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Selective and high affinity labeling of neuronal and recombinant nociceptinreceptors with the hexapeptide radioprobe [3H]Ac-RYYRIK-ol

Engin Bojnik a, Judit Farkas a, Anna Magyar b, Csaba Tomboly a, Umit Guclu a,Ozge Gunduz a,c, Anna Borsodi a, Maıthe Corbani d, Sandor Benyhe a,*a Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, 6726 Szeged, Temesvari krt 62, Hungaryb Research Group of Peptide Chemistry, Hungarian Academy of Sciences and Eotvos Lorand University, P.O. Box 32, 1518 Budapest, 112, Hungaryc Yeditepe University, Faculty of Pharmacy, 26 Agustos Campus, 34755 Istanbul, Turkeyd Institut de Genomique Fonctionnelle, UMR5203-CNRS, INSERM U661, Universite Montpellier I, Universite Montpellier II, Montpellier, France

1. Introduction

The neuropeptide nociceptin/orphanin FQ (N/OFQ) was simul-taneously identified as endogenous ligand for an orphan opioid-like receptor (Mollereau et al., 1994) by pioneer applications of thereverse pharmacology approach (Meunier et al., 1995; Reinscheid

et al., 1995). N/OFQ is a heptadecapeptide (FGGFTGARKSARK-LANQ) sharing some sequence similarities with the endogenouskappa-opioid receptor (KOPr) agonist peptide dynorphin A.Likewise, the highest primary sequence identity/homology ofthe NOP receptor are observed with the KOPr. A unique anddistinguishing difference between opioid peptides and N/OFQ isthat the N-terminal message motif is YGGF in the opioids(Schwyzer, 1986), while the corresponding N-terminal tetrapetidesegment in N/OFQ is composed of FGGF. This difference may besufficient to prevent N/OFQ binding to opioid receptors. Theaddress domain of N/OFQ is relatively long and involves centrallylocated positively charged amino acids (Tancredi et al., 2005). C-Terminal truncation studies have shown that the tridecapeptide

Neurochemistry International 55 (2009) 458–466

A R T I C L E I N F O

Article history:

Received 1 March 2009

Received in revised form 22 April 2009

Accepted 24 April 2009

Available online 3 May 2009

Keywords:

Orphanin FQ

Hexapetide ligand

Radiolabeling

Rat brain

CHO cells

Human NOP receptor

Binding assay

A B S T R A C T

The synthetic hexapeptide Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) represents a highly potent and

selective partial agonist ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (nociceptin

receptor, NOPr). Ac-RYYRIK-ol has been labeled with tritium yielding [3H]Ac-RYYRIK-ol with

exceptionally high specific radioactivity of 94 Ci/mmol. The radioprobe is chemically stable even at

24 8C in ethanol solution for at least 4 days. No significant decomposition of the [3H]ligand occurred

under the condition of the binding experiments indicating a fine enzymatic stability of the peptide.

Radioreceptor binding studies were conducted using native neuronal NOPr preparation of rat brain

membrane fractions and recombinant human nociceptin receptor (hNOPr) preparations from cultured

Chinese Hamster Ovary (CHO) cells stably expressing hNOPr. Specific binding of the compound was

reversible, saturable and of high affinity. No cross-reaction with the opioid receptors was observed

suggesting superior NOPr selectivity of the ligand. Monophasic isotherm curves obtained in radioligand

binding saturation and homologous displacement experiments indicated the presence of single binding

sites in both preparations. Average densities of the [3H]Ac-RYYRIK-ol recognition sites were 237 and

749 fmol/mg protein in rat brain and transfected cells, respectively. Equilibrium affinity values (Kds)

were determined by three independent way providing identical results. In rat brain membranes Kds of

0.3–1.3 nM were found depending upon the assay type. In homologous competition studies performed

on hNOP-CHO cell membranes almost the same binding affinities were measured for Ac-RYYRIK-ol either

with [3H]Ac-RYYRIK-ol (Ki 2.8 nM) or with [3H](Leu14)nociceptin (2.3 nM). A number of NOPr and opioid

ligands were screened in heterologous displacement experiments and displayed a rank order of affinity

profile being consistent with fairly good NOPr selectivity of the sites labeled by [3H]Ac-RYYRIK-ol. Taken

together, the high molar activity, improved chemical and biological stability and the capability of the

selective and high affinity labeling make this novel radioprobe available for further exploring the

biochemical pharmacology and receptor-ligand interaction of the NOP receptor.

� 2009 Elsevier Ltd. All rights reserved.

Abbreviations: N/OFQ, nociceptin/orphanin FQ receptor; NOP, nociceptin; Ac-

RYYRIK-ol, Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol.

* Corresponding author at: Institute of Biochemistry, Biological Research Centre,

Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary.

Tel.: +36 62 432 099; fax: +36 62 433 432.

E-mail address: benyhe@brc.hu (S. Benyhe).

Contents lists available at ScienceDirect

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journa l homepage: www.e lsev ier .com/ locate /neuint

0197-0186/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2009.04.014

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N/OFQ-1–13 still carries the activity of the full-size nociceptin(Butour et al., 1997). N/OFQ and its receptor are involved in a widerange of physiological functions both in the central and peripheralnervous system, the cardiovascular system, the airways, thegastrointestinal and the urogenital tracts, as well as in the immunesystem (Lambert, 2008).

