orphanin FQ: Identification of highly potent agonists and antagonists of its receptor

Regulatory Peptides 130 (2005) 116 – 122 www.elsevier.com/locate/regpep

Structure–activity studies on different modifications of nociceptin/orphanin FQ: Identification of highly potent agonists and antagonists of its receptor Min Chang1, Ya-li Peng1, Shou-liang Dong, Ren-wen Han, Wei Li, Ding-jian Yang, Qiang Chen, Rui Wang* Department of Biochemistry and Molecular Biology, School of Life Science, Lanzhou University, 222 Tian Shui South Road, Lanzhou, 730000, PR China Received 15 February 2005; received in revised form 14 April 2005; accepted 26 April 2005 Available online 1 June 2005

Abstract Nociceptin/orphanin FQ (N/OFQ) and its receptor system modulate a variety of biological functions and further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor. Two series of N/OFQ related analogues were synthesized to investigate the relationship of different modifications. We combined modifications including: (a) Phe4Y(pF)Phe4; (b) Ala7, Ala11YAib7, Aib11; (c) Leu14, Ala15YArg14, Lys15. Compared with the first series, N-terminus of the second series was changed from Phe1 to Nphe1. All the analogues were amidated at C-terminus. These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. Results indicated that the compounds of the first series showed higher affinity and potency than N/OFQ (pK i = 9.33; pEC50 = 7.50). In particular, [(pF)Phe4, Aib7, Aib11, Arg14, Lys15] N/OFQ-NH2 was found to be a highly potent agonist with pK i = 10.78 in binding studies and pEC50 = 9.37 in mouse vas deferens assay. The second series all competitively antagonized the effects of N/OFQ in mouse vas deferens assay. [Nphe1, (pF)Phe4, Aib7, Aib11, Arg14, Lys15] N/OFQ-NH2 was the best antagonist with pA 2 = 8.39 and showed high binding affinity with pK i = 9.99. Thus modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist. D 2005 Elsevier B.V. All rights reserved. Keywords: Nociceptin/orphanin FQ (N/OFQ); Nociceptin/orphanin FQ receptor (NOP); Rat brain membranes; Mouse vas deferens; Analogues

1. Introduction Neuropeptide nociceptin/orphanin FQ (N/OFQ) [1 –3] is the endogenous ligand of the opioid receptor-like 1 receptor (NOP) [3 –6], the G-protein coupled receptor which shows overall 60% homology with the classical opioid receptors. N/OFQ is a 17-amino acid peptide (H2N-FGGFTGARKSARKLANQ-COOH) and shows structure similarity to opioid peptide Dynorphin A, but it has very low affinity to A, y and n opioid receptors. A number of studies have demonstrated that N/OFQ/NOP system modulates a variety * Corresponding author. Tel.: +86 931 8912567; fax: +86 931 8912561. E-mail address: [email protected] (R. Wang). 1 Both authors contributed equally to this work. 0167-0115/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2005.04.005

of biological functions including pain threshold, morphine analgesia, neurotransmitter release, food intake, anxiety, locomotor activity, memory processes, cardiovascular and gastrointestinal functions [7,8]. Further understandings of physiological and pathological roles of the N/OFQ/NOP system require new potent agonists and antagonists of NOP. Several structure –activity relationship studies have been performed on N/OFQ. These studies increased our knowledge of structure – activity relationship. Modifications of the N/OFQ N-terminus led to the design of [Nphe1]N/OFQ(1 – 13)-NH2 by transposition of the Phe1 side chain from the acarbon of Phe1 to the N-terminal nitrogen [9]. This peptide was the first pure NOP peptide antagonist and it had low potency (pA 2 values 6.0 – 6.4) but was devoid of any residual agonist activity [10].

