 4A). The Hill-slope of[Arg14,Lys(DTPA)15]nociceptin-NH2 concentration–respo-nse curve was similar to that of nociceptin (nH were 1.18 and0,87, respectively) whereas the slope for the cross-linkedpeptide was slightly lower (nH 0,66). The onset and washout

Table 3IC50 values of nociceptin derivatives in field-stimulated mouse vas deferens

Agonist (no. of experiments) IC50 (nM)

Nociceptin-OH (16) 121± 14[Arg14,Lys(DTPA)15]Noc(1–17)-NH2 (4) 32.4± 4.6Cross-linked dimer (4) 37.3± 12.8

times were considerably longer in the case of dimeric analogas compared to the other two agonists. The IC50 values forthe agonists on the isolated organ preparation are given inTable 3. The concentration–response curves of nociceptin,[Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2 (Fig. 4B) andthe cross-linked dimer (not shown) were shifted to theright in a parallel fashion by a hexapeptide NOP receptorantagonist, Ac-RYYRIK-NH2 (10−6 M).

4. Discussion

Nuclear molecular imaging started to progress more withthe development of new radiopharmaceuticals especiallythe natural peptides labeled with radionuclides which aredirected towards detecting and monitoring the normal andphysiopathological processes[1]. Small peptide radiophar-maceuticals have advantages over monoclonal antibodieswhich are relatively large in size resulting in slow clearanceand suboptimal targeting of diseased tissue. Radiolabeledsomatostatin analogues targeted to somatostatin typereceptor (SSTR) are notable examples of peptide radiophar-maceutical imaging[4]. Technetium and Indium isotopes,such as99mTc, and111In can be used as radionuclides inimaging by Single Photon Emission Computed Tomography( y ofT rea orpo-rt tegi-c ct tor ndw A)w ups,u ther hasb mongt spitet ideb

pli-c brali hasb pato-c ofn ter-a cancerc phic

SPECT). Relatively low cost and widespread availabilitechnetium-99m makes99mTc-radiopharmaceuticals moccessible. Since transition metals cannot easily be incated into small-molecule receptor ligands,99mTc might berapped by chelation. Radiometal must be placed straally at a sterically tolerated part of the ligand, with respeeceptor binding[14]. Radioactive metal ions can be bouith a peptide via bifunctional chelating agents (BChich function as a cross-linker between amine grosually in a lysine residue, in the carrier molecule andadiometal. Diethylenetriaminepentaacetic acid (DTPA)een used extensively as a metal chelator and known a

he strongest chelators for a large number of metals, dehe loss of carboxyl group in the formation of the amond[13].

Beside many others, the role of nociceptin has been imated in neuropathic pain, inflammation, epilepsy, cereschemia and neurogenic bladder. Recently nociceptineen reported to be involved in Wilsons disease and heellular carcinoma[11,33]. The mechanisms and the roleociceptin in liver pathologies still need to be clarified. Altions of the receptor system in disease state such asan be exploited by novel imaging techniques. Scintigra

M. Ligeti et al. / Peptides 26 (2005) 1159–1166 1165

imaging is a powerful diagnostic tool in the management ofpatients with infectious or inflammatory diseases[5]. Noci-ceptin is also suggested to be involved in the physiopathologyof inflammation, cardiac or brain circulatory ischemia and ar-terial hypertension[18].

Following the discovery of the endogenous ligand noci-ceptin, there has been a tremendous effort to develop specificligands for understanding more clearly the pharmacologicaleffects. Until now several powerful agonists and antagonistcompounds[8,26] were identified by different groups.[Arg14,Lys15]nociceptin was introduced as one of the mostpotent synthetic nociceptin agonist[25]. In this study wesynthesized novel nociceptin analogues containing chelatinggroup in order to get a successful candidate compound forlabeling with a metal radionuclide. Incorporation of DTPAby amide bond requires a free amino group in the peptide. Innative nociceptin three free amino groups exist: N-terminalamino group of Phe1, and the two�-amino groups of Lys9

and Lys13. It is known that the positively charged N-terminalamino group is crucial for the binding presumably with theAsp residue of the third receptor domain[29]. The negativelycharged DTPA is likely not to be tolerated in the N-terminalof the peptide. Also Arg8-Lys9 and Arg12-Lys13 basicamino acid pairs are necessary for the tight interactions withthe second extracellular loop of the receptor. Since theseresidues are important in activating the receptor[7,27], wed otherA tc rmg LCpa at inD gentr