Specific ligands acting at the NOP receptor were primarilysynthetic peptide analogues (Zaveri, 2003), though increasingnumbers of non-peptide ligands have also been reported (Zaveriet al., 2005). Among N/OFQ structural analogues UFP-102[(pF)Phe4, Arg14, Lys15]-N/OFQ-NH2 (Carra et al., 2005a) andUFP-101 [NPhe1, Arg14, Lys15]-N/OFQ-NH2 (Calo’ et al., 2002)represent highly potent and selective agonist and antagonistpeptides, respectively. Non-peptide NOPr agonists triazaspirode-canone Ro 64–6198 (Wichmann et al., 2000), dihydrospiro[iso-quinoline-4(3H),40-piperidin]-3-ones (Mustazza et al., 2008) and aseries of hexahydro-pyrrolopyrroles are among others (Kolczewskiet al., 2003). The first non-peptide NOPr antagonist to be reportedwas J-113397 belonging to benzimidazolinones (Kawamoto et al.,1999). Among other non-peptide antagonists phenylpiperidines(Zaratin et al., 2004), naloxone-benzoylhydrazone (Noda et al.,1998) and a 4-aminoquinoline derivative JTC-801 can be men-tioned (Shinkai et al., 2000). Latter is the only small-molecule NOPrantagonist that has demonstrated analgesic activity in vivo.However, it has been difficult to identify any general structuralelements common to all of these heterocyclic organic molecules.

In the last decade a novel group of short artificial peptidesequences structurally unrelated to the N/OFQ peptide emerged asselective ligands targeting the NOP receptor. Dooley et al. (1997)reported the identification of acetylated hexapeptides withsubnanomolar binding affinity for the NOPr, using a syntheticcombinatorial library approach. These peptides with the generalformula of Ac-RYY-R/K-W/I-R/K-NH2 display only minimal struc-tural similarity to N/OFQ. Many of them turned out to be partialagonists at the NOP receptor in [35S]GTPgS binding- and cAMPaccumulation experiments and in mouse vas deferens bioassay. Thepeptides are positively charged, which could enable them to bindto the negatively charged second extracellular loop thought to be alikely binding site for N/OFQ. One of the lead compounds withinthe series is Ac-RYYRIK-NH2 exhibiting agonistic and antagonisticproperties in various assays (Berger et al., 2000). Ac-RYYRIK-NH2

and Ac-RYYRWK-NH2 have been templates for many otheranalogues in this family (Thomsen et al., 2000; Kawano et al.,2002; Judd et al., 2004; Carra et al., 2005b; Ambo et al., 2007; Liet al., 2008).

Recently a close derivative with a reduced C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) has been described by ourgroup as a partial agonist at the NOP receptors using various in vitro

and in vivo studies (Kocsis et al., 2004; Gunduz et al., 2006, 2006/2007). Being a partial agonist with intrinsic antagonist potency,this hexapeptide alcohol is capable of inhibiting competitivelysome effects mediated by full agonists at the NOP receptor. In thepresent study the effective radiolabeling and detailed receptorbinding properties of [3H]Ac-RYYRIK-ol to native and recombinantNOP receptors is described.

2. Materials and methods

2.1. Chemicals

[Leucyl-3H]Nociceptin (TRK1047; specific activity 162 Ci/mmol were purchased

from Amersham, U.K. [Phenylalanyl-3H]Nociceptin-amide (25 Ci/mmol) was

synthesized in our BRC Radiolab, Szeged, Hungary (Wollemann et al., 2008).

Polyethyleneimine (PEI) and bovine serum albumin (Protease-free BSA, fraction V)

were products of Sigma–Aldrich (Budapest, Hungary). N/OFQ used in this study was

purchased from Bachem Feinbiochimica, Bubendorf, Switzerland. Hexapeptide

analogues were prepared as previously reported (Gunduz et al., 2006/2007) and

purified by semi-preparative HPLC methods. Their structures were confirmed by

mass spectrometry. The NOP receptor antagonists [Nphe1, Arg14, Lys15]nociceptin-

NH2 (UFP-101) and (1-[(3R, 4R)-1-cyclooctylmethyl-3-hydroxy-methyl-4-piper-

idyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; J-113397) were kindly pro-

vided by Dr. Girolamo Calo’ (Ferrara, Italy). Naloxone hydrochloride was kindly

provided by Du Pont de Nemours and buprenorphine was obtained from ICN-

Alkaloida Inc. (Tiszavasvari, Hungary).

2.2. [3H]Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol

The peptide alcohol, Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) was synthe-

sized as described (Kocsis et al., 2004), that was dissolved in 50 mM Tris–HCl buffer

(pH 7.2) and it was iodinated with sodium iodide in the presence of chloramine T.

The mixture of the multi-iodinated peptides was purified by HPLC, and their

structures were verified by mass spectrometry. The iodohexapeptides was

dissolved in dimethylformamide containing 4 equiv. triethylamine, followed by

the addition of the catalyst Pd/BaSO4 (10% Pd). The catalytic reduction was

performed at ambient temperature for 60 min with tritium gas (Technobexpo,

Russia). Then the crude labeled peptide was purified by HPLC (Merck) on an Alltima

C18 column resulting in the radioligand with a radiochemical purity of 98% and with

a specific activity of 3.48 TBq/mmol (94 Ci/mmol).

2.3. Animals

Inbred Wistar rats (250–300 g body weight) were housed in the local animal

house of the Biological Research Center (BRC, Szeged, Hungary). Rats were kept in

groups of four, allowed free access to standard food and tap water and maintained

on a 12:12-h light/dark cycle until the time of killing. Animals were handled

according to the European Communities Council Directives (86/609/ECC) and the

Hungarian Act for the Protection of Animals in Research (XXVIII.tv. 32.§).

Accordingly, the number of animals and their suffering were minimized.