M. Chang et al. / Regulatory Peptides 130 (2005) 116 – 122

Studies on N/OFQ and N/OFQ(1 – 13)-NH2 (the minimal sequence that maintains full agonistic activity and is protected from degradation at C-terminus [11]) have also produced peptide agonists more potent than N/OFQ itself. Okada et al. [12] reported the synthesis of [Arg14, Lys15]N/OFQ, which had 3-fold higher binding affinity than N/OFQ itself at human NOP and was 17 times more potent in the GTPgS functional assay. In in vivo experiments, this agonist was 30-fold more potent than N/OFQ in producing pronociceptive effects in the mouse tailwithdrawal assay and produced longer-lasting effects compared to N/OFQ [13]. Guerrini et al. [14] focused their study on the Phe4 residue and found that parasubstituted electron-withdrawing groups such as p-F increased binding affinity 5-fold. The pharmacological profile of [(pF)Phe4]N/OFQ(1 – 13)-NH2 has been evaluated in details both in vitro and in vivo [15,16]. Zhang et al. [17] found potent peptides containing a-helix-promoting conformational constraints, such as [Aib7, Aib11]N/ OFQ-NH2, which had 5-fold greater binding affinity than N/OFQ itself at human NOP. Combining the structure modification that led to potent agonist activity (Arg14, Lys15) and that which led to pure antagonism (Nphe1), Calo et al. [18] recently reported a potent, selective antagonist, [Nphe1, Arg14, Lys15]N/OFQ-NH2, also called UFP-101, whose potency was at least one order of magnitude greater than [Nphe1]N/OFQ(1 – 13)-NH2. More recently, Guerrini et al. [19] combined in the N/OFQ-NH2 structure the chemical modifications that reduce ([Phe1C(CH2NH) Gly2]) or eliminate ([Nphe1]) agonist efficacy with those that increase agonist potency. They found the full agonist [(pF)Phe4,Arg14,Lys15]N/OFQ-NH2, the partial agonist [Phe 1C(CH 2NH)Gly2 ,(pF)Phe4 ,Arg 14,Lys 15]N/ OFQ-NH 2, and the pure antagonist [Nphe1 ,Arg 14 , Lys15]N/OFQ-NH2, which represent the most potent peptide ligands for NOP. The present investigation was undertaken to explore the relationship of combinations of these different modifications and find their contributions to binding affinity and biological activity, respectively. Here we report the effects of combining these different modifications. Also, several peptides were found to be highly potent agonists and antagonists of NOP.

2. Materials and methods 2.1. Materials Protected amino acids and chemicals were purchased from ACT, Fluka (USA). Rink-Amide-MBHA resin was purchased from Tianjin Nankai Hecheng Science and Technology Co., Ltd (China). Stock solutions of peptides were made in distilled water and kept at 20 -C until use.