andb asd agent nityi edb re.T eric[b erensb s oft flatds nlyw ver,tb ionw of� thed ys ratb ease

in the efficacy (250% maximal stimulation over the basal) ofthe cross-linked dimer that substantially exceeded the levelof stimulation induced by native nociceptin or the DTPA-conjugate monomer peptide. Dimeric structure that makesthis compound as bifunctional peptide ligand might explainits atypical properties. Indeed, neither the [35S]GTP�S norMVD dose–response curves could be described with single-site models. The possibility of additional components in thebinding or at the site(s) of actions was reflected also by shal-low curves with low Hill coefficient values. Bivalent opioidpeptide ligands derived from enkephalins or dermorphin havebeen reported to possess decreased or increased affinities overthe monomer forms depending upon the chemical nature andsize of the interconnecting spacer structure[17,20,30,31].Bivalent ligands can also trigger receptor-receptor interac-tions[35] and are considered to have interesting possibilitiesin terms of increasing potency or enhanced activation of G-proteins. It is assumed that the above changes in the propertyof the DTPA-conjugated dimer nociceptin are not a spuriouseffect of the presence of the DTPA linker but rather mayrepresent an enhanced effect due to the bivalent nature of theligand.

In summary, [Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2and its covalently cross-linked dimer displayed higher orsimilar in vitro binding affinities comparing with noci-ceptin in [3H]nociceptin(1–17)-NH binding assays. Thesec latedr em-b atelyt thann Ther tag-o -s no-c ucha ec toolo ticalp

A

11( V.)f 04-D rcha en-t 01( B.a rcha n,H oupo nA ingp

ecided to use the nociceptin analog which contains anrg-Lys repetition on the 14–15 position[25]. As a parenompound, [Arg14,Lys15]nociceptin(1–17)-NH2 was furtheodified by incorporating DTPA as a BCA into�-aminoroup of Lys15. Two compounds were obtained by HPurification: [Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2nd a cross-linked dimer. We have demonstrated thTPA-peptide conjugates the bifunctional chelating a

etains its ability to chelate115In3+.The new compounds were examined by radiolig

inding assays, [35S]GTP�S binding tests and mouse veferens bioassays. Both analogs containing chelating

urned out to be very effective showing that the high affin [3H]nociceptin-NH2 binding experiments is preservy the incorporation of DTPA into the peptide structuhe highest affinity was obtained with the monom

Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2. This peptideehaved as pure agonist ligand in the mouse vas defioassay. Biochemical and pharmacological feature

he dimer are more complex, e.g., it showed ratherose–response curve in the bioassay. In the [35S]GTP�Stimulation experiments sigmoid fitting was fine oith the variable slope option of the analysis. Moreo

he dimer was capable of inhibiting�-opioid radioligandinding with moderate affinity indicating that interactith �-opiod sites may also occur. However, the lack-opioid receptor mediated G-protein stimulation byimer in�CHO cells supports that�-receptors did not plaignificant role in mediating the actions of this peptide. Inrain [35S]GTP�S assays we observed an unusual incr

t

2ompounds dose-dependently and effectively stimueceptor-mediated G-protein coupling in rat brain mranes. Both compounds turned out to be approxim

hree to four times more potent NOP receptor agonistsociceptin in the field-stimulated mouse vas deferens.eceptor specificity of agonism was proved by the annism by Ac-RYYRIK-NH2. In the light of results preented here it is our further aim to prepare radiolabelediceptin derivatives by chelating metal radionuclides, ss 99mTc or 111In. The availability and stability of theshelating metals would allow their use as researchr radiopharmaceuticals for diagnostical and therapeuurposes.

cknowledgements

This work was supported by grants OTKA T-0257S.B.), T-032736 (E.K.), T- 033078 (A.M.), T-047047 (I.rom the Hungarian Scientific Research Fund; RET-20NT (S.B., A.B. and G.T.) from National Office for Reseand Technology; ETT 262/2001 (A.Z.R.) from the Sci

ific Council of the Ministry of Welfare; Medichem 047/20F.H.) from the Ministry of Culture; NKFP 027/2001 (A.nd G.T.) and OMFB-00656/2003 (A.B.) from the Reseand Development Division of the Ministry of Educatioungary. We are grateful to L. Kocsis (Research Grf Peptide Chemistry, Eotvos University and Hungariacademy of Sciences, Budapest, Hungary) for provideptide Ac-RYYRIK-NH2.

1166 M. Ligeti et al. / Peptides 26 (2005) 1159–1166

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