2.4. Cell membrane preparations for ligand binding studies

Membranes from rat brain and Chinese Hamster Ovary (CHO) cells stably

expressing the wild-type human nociceptin (hNOP) receptor were used. Crude

membrane fractions from Wistar rat forebrains were prepared as described (Bojnik

et al., 2009) and kept in frozen aliquots at �80 8C. hNOP-CHO cell cultures were

maintained in standard sterile tissue culture laboratory. Cells were cultured in a

medium containing Nut Mix F-12 (HAM) with L-glutamine (GIBCO-Invitrogen) and

25 mM Hepes, 10% fetal calf serum, 100 UI/ml penicillin, 100 mg/ml streptomycin,

and 0.4 mg/ml G418 at 37 8C in a humidified atmosphere consisting of 5% CO2 and

95% air. Cells were sub-cultured in every third day. Cells were harvested with

trypsin versen (0.05% trypsin, 0.02% EDTA) in phosphate buffered saline by washing

two times and then centrifuged at 3000 rpm (Sorvall RC5C centrifuge, SS34 rotor)

and homogenized in TEM buffer (50 mM Tris, 1 mM EGTA, 5 mM MgCl2, pH 7.4)

with a glass/glass hand homogenizer (Wheaton, USA). Homogenates were

centrifuged at 18,000 rpm for 20 min at 4 8C. Pellets were suspended in TEM pH

7.4 buffer for radioligand binding assays and stored in aliquots at �80 8C until use.

2.5. Receptor binding assays

All binding assays were performed at 24 8C for 45 min in 50 mM Tris–HCl buffer

(pH 7.4) in a final volume of 1 ml, containing 1 mg BSA and 0.2–0.4 mg/ml (rat

brain) or 0.02 mg/ml (CHO cells) membrane protein. Samples were made in

disposable plastic reaction tubes (diameter: 0.7 cm, volume 10 ml; Sarstedt Co.,

Numbrecht, Germany). The time course of association was measured by incubating

[3H]Ac-RYYRIK-ol with the membrane fractions for the indicated times. In

dissociation experiments the radioligand was preincubated with the membranes

until equilibrium achieved, and the dissociation of the receptor ligand complex was

initiated by the addition of unlabeled Ac-RYYRIK-ol, and the dissociation of the

radioligand was subsequently assessed for 45 min. Saturation binding experiments

were performed with 0.05–7 nM [3H]Ac-RYYRIK-ol in rat brain membranes. Non-

specific binding was determined in the presence of 10 mM unlabeled Ac-RYYRIK-ol.

Reaction was terminated and bound and free radioligands were separated by rapid

filtration under vacuum through Whatman GF/C (pre-soaked with the PEI for

30 min before washing) glass fiber filters by using Brandel M24R Cell Harvester.

Filters were washed three times with 5 ml ice-cold Tris–HCl buffer (50 mM, pH 7.4).

Dried filter disks were inserted into UltimaGoldTM scintillation fluid (Packard) then

rapidly removed and placed into individual counting vials (transparent glass,

Packard). Bound radioactivity was determined in a Packard Tricarb 2300TR Liquid

Scintillation counter.

2.6. Data analysis

Experimental data were analyzed and graphically processed by GraphPad Prism

software package for research (version 4.00 for Windows, GraphPad Software, San

Diego, California, USA, www.graphpad.com). Reversible binding kinetics was

analyzed on the basis of the following equation:

½�L�f þ ½R�u @kon

koff

½�LR�b; (I)

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where [*L]f is the free radioligand concentration, [R]u is the concentration of the

membrane-bound unoccupied receptor protein, [*LR]b is the concentration of the

bound ligand-receptor complex, while kon and koff are the association and

dissociation rate constants, respectively. Based on Eq. (I) the formation of the

binary complex follows bimolecular association defined by the second-order rate

constant kon. Considering the high excess of the ligand concentration over the

receptor molar concentrations and that [*L]f remains practically unchanged, the

association kinetics can be simply described by first-order reaction providing a

pseudo-first-order association rate constant. Real rate is thereafter calculated by

the equation:

kon ¼kobs � koff

½�L�f(II)

where kobs is the observed pseudo-first-order rate constant. The association rate

constant kon was therefore determined by plotting the individual kobs values as a

function of [*L]f in repeated association experiments performed at various

radioligand concentrations. The slope of the line obtained by linear regression

gives kon, while the intercept on the ordinate (Y-axis) corresponds to koff.

Alternatively, the koff values were also calculated by directly fitting one-phase

exponential dissociation curves in dissociation experiments.

In kinetic studies the dissociation constant Kd for the complex is calculated by

ratio of koff to kon:

Kd ¼koff

kon(III)

In equilibrium binding experiments Kds were determined by non-linear

regression analysis of the saturation curves. Note that the equation below is a

rearrangement of Eq. (I):

Kd ¼½R�u½

�L�f½R�L� (I0)

Heterologous competition curves were analyzed by non-linear regression using

the one-site competition option of GraphPad Prism with no ligand depletion.