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2.2. Peptide synthesis and purification Peptides were prepared by manual solid-phase synthesis using standard Fmoc chemistry [20]. The following schedule was employed: (1) DMF wash ( 3); (2) 20% piperidine/ DMF ( 3, 4 min); (3) DMF wash ( 3); (4) Na-FmocAmino Acid (2.5 eq.)/HBTU (2.5 eq.)/HOBt (2.5 eq.)/ DIPEA (5 eq.) in DMF ( 1), 1 h; (5) DMF wash ( 3); (6) Kaiser Test. Aib residue coupling was repeated 2  1 h. The protected peptide-resin was treated with reagent K (TFA/ H2O/phenol/ethanedithiol/thioanisole, 82.5:5:5:2.5:5; v/v; 10 mL/0.2 g of resin) for 2 h at room temperature. After filtration of the exhausted resin, the solution was concentrated in vacuo, and the residue was triturated with ether. The crude peptide was purified by preparative reverse phase HPLC to yield a white powder after lyophilization. Crude peptides were purified by reversed-phase HPLC using a Water Delta 600 system with a Waters Delta-pak semi˚ , 15 Am). preparative column C18 (7.8 mm  300 mm, 300 A Analytical HPLC analyses were performed on a Waters Delta 600 system with a Delta-pak Analytical column C18 (3.9 ˚ , 5 Am). Analytical purity and retention mm  150 mm, 300 A time (tR) of the peptides were determined at a flow rate of 1 mL/min using the two linear gradients: 10 –40% acetonitrile (0.05% TFA) in 30 min and 0 – 35% acetonitrile (0.05% TFA) in 40 min. All analogues showed > 95% purity when monitored at 220 nm. Molecular weights of compounds were determined by an ESI-TOF (electrospray ionization time-offlight) mass spectrometer. Values are expressed as MH+. 2.3. Binding studies with rat brain membranes Male Wister rats weighing 250 – 300 g were used, and binding experiments were performed as reported previously [11,21]. The rats were decapitated, and the forebrain was dissected on ice. The tissue was disrupted in a homogenizer in 20 volumes of 50 mM Tris – HCl, 2 mM EDTA, 100 AM PMSF at pH 7.4. The homogenate was centrifuged at 40 000g for 10 min, and the pellet was resuspended in the same buffer. After a 30-min incubation at 37 -C, the membranes were centrifuged, and the resulting pellets were stored at 80 -C. Prior to freezing, an aliquot of the homogenate was removed for protein assay using the BioRad method with bovine albumin as a reference standard. The final pellet was resuspended in the same incubation buffer at a concentration of 167 Ag/mL, and this homogenate was used in the binding assay. Displacement experiments were carried out in duplicate in a final volume of 500 AL in test tubes containing 0.15 nM [3H]N/OFQ (Amersham, 154 Ci/mmol), 50 mM Tris– HCl buffer, 2 mM EDTA, 100 AM PMSF at pH 7.4, rat forebrain membranes (50 Ag protein/assay), and different concentrations of the ligand under study. Nonspecific binding was defined as that in the presence of 10 AM N/OFQ. Based on the previous studies the incubation time was 60 min at 25 -C. Binding reactions were terminated by filtering the assay mixture

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Table 1 Modifications of peptides and receptor affinities of peptides 1 – 10 in rat forebrain membranes No.

Peptide

pK i (CL95%)a

1 2 3 4 5 6 7 8 9 10

Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-OH Phe-Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Aib-Arg-Lys-Leu-Ala-Asn-Gln-NH2 Phe-Gly-Gly-(pF)Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln-NH2 Phe-Gly-Gly-Phe-Thr-Gly-Aib-Arg-Lys-Ser-Aib-Arg-Lys-Arg-Lys-Asn-Gln-NH2 Phe-Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Aib-Arg-Lys-Arg-Lys-Asn-Gln-NH2 Nphe-Gly-Gly-Phe-Thr-Gly-Aib-Arg-Lys-Ser-Aib-Arg-Lys-Leu-Ala-Asn-Gln-NH2 Nphe-Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Aib-Arg-Lys-Leu-Ala-Asn-Gln-NH2 Nphe-Gly-Gly-(pF)Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln-NH2 Nphe-Gly-Gly-Phe-Thr-Gly-Aib-Arg-Lys-Ser-Aib-Arg-Lys-Arg-Lys-Asn-Gln-NH2 Nphe-Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Aib-Arg-Lys-Arg-Lys-Asn-Gln-NH2

9.33 10.08 10.19 10.33 10.78 9.08 9.23 9.50 9.58 9.99

a

(0.23) (0.15) (0.37) (0.16) (0.19) (0.27) (0.41) (0.39) (0.23) (0.25)

pK i is the negative logarithm to base ten of the inhibitory binding constant, K i.

through glass-fiber filters (presoaked in 0.5% polyethylenamine) using a cell harvester. Filters were washed three times with 1 mL of ice-cold incubation buffer. Filters were subsequently dried at 80 -C for 1 h. Filter-bound radioactivity was counted using Wallac MicroBetai 1450. The inhibitory binding constant, K i, was calculated from the IC50 value using the Cheng and Prusoff equation. Under the experimental conditions described here we demonstrated that rat forebrain membranes expressed a single class of binding sites for [3H]N/OFQ with a K d value of 0.27 nM.

pEC50 = the negative logarithm to base 10 of the molar concentration of an agonist that produces 50% of the maximal possible effect [24]. Apparent affinities of antagonists are given in terms of pA 2 which were calculated using the Gaddum Schild equation: pA 2 = log((CR 1) / [antagonist]) assuming a slope value equal to unity, where CR is the ratio between equieffective concentrations of agonist in the presence and absence of the antagonist. Ligand affinities obtained in binding competition experiments are given as pK i = the negative logarithm to base 10 of the inhibition equilibrium constant [24].

2.4. Mouse vas deferens studies Kunming male mice weighing 25 –30 g were used, and bioassay experiments were performed as reported previously [22,23]. Briefly, the mouse vas deferens (MVD) was prepared according to Hughes et al. [23] and suspended in 10 mL organ baths containing Mg2+-free Krebs solution at 33 -C. Krebs solution (gassed with 95% O2 and 5% CO2, pH 7.4) was of the following composition (in mM): NaCl (118.5), KCl (4.7), KH2PO4 (1.2), NaHCO3 (25), CaCl2 (2.5), and glucose (10). The tissues were stimulated through two platinum ring electrodes with supramaximal rectangular pulses of 1-ms duration and 0.05-Hz frequency. Resting tension was maintained at 0.3 g. Isometric responses were recorded using a strain gauge transducer (Machine Equipment Corporation of GaoBeiDian, China) linked to a recorder system (model BL-420E+, Taimeng Technology Corporation of Chengdu, China). After an equilibration period of about 2 h the contractions induced by electrical field stimulation were stable. At this time, cumulative concentration – response curves (crc) to N/OFQ and N/OFQ related peptides were performed. When required, antagonists (1 AM, 0.1 AM and 0.01 AM for peptide 6– 9; 0.1 AM, 0.01 AM and 0.001 AM for peptide 10) added to the Krebs solution 15 min before performing crc for agonists. 2.5. Data analysis and terminology Data are expressed as a mean of n experiments. For pK i, pEC50, pA 2 and values, the confidence limits at 95% (CL95%) are given. Agonist apparent affinities are given as

3. Results 3.1. Modifications of peptides Nine analogues of N/OFQ were synthesized with the following modifications: (a) exchange of Phe4 for (pF)Phe4; (b) replacements of Ala7, Ala11 by Aib7, Aib11; (c)Leu14, Ala15 were substituted with basic amino acid Arg14, Lys15; (d) N-terminus changed from Phe1 to Nphe1; (e) analogues were amidated at C-terminus. We combined these modifiTable 2 Analytical results corresponding to all synthesized peptides 2 – 10 No.

2 3 4 5 6 7 8 9 10

Analytical HPLC t R (min)

MH+

Method 1

Calcd

Found

1854.1 1926.2 1936.2 1954.2 1836.3 1854.1 1926.2 1936.2 1954.2

1854.4 1926.0 1935.2 1954.1 1836.8 1854.3 1926.5 1936.1 1955.1

a

15.77 18.12c 22.08c 11.07a 15.24a 17.93a 19.35c 9.90a 11.20a

Method 2 b

27.37 20.84b 23.60b 25.21b 28.30b 30.39b 20.80a 25.32b 24.86b

a 10 – 40% acetonitrile (0.05% TFA) (30 min), 1 mL/min, 220 nm, Delta Pak C18, 5 Am, 150  3.9 mm. b 0 – 35% acetonitrile (0.05% TFA) (40 min), 1 mL/min, 220 nm, Delta Pak C18, 5 Am, 150  3.9 mm. c 0 – 30% acetonitrile (0.05% TFA) (30 min), 1 mL/min, 220 nm, Delta Pak C18, 5 Am, 150  3.9 mm.

M. Chang et al. / Regulatory Peptides 130 (2005) 116 – 122

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Table 3 Effects of peptides 1 – 10 in the electrically stimulated mouse vas deferens No.