Sigmoid fitting resulted in log IC50, or pIC50 values with the respective standard

errors. Log IC50 can be converted into IC50 value that is the concentration of the

competing ligand at 50% inhibition of the radioligand specific binding. Whereas the

IC50 value for a compound may vary between experiments depending on the

radioligand concentration, the equilibrium inhibition constant (Ki) is an indepen-

dent value. Ki values were evaluated according to the Cheng–Prusoff equation

(Cheng and Prusoff, 1973):

K i ¼IC50

1þ ð½�L�=KdÞ(IV)

Homologous competition is defined when the radioligand being displaced by its

own unlabeled form. In such assays Ki = Kd and the value can be calculated by

subtracting the radioligand concentration from the IC50:

K i ¼ Kd ¼ IC50 � ½�L�f (V)

3. Results

3.1. Molar activity, purity and stability of [3H]Ac-RYYRIK-ol

Iodination of the Ac-RYYRIK-ol resulted in a mixture ofiodinated peptides containing 3–4 iodine atoms per molecule inaverage. All species of this iodinated peptide mixture yielded thesame peptide in the catalytic dehalotritiation reaction. The averagespecific activity of the [3H]Ac-RYYRIK-ol isotopomer mixture wasfound to be 3.48 TBq/mmol (94 Ci/mmol) that was consistent withthe content of 3–4 tritium atoms per peptide molecule in average.Fig. 1A exhibits the radio-HPLC chromatogram of the purifiedradioligand indicating that the radiochemical purity of the tritiatedpeptide is >98%. No radiolysis of the labeled ligand was observedwhen the ethanolic solution of the ligand with radioactivityconcentration of 37 MBq/ml (1 mCi/ml) was incubated at roomtemperature for 4 days (data are not shown). Further, theenzymatic stability of [3H]Ac-RYYRIK-ol was studied in a rat brainmembrane preparation. An amount of 0.18 MBq (5 mCi) of [3H]Ac-RYYRIK-ol was incubated with 1 ml of the diluted membranepreparation (0.3 mg/ml protein) in the absence of peptidaseinhibitors at 24 8C for 60 min. The proteolytic reaction wasterminated with the addition of glacial acetic acid followed by acentrifugation at 15,000 rpm (Eppendorf). The supernatant was

analyzed by a radio-HPLC equipped with an on-line radioactivitydetector (Packard 500TR). It revealed that 23% of the [3H]Ac-RYYRIK-ol was degraded during the incubation (Fig. 1B).

3.2. Kinetics of [3H]Ac-RYYRIK-ol binding

All kinetic experiments were conducted at 24 8C using rat brainmembrane preparations. The association of the ligand-receptorcomplex occurred rapidly and the steady-state level of specificbinding was achieved in 10–12 min (Fig. 2C). Initial bindingassociation was described by a half-life time of 1.43 min (95% CI1.13–1.94). Non-specific binding, determined in the presence of10 mM unlabeled Ac-RYYRIK-ol displayed no changes with theincubation time and was therefore done by horizontal straightline (not shown). Ligand-concentration dependence of theassociation is illustrated at three different radioligand concen-tration (Fig. 2A). Kinetic curves were fitted by non-linearregression analysis using the ‘one-phase association’ option ofPrism 4.0 providing pseudo-first-order rate constant (kobs) values.Replotting of individual kobs values as a function of ligandconcentration resulted in a straight line (Fig. 2B). A slope factorrepresenting kon of 0.210 min�1 nM�1, and Y-axis intercept of0.498 min�1 that equal to koff were obtained by linear regression(goodness of fit, R2 = 0.99). The dissociation rate constant, koff, wasalso calculated by analyzing dissociation curves. Dissociation ofthe receptor-ligand complex follows first-order kinetics and canbe described with decreasing one-phase exponential curve.Dissociation was experimentally initiated by the addition ofunlabeled Ac-RYYRIK-ol (10 mM final concentration) in equili-brium conditions (Fig. 2D). A half-life time of 6.9 min (95% CI 5.60–8.97) for the binary complex was calculated. Dissociationconstant, Kd from kinetic experiments was calculated as ratio ofkoff to kon (see Eq. (III) in Section 2). Rate constant and dissociationconstant values are summarized in Table 1.

3.3. Equilibrium binding assays

Based upon kinetic experiments 30 min incubation time waschosen for all equilibrium binding studies. Ligand concentration

Fig. 1. Radio-HPLC chromatograms of [3H]Ac-RYYRIK-ol. A Jasco Lichrosphere 218

TP54 column was applied with a linear gradient of acetonitrile in 0.1% aqueous

trifluoroacetic acid from 5% to 45% over 40 min at a flow rate of 1 ml/min. (A)

Radiochromatogram of the radioligand alone. Pure [3H]Ac-RYYRIK-ol was identified

as single peak at the retention time of 10.2 min. (B) Radiolysis of the labeled

compound after incubation with brain membranes at 24 8C for 60 min.

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dependence of specific binding, also called as ‘hot ligandsaturation’ was measured in rat brain membranes at 24 8C.Binding isotherms of a representative assay reveal curvilinearplot for the total- and linear plot for the non-specific binding,respectively (Fig. 3A). Total to non-specific binding ratios were 3.8at 0.4 nM and 1.9 at 4.0 nM free radioligand concentrationindicating increasing proportion of the non-specific binding athigher ligand concentrations. Bound to free radioactivity percen-tage was typically less than 4% under the condition used (50 mMTris–HCl buffer at pH 7.4, containing 1 mg/ml BSA), indicating thatno significant ligand depletion had occurred. Specific binding of[3H]Ac-RYYRIK-ol, defined as the difference of the total and non-specific binding values, was saturable and described by arectangular hyperbola (Fig. 3B). Non-linear regression analysisof specific binding data reveals interactions with a single set ofhomogenous binding sites (n = 7, R2 = 0.98). Consequently, a linearScatchard plot was obtained (Fig. 3B, inset). The equilibriumdissociation constant, Kd was 0.35 nM, while the maximal numberof the binding sites (Bmax) was 237 fmol/mg protein in rat brainmembranes.