Abbreviated name

Agonist

Antagonist a

pEC50 (CL95%) 1 2 3 4 5 6 7 8 9 10

N/OFQ [p(F)Phe4,Aib7,Aib11]N/OFQ-NH2 [p(F)Phe4,Arg14,Lys15]N/OFQ-NH2 [Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 [p(F)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 [Nphe1,Aib7,Aib11]N/OFQ-NH2 [Nphe1,p(F)Phe4,Aib7,Aib11]N/OFQ-NH2 [Nphe1,p(F)Phe4,Arg14,Lys15]N/OFQ-NH2 [Nphe1,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 [Nphe1,p(F)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2

7.50 8.49 9.01 9.08 9.37

pA 2(CL95%)b

E max (%) 78 T 3 76 T 4 77 T 7 78 T 4 82 T 5

(0.21) (0.25) (0.41) (0.27) (0.34)

7.14 7.75 7.78 8.01 8.39

(0.51) (0.44) (0.17) (0.23) (0.26)

a

pEC50 is the negative logarithm to base 10 of the molar concentration of agonist that produces 50% of the maximal effect. pA 2 is the negative logarithm to base 10 of the molar concentration of an antagonist that makes it necessary to double the concentration of agonist needed to elicit the original submaximal response; the antagonistic properties of these compounds were tested using N/OFQ as agonist. b

cations and the peptides were divided into two series (Table 1). Compared with the first series, N-terminus of the second series was changed from Phe1 to Nphe1. 3.2. Analytical results corresponding to all synthesized peptides Peptides 1 –10 were prepared by solid-phase peptide synthesis. Chemical characteristics of these new compounds are presented in Table 2. Retention time values were determined in two (methods 1 and 2) solvent systems to assess the purity of each compound and mass ion values (MH+) are also reported. 3.3. Receptor affinities of peptides and effects of peptides in the electrically stimulated MVD Peptides 1 – 10 were tested in a receptor binding assay performed using rat forebrain membranes and [3H]N/OFQ

A

2

N/OFQ+0.1u M peptide10 N/OFQ+0.01u M peptide10 N/OFQ+0.001 uM peptide10

90 80

1.5 1

log(CR-1)

70

%Twitch

B

N/OFQ

100

as a radioligand. Binding data are presented as pK i. These analogues were also tested biological activity in the electrically stimulated mouse vas deferens. If these compounds were found to be inactive as agonists, they were assayed as antagonists against the reference agonist N/OFQ. Results of this biological assay are presented as pEC50 and E max to describe agonist potency and pA 2 to describe antagonist potency. The results of these experiments are presented in Tables 1 and 3. On the basis of the assay results summarized in Tables 1 and 3, the compounds of the first series showed higher affinity and potency than N/OFQ. The measured binding affinities (pK i) for peptides 2 –5 ranged from 10.08 to 10.78. These peptides showed 6 – 27-fold higher affinity than N/ OFQ (pK i 9.33). In MVD assay the peptides 2 – 5 showed full NOP agonism which exhibited higher potency (pEC50 8.49 – 9.37) than N/OFQ (pEC50 7.50). In particular, [(pF)Phe4, Aib7, Aib11, Arg14, Lys15]N/OFQ-NH2 was found to be a highly potent agonist with pK i = 10.78 in

60 50 40

0.5 0

30

y = -0.99x + 8.3927 R2 = 0.9984

-0.5 20 -1

10 10

9

8

7

6

-log[N/OFQ]

5

4

6

7

8

9

10

-log[peptide10]

Fig. 1. Electrically stimulated mouse vas deferens. (A) Concentration – response curve to N/OFQ obtained in the absence (control) and presence of increasing concentrations of peptide 10. Corresponding Schild plot is shown in (B). Points indicate the means and vertical lines the S.E.M. of at least six experiments.

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binding studies on rat brain membranes and pEC50 = 9.37 in MVD assay. Compounds of the second series (6 –10) with exchange of Phe1 for Nphe1 showed antagonistic activity. In MVD assay, all these compounds competitively antagonized the effect of N/OFQ with pA 2 in the range of 7.14 –8.39. These peptides up to 10 AM did not show any residual agonist activity in this preparation. In receptor binding assay, the pK i values of peptides 6 – 10 were 9.08 – 9.99. Similarly, all of these peptides containing Nphe1 exhibited lower affinities than those peptides of the first series and the values showed average of a 0.77 decrease in pK i. In addition, [Nphe1, (pF)Phe4, Aib7, Aib11, Arg14, Lys15]N/OFQ-NH2 was the best antagonist with pA 2 = 8.39 (Fig. 1) and showed high binding affinity with pK i = 9.99. The order of potency in MVD assay of the first series was found to be modification (a) + (b) + (c) (peptide 5) > modification (b) + (c) (peptide 4) > modifications (a) + (c) (peptide 3) >modifications (a) + (b) (peptide 2). Binding data of these peptides were in good agreement with those of the tissue bioassay. Compounds of the second series showed similar results in binding data and order of antagonistic potency in MVD assay also was in accordance with the above results.

4. Discussion For many peptide hormones and neurotransmitters, the peptide can be thought of as being composed of a Fmessage_ region and an Faddress_ region [25 – 27]. The message sequence is the part of the peptide structure that is necessary for agonist activity and signal transduction. The address region is the part of the structure that is primarily important for recognizing (binding to) the receptor. Structure – activity relationship (SAR) studies demonstrated that the N/OFQ sequence can also be divided into a N-terminal tetrapeptide Fmessage_ crucial for receptor activation and a C-terminal Faddress_ important for receptor binding [28]. The past 5 years have witnessed tremendous advances in the design and discovery of very potent and selective peptide agonist and antagonist ligands at NOP. Several modifications in Faddress_ region of N/OFQ increase the potency of the agonist. For modification (a), exchange of Phe4 for (pF)Phe4 , electronic changes in the aromatic ring of Phe4 induced by p-substitution with electron-withdrawing groups might facilitate the interaction with the two aromatic residues of the receptor (Phe220/Tyr131) [14]. The double backbone constraint of modification (b) is likely to induce significant helical character into the secondary structure of the Faddress_ segment of this peptide [17]. Recently, a solution conformational study of N/OFQ and its fragments using CD and 2D NMR found in most conformations of the active peptides was a helical character of fragments 8 – 13 [29]. Another study found that the pattern of NOEs indicated formation of a relatively good helix conformation

from about A7 to Q17 and the surface of one face of the helix was highly positively charged with two pairs of RK residues (R8, K9 and R12, K13) [30]. The presence of Arg14, Lys15 further cap the cationic groups such as guanidino and amino groups, eliciting an additional interaction with the cluster of acidic amino acids in the second extracellular loop (EL2) of NOP. These extra interactions may stabilize the total ligand/receptor interaction to induce a reinforced activation [12]. Moreover, amidation at C-terminus may mask the charge such that it is more complementary to the electrostatic environment posed by the EL2 domain of NOP and is less susceptible to enzymatic degradation [17,31]. All the compounds of the first series have higher affinity and potency than N/OFQ. In particular, peptide 5 was found to be about 70-fold more potent than N/OFQ in MVD assay. Our results exhibit that modifications which increase the potency of agonist have synergistic effect on biological activity. The effect of these modifications in the address region suggests that interactions of ligand and receptor at different positions can cooperate in harmony. For these modifications, each one is independent of the others. Not only a single modification but also combination of modifications can result in a much improvement in receptor binding and biological activities. The results that peptide 5 showed higher affinity and potency than peptide 3 indicate that the backbone constraint of modification (b) has no influence on the reinforced activation of the others and even enhances activation. This finding supports a hypothesis that receptor-bound form of N/OFQ might adopt a helix in the address region. Very recently, the Calo’s group of research published a rather detailed pharmacological characterization of [(pF)Phe4,Arg14,Lys15] N/OFQ-NH2 (which is identical to peptide 3), which showed similar maximal effects but higher potency (2– 48-fold) relative to N/OFQ [32]. Our experimental data were in accordance with these results. Similarly, the order of these three modifications for contributions to affinity and potency is probably modification (c) > modification (b) > modification (a). In other words, the order is increase of cationic groups > conformational constraints of cationic groups > electron withdrawal properties presented in the p-position of Phe4. Peptides 6– 10 with exchange of Phe1 for Nphe1 all showed antagonistic activity. The CYN shift of the Phe1 side chain leads to complete elimination of efficacy thus giving pure antagonists. The modifications (a), (b) and (c) only increase the analogues affinity for receptor and antagonistic potency without modifying their pharmacological activity (agonist or antagonist). This further confirms that N-terminal of N/OFQ is a message region. These data may be useful toward a model of antagonist designing: combination of shift of pharmacological activity in message region and reinforced modifications in address region. According to a comparison between the first series and the second series, it is apparent that the introduction of Nphe leads about a 6-fold decrease in affinity. But [Nphe1]N/