Binding affinity and capacity values were also estimated byequilibrium homologous competition experiments using [3H]Ac-RYYRIK-ol at given (‘tracer’) concentration and serial dilutions ofunlabeled Ac-RYYRIK-ol as competing ligand (Table 2, curves areshown in Fig. 5). In theory, ‘hot’ saturation and ‘cold’ competitionexperiments differ mainly in the way how proportion of the boundradioligand is determined (Munson and Rodbard, 1980). Non-linear regression analysis of the sigmoid competition curves alsoindicated the presence of one binding site in rat brain membranes(n = 8, R2 = 0.97). Accordingly, the average Hill slope of the curveswas not far from the unity, being 0.85 � 0.12 (mean � S.D.). Inhomologous displacement studies an affinity (Kd = Ki) of 1.3 nM, andreceptor density (Bmax) of 266 fmol/mg protein values werecalculated. Thus, nearly identical receptor capacities and quitematching affinities were obtained in kinetic, saturation and homo-logous displacement experiments (Tables 1 and 2).

[3H]Ac-RYYRIK-ol binding was also studied in cultured ChineseHamster Ovary cells stably transfected with recombinant humanNOP receptor. Equilibrium saturation and competition assaysconfirmed the presence of single high affinity binding sites. Theapparent Kd of [3H]Ac-RYYRIK-ol in saturation binding assays(n = 3, R2 = 0.84) was 0.44 nM, and the maximal number (Bmax) ofthe hexapeptide binding sites was found to be 749 fmol/mgprotein in hNOP-CHO cell membranes (Table 2). Competitionstudies were performed with [3H]Ac-RYYRIK-ol and [3H]N/OFQ(1–

17) (Amersham) permitting comparisons for the NOPr sites labeledby the hexapeptide and heptadecapeptide radioligands. Results ofthe cross- and self-displacement studies (‘competition tetradanalyses’) are shown in Fig. 4. Hill coefficient values (slope factor,nH) for [3H]Ac-RYYRIK-ol competition binding data were neitherdiffering from one another, nor altered from the unity (Table 2),whereas pIC50 values for Ac-RYYRIK-ol (8.31 � 0.07) and N/OFQ(8.06 � 0.08) were statistically different (P = 0.028, F-test compar-ison, Fig. 4A). Thus, the equivalent Ki value was 2.81 nM for the

Fig. 2. Kinetic studies with [3H]Ac-RYYRIK-ol in rat brain membranes. (A) Rates of association in the presence of various concentrations of [3H]Ac-RYYRIK-ol at 24 8C. Pseudo-

first-order association rate constant values (kobs) were determined by non-linear regression analysis selecting the one-phase association model. (B) Determination of the real

association rate constant (kon, slope) and the dissociation rate constant (koff, Y-intercept) by plotting kobs values as a function of free radioligand concentration. (C) Association

experiments (n = 3) in the presence of 0.5 nM radioligand. Regression coefficient, R2 = 0.81. (D) Dissociation experiments at 24 8C (n = 3) initiated by the addition of excess

unlabeled hexapeptide (10 mM). Only specific binding data are shown (R2 = 0.98).

Table 1Kinetic constants of [3H]Ac-RYYRIK-ol in rat brain membranes.

Parameter Value (�S.E.) Remark

Observed pseudo-first-order

association rate constant,

kobsa (min�1)

0.521 � 0.07 [*L]f: 0.10 nM

0.585 � 0.08 [*L]f: 0.44 nM

0.644 � 0.18 [*L]f: 0.68 nM

Association rate

constant, kon (nM�1 min�1)

0.21 � 0.02 Slope in Fig. 2B

Dissociation rate

constant, koff (min�1)

0.498 � 0.008 Y-intercept in Fig. 2B

0.100 � 0.011 From dissociation expts.

‘Kinetic’ dissociation

constant, Kd (nM)

0.5–2.3 koff/kon

a kobs varies with the concentration of the radioligand: [*L]f.

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hexapeptide, whereas N/OFQ displayed around two times loweraffinity (Ki 4.96 nM) in [3H]Ac-RYYRIK-ol displacement experiments.The maximal number of the binding sites measured by homologouscompetition was 1116 fmol/mg protein in hNOP-CHO cell membranes(Table 2). In [3H]N/OFQ(1–17) competition assays homologous andheterologous pIC50 values were not statistically different (P = 0.44, F-test) making the respective curves apparently indistinguishable(Fig. 4B). pIC50 was 8.47 � 0.12 for N/OFQ (n = 5, R2 = 0.87), while8.69 � 0.13 for unlabeled Ac-RYYRIK-ol (n = 4, R2 = 0.88) indicatingthat the affinities of the competitors were similarly high as measuredwith the full-size radioligand. Again, both Hill coefficients were closeto the unity displaying nH values of 0.91 � 0.20 and 0.92 � 0.21 forhomologous and heterologous competition, respectively.

3.4. Heterologous competition experiments

Competition assays were performed using various NOPreceptor related ligands such as the full-size nociceptin (N/OFQ(1–17)), Tyr1-N/OFQ(1–17), the NOP receptor antagonist peptideUFP-101, the non-peptide antagonist J-113397, several acetyl-hexapeptides, e.g., Ac-RYYRIK-ol, Ac-RYYRIK-NH2 (parent com-pound, or Dooley’s-peptide), the Arg6-derivative Ac-RYYRIR-ol, thecitrulline-substituted (Cit4) analogues Ac-RYYCitIK-NH2 and Ac-RYYCitIK-ol (Gunduz et al., 2006/2007). Some opioid compoundsincluding the general opioid receptor antagonist naloxone, thekappa-opioid (KOPr) agonist/mu-opioid (MOPr) antagonist bupre-norphine, naloxone-benzoylhydrazone, an antagonist compoundknown to interact with both KOPr and NOPr, the MOP receptorselective peptide agonist [D-Ala2, NMePhe4, Gly5-ol]enkephalin(DAMGO) and the KOPr agonist peptide D-Ala3-dynorphin(1–11)

were also studied to determine their displacing capability in ratbrain membranes. All NOPr ligands efficiently displaced [3H]Ac-RYYRIK-ol binding with varying affinities, in concentrationdependent manner (Fig. 5 Left Panel). Opioid compounds exhibitedless potency in competing reversibly for the [3H]Ac-RYYRIK-ol

recognition sites, although moderate affinities were obtained withbuprenorphine, naloxone-benzoylhydrazone and D-Ala3-dynor-phin(1–11) (Fig. 5 Right Panel). Rank order of the affinity was:Ac-RYYRIK-ol > N/OFQ > Tyr1-N/OFQ(1–17) > Ac-RYYRIK-NH2 >

Ac-RYYRIR-ol > UFP-101 > Ac-RYYCitIK-ol > Ac-RYYCitIK-NH2 >

J-113397 > naloxone-benzoylhydrazone > buprenorphine >D-Ala3-dynorphin(1–11) > I2Tyr1-N/OFQ(1–17)-NH2 > naloxone andDAMGO. The latter two opioids were unable to produce even20% inhibition of the specific [3H]Ac-RYYRIK-ol binding. Theequilibrium inhibition constant (Ki) values are summarized inTable 3.

4. Discussion

The synthesis and in vitro characterization of a novelradiolabeled hexapeptide analogue, selective for the nociceptin/orphanin FQ receptor has been described. The amino acid sequenceof the peptide Ac-RYYRIK-ol is unrelated to that of the endogenousligand N/OFQ (Meunier et al., 1995; Reinscheid et al., 1995),although it contains three basic amino acids – Arg1, Arg4 and Lys6.Thus, the basicity and the possible ionic interactions with thereceptor protein are somehow similar to the core region of N/OFQ(. . .RKSARK. . .) where four basic amino acids, the two Arg-Lysrepeats are present. The sequence motif for the radioligand hasbeen found by screening combinatorial peptide libraries designedfor NOPr interacting oligopeptides (Dooley et al., 1997). One of thereported N

a-acetyl-hexapeptides, Ac-RYYRIK-NH2 was chosen for

additional modifications (Gunduz et al., 2006/2007). Reducing itsC-terminal carboxamide group to a primary alcohol resulted in thepartial agonist Ac-RYYRIK-ol with improved biochemical stability(Kocsis et al., 2004; Gunduz et al., 2006). The same reductivemodification exists in the structure of the MOPr selective agonist[D-Ala2, NMePhe4, Gly5-ol]enkephalin (DAMGO) bearing a glycinolresidue at the C-terminus. Another example within the opioidpeptide family is the synthetic tetrapeptide analogue endomor-phin-1-ol (YPWP-ol; Tyr-Pro-Trp-Pro-ol) exhibiting enhancedefficacy in the mouse vas deferens bioassay (Al-Khrasani et al.,2001).

Radioligands are essential tools in the G-protein coupledreceptor (GPCR) research, especially the 3H and 125I labeledligands. Both radionuclides have certain advantages and draw-backs. The g emission of the 125I makes it detectable at extremelylow concentrations, and its short half-life results in ligands withhigh specific radioactivities. This latter issue also affects theavailability of these ligands, which are generally freshly preparedradioligands. The most important factor that restricts theapplication of radio-iodinated ligands is the steric effect of theiodine atom(s) incorporated. Such bulky substituents can

Fig. 3. Saturation analysis of [3H]Ac-RYYRIK-ol binding in rat brain membranes at 24 8C. Samples were incubated with increasing concentrations of [3H]Ac-RYYRIK-ol in the

absence (‘total binding’) or the presence of 10 mM Ac-RYYRIK-ol (‘non-specific’ binding). Real concentrations of the radioligand were determined by counting the

radioactivity of aliquot drops directly taken from the radioligand dilution series. (A) Isotherms for total and non-specific binding in a single experiment performed in

triplicate. (B) Concentration dependence of [3H]Ac-RYYRIK-ol specific binding. Points represent the means � S.E.M. of six independent experiments each performed in triplicate.

The corresponding Scatchard plot is given as inset.

Table 2Equilibrium binding parameters of [3H]Ac-RYYRIK-ol.

Preparation, assay typea Affinity,

Kd (nM)

Hill

coefficient

Capacity, Bmax

(fmol/mg)

Rat brain membranes

Radioligand saturation (7) 0.35 � 0.02 – 237 � 11

Homologous competition (8) 1.3 � 0.3 �0.85 266 � 24

hNOP-CHO cell membranes

Radioligand saturation (4) 0.44 � 0.06 – 749 � 46

Homologous competition (6) 2.8 � 0.7 �1.07 1116 � 407

a Number of experiments are given in parenthesis.

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adversely interfere with important amino acid side chains in thebinding pocket involved in the docking process. In contrast, tritiumlabeling results the same chemical species with almost the samechemical properties as the unlabeled ligand. Furthermore, hydro-gen is the most abundant element in organic compounds, althoughits abundance by mass is only 10% within the human body. Thegentle b-emission of 3H decay is relatively safe (5.7 keV, e�) andbeing detected by liquid scintillation techniques. The longer half-life time of tritium results in lower specific radioactivity.Incorporation of one 3H atom per molecule provides 1.03 TBq/mmol (28 Ci/mmol) molar activity, permitting the detection offemto- to picomolar level of ligand-receptor complexes. The more

number of 3H nuclide in the molecule the higher specificradioactivity of the compound. For these reasons tritium labelingof neuropeptides has been long tradition, and still being areasonable method to obtain labeled compounds (Toth et al.,1997).

Binding characteristics and anatomical localization of the NOPreceptors has been studied by using a set of radioligands (reviewedby Dooley and Houghten, 2000). Since the natural N/OFQ sequencedoes not contain tyrosine side chain that is typical target for bothiodination and tritiation, other labeling approaches were preferred,such as introducing diiodo-tyrosine into positions known to be notcritical for the specific recognition. Among these Tyr-substituted

Fig. 4. Cross- and self-displacement studies with two radioligands in Chinese Hamster Ovary cells expressing recombinant human nociceptin receptor (hNOP-CHO). (A)

[3H]Ac-RYYRIK-ol binding (0.2–0.3 nM, 30 min, 24 8C). (B) [3H](Leu14)N/OFQ(1–17) (nociceptin) binding (0.05–0.15 nM, 30 min, 24 8C).

Fig. 5. Equilibrium competition binding of various nociceptin (Left Panel) and opioid (Right Panel) ligands at the [3H]Ac-RYYRIK-ol sites in rat brain membranes. [3H]Ac-

RYYRIK-ol (0.2–0.4 nM) was incubated in the presence of increasing concentrations of homologous or heterologous competitors, compound names are indicated in the figure.

Points represent the means � S.E.M. of at least three different experiments each performed in duplicate. Homologous competition curves are shown in both panels for comparison.

Table 3Competition of [3H]Ac-RYYRIK-ol binding in rat brain membranes.

Compound Property [3H]Ac-RYYRIK-ol [3H]N/OFQ-NH2

pIC50 � S.E.M Ki (nM)a Ki (nM)

N/OFQ(1–17) NOPr agonist (endogenous sequence) 8.56 � 0.09 2.3 1.6

Tyr1-N/OFQ(1–17) Tyr1-substituted analogue 8.50 � 0.07 2.6 2.6

I2Tyr1-N/OFQ(1–17)-NH2 Iodinated analogue 5.04 � 0.40 7726 >3000

Ac-RYYRIK-ol NOPr partial agonist hexapeptide 8.77 � 0.07 1.3 0.9

Ac-RYYRIK-NH2 8.22 � 0.07 5.1 2.6

Ac-RYYRIR-ol NOPr hexapeptide analogues 8.14 � 0.03 6.1 –

Ac-RYYCitIK-ol 7.43 � 0.04 31 50

Ac-RYYCitIK-NH2 6.83 � 0.04 126 234

UFP-101 NOPr antagonist peptide 7.71 � 0.04 16 –

J-113397 Non-peptide NOPr antagonist 6.61 � 0.05 206 –

Naloxone-benzoylhydrazone Partial opioid agonist/NOPr antagonist 5.85 � 0.07 1196 –

Buprenorphine Opioid agonist-antagonist 5.34 � 0.11 3827 –

D-Ala3-dynorphin(1–11) KOPr selective opioid agonist peptide 5.12 � 0.11 6399 –

DAMGO MOPr selective opioid agonist peptide n.d.b n.d.b –

Naloxone Opioid antagonist n.d.b n.d.b –

a Calculated by the Cheng–Prusoff equation.b n.d.: not determined because of ineffectiveness.

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peptides mostly [125I](Tyr14)N/OFQ analogues (Shimohigashiet al., 1996; Ardati et al., 1997; Foddi and Mennini, 1997; Mathiset al., 1997; Neal et al., 1999; Hashiba et al., 2002; Bridge et al.,2003), and in a lesser extent [125I](Tyr10)N/OFQ(1–11) (Letchworthet al., 2000) were investigated in receptor binding and radio-autography studies. Tritium-labeled nociceptin analoguesincluded the full-size [3H](Tyr14)N/OFQ (Adapa and Toll, 1997),[3H](Phe1, Phe4)N/OFQ (Dooley and Houghten, 1996; Butour et al.,1997; Florin et al., 2000) and [3H](Phe1)N/OFQ-NH2, an amidatedheptadecapeptide which is partially protected from degradation(Varani et al., 1998; Benyhe et al., 1999; Wollemann et al., 2008).[3H](Phe1)N/OFQ(1–13)-NH2 represents a tridecapeptide radioli-gand still carrying the full biological activity (Slowe et al., 2001;Hashiba et al., 2002). [3H]UFP-101 has been the first NOP receptorfull antagonist peptide to be radiolabeled (Ibba et al., 2008).Among the partial agonist acetyl-hexapeptides only [3H]Ac-RYYRWK-NH2 has been investigated in binding assays so far(Thomsen et al., 2000).

[3H]Ac-RYYRIK-ol examined in this study possesses 94 Ci/mmolspecific activity indicating the presence of 3-4 tritium atom perpeptide molecule in average. The molar radioactivity of thishexapeptide alcohol is higher than reported for [3H]Ac-RYYRWK-NH2 (Thomsen et al., 2000). Radiochemical purity and integrity ofthe peptide are accompanied by an enhanced stability in thepresence of rat brain membranes (Fig. 1). Biotransformation ofpeptides is an inevitable concern that has to be taken into accountwhile conducting radioligand binding assays. N/OFQ itselfexhibited significant decomposition under various experimentalconditions (Yu et al., 1996; Montiel et al., 1997; Quigley et al.,2000; Terenius et al., 2000). The degradation half-life of unlabeledAc-RYYRIK-ol was two times higher than Ac-RYYRIK-NH2 in mousebrain homogenates and similar to that of the naturally occurringligand N/OFQ (Gunduz et al., 2006). The improved metabolicstability of [3H]Ac-RYYRIK-ol observed in our experiments is ofimportance because acetyl-hexapeptides were found to be quicklydegraded in vivo (Zaveri et al., 2005). The relative resistance of Ac-RYYRIK-ol against biodegradation can be attributed to theprotected N- and C-termini. Altogether, our study underscoresthe importance of monitoring the integrity of the trace ligand beingused in a given binding assay.

The kinetic experiments showed that the association ofligand–receptor complexes occurred quite rapidly displayingequilibrium around in 10 min at room temperature. Ligand-induced dissociation of the radioprobe proceeded also inmonophasic manner, proving the reversibility of the bindinginteraction. In equilibrium binding studies both saturation andhomologous competition curves could be sufficiently fitted usingthe one-site model of the binding process. The presence of ahomogenous set of specific binding sites was also suggested bythe linear Scatchard plots and the Hill coefficient values close tothe unity. The capacities of the [3H]Ac-RYYRIK-ol recognitionsites (Bmax) were two-three times higher in hNOP-CHO cellmembranes than in rat brain homogenates, reflecting simply theoverexpression of NOPr in transfected epithelial cells. Nosignificant differences in the equilibrium binding affinities (Kd

and homologous Ki values) were obtained in these two systems.Kis for [3H]Ac-RYYRIK-ol were as high as for [3H](Leu14)N/OFQevidenced by cross- and self-competition studies (Fig. 4, Table 2).Affinities of native and recombinant receptors are reportedlyclose to one another (Ardati et al., 1997; Ibba et al., 2008).Heterologous competition assays with various nociceptin analo-gues and some opioid compounds revealed that [3H]Ac-RYYRIK-ol specific binding is effectively inhibited by NOPr-selectiveligands, whereas opioids displayed moderate to negligibleaffinities. The highest affinities were obtained with the homo-logous ligand Ac-RYYRIK-ol and N/OFQ, although the affinity of

the Tyr1-replaced derivative (Tyr1)N/OFQ was also very good. Theiodinated analogue (I2Tyr1)N/OFQ, however, displayed onlymodest affinity (Table 3) confirming the steric effect of theiodine atoms discussed above. Among the opioids tested,naloxone-benzoylhydrazone was the most efficient (Ki � 1 mM),mM), while the general opioid antagonist naloxone and the MOPragonist DAMGO were completely ineffective. The relativepotency of naloxone-benzoylhydrazone in competing reversiblythe binding of [3H]Ac-RYYRIK-ol is in line with the peculiarfeature of this rather promiscous opiate (Cox et al., 2005; Olianaset al., 2006; Connor and Kitchen, 2006). It should also be notedthat some non-peptide compounds such as buprenorphine and J-113397 displayed considerably lower affinities than expected.This might be due to a potentially different, or perhapsoverlapping binding site(s) for N/OFQ and the hexapeptideswithin the receptor as discussed below.

One can assume that the hexapeptides interact with thereceptor in a similar manner as N/OFQ, i.e., with the negativelycharged side chains of the second extracellular loop (EL-II) of theNOPr. However, photo-affinity labeling of the NOPr with radio-iodinated probes have indicated that the hexapeptides actuallyinteracted with a region of Gln107-Gly-Thr-Asp-Ile-Leu-Leu113

within the C-terminus of the second transmembrane domain (TM-II) in the NOP receptor (Bes and Meunier, 2003), whereas N/OFQinteracted with the region of Thr296-Ala-Val-Ala-Ile-Leu-Arg302,spanning the C-terminus of EL-III and the N-terminus oftransmembrane helix VII (Mouledous et al., 2000). Monophasicdisplacement of [3H]nociceptin is reasonable only when Ac-RYYRIK-ol occupies the same receptor site for [3H]nociceptin inthe NOPr. Thus, if the segments NOPr107–113 and NOPr296–302 arespecific for the hexapeptides and N/OFQ, respectively, the bindingsite shared by these peptides might exist in region(s) other thanthese portions in NOPr. Recently, receptor-bound conformationsof hexapeptide – NOPr and N/OFQ – NOPr complexes have beencalculated by computer aided structural modeling (Akuzawaet al., 2007). The models explain why both the ligands activateNOPr and suggest that their receptor-bound conformations havesimilar three-dimensional structures. The conformation of thereceptor-bound Ac-RYY fragment showed a quite intriguingspatial correlation with that of the N-terminal FGGF fragmentof N/OFQ.

The results obtained in the present study revealed that the newtritiated peptide analogue [3H]Ac-RYYRIK-ol recognized andlabeled NOP receptors specifically in rat brain membranepreparations and in cellular system expressing the recombinanthuman NOPr. Importantly, this radioligand satisfied the criteria ofreversibility, saturability and relatively low non-specific bindingnecessary for valuable radioprobes. These characteristics of[3H]Ac-RYYRIK, together with its improved chemical and biologicalstability, and high specific radioactivity, make this radioligand aninspiring tool for analyzing the properties and function of the NOPreceptor.

Acknowledgments

This work was supported by a DNT-RET-2004 grant (SB) fromthe National Office for Research and Development (NKTH), and twogrants from the National Scientific Research Fund (OTKA),Budapest, Hungary: K-77783 (CST) and CK-78566 (SB). AFrench-Hungarian bilateral mobility grant provided by the NKTH,OMFB-F-33/2006 for MC and SB is also acknowledged. EB isapplying for the conditional support to young scientists providedby the Dr. Rollin D. Hotchkiss Foundation. Appreciation would bedue to that organization if the applicant is awarded. EB would liketo express his gratefulness to Valeria Biscotti (Forli, Italy) forproviding the fruitful atmosphere.

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