M. Chang et al. / Regulatory Peptides 130 (2005) 116 – 122

OFQ(1 –13)-NH2 shows lower affinity (100-fold ) than N/ OFQ(1 –13)-NH2. Thus the factors of increase binding affinity can compensate for disadvantage of Nphe. Moreover, Guerrini et al. [19] stated that [Nphe1,(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 displayed high potency but clear residual agonist activity. However, we did not find the similar results in MVD assay. This may result from the ability of the MVD bioassay to detect residual agonist activity of NOP ligands. Previous studies reported that the residues are in the Nterminal ‘‘address’’ portion of the molecule known to be critical for NOP binding [28,33]. Phe1 can be replaced with well-positioned aromatic (Tyr or Dmt) or aliphatic (Cha, Leu) residues without loss of activity, while any spatial displacement of the aromatic group (D-Phe) or spatial encumbrance (Tic) leads to inactivity [31]. Alanine substitutions at positions 1 and 4 of N/OFQ produced some of the most dramatic decreases in receptor binding, as well as signaling, as measured by calcium release [30]. Moreover, after a thorough analysis of the shared features of small molecule, Chen et al. [34] have developed a pharmacophore for the NOP ligands. This pharmacophore contains a large hydrophobic group connected to a heteroatom-containing ring system. Recently a publication demonstrates nonpeptide/peptide chimeric ligands for the NOP [35]. Hence N-terminal and aromatic ring of Phe1 are crucial for binding to and activating NOP. Now the reason that the CYN shift of the Phe1 side chain can lead to complete elimination of efficacy thus giving pure antagonists has not been found. A computational analysis of the NOP predicts a binding cavity for the N-terminal part of N/OFQ within transmembranes(TMs) 3, 5, 6, and 7, a region highly conserved across the other opioid receptors [36]. Amino acid residues Asp130, Tyr131, Phe220, Phe224, ˚ of the Nand Trp276 are all predicted to lie within 5 A terminal tetrapeptide of N/OFQ. Mutation of any one of these residues has been observed to affect the ability of N/ OFQ to bind to and activate the NOP [37]. Moreover, alkaloid opioid receptor antagonists (such as naloxone) are thought to shift slightly deeper in the binding pocket than agonists, thereby sterically hindering a shift of TM3 and TM7, and consequently preventing an active receptor confirmation, thus leading to functional antagonism [38]. According to a molecular model [36], N-terminal of N/OFQ is likely to be oriented toward a gap of TM3 and TM7 and benzyl of Nphe1 produces an effect on NOP which possibly is similar to naloxone. In conclusion, in the present investigation we explore the relationship of combinations of these different modifications and find their contributions to binding affinity and biological activity. Our results exhibit that modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist. It may be a useful model that antagonist can be designed by combination of shift of pharmacological activity in message region

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and reinforced modifications in address region. Moreover we report several peptides which behave as highly potent agonists and antagonists of NOP. [(pF)Phe4, Aib7, Aib11, Arg14, Lys15]N/OFQ-NH2 is the NOP agonist more potent than the natural ligand N/OFQ and [Nphe1, (pF)Phe4, Aib7, Aib11, Arg14, Lys15] N/OFQ-NH2 is one of the most potent antagonists for NOP. These analogues can be considered novel pharmacological tools for the investigation of the neurobiology of the N/OFQ/NOP system.

Acknowledgement This work was supported by the grants from the National Natural Science Foundation of China (Nos. 3030061, 20372028, 20472026), the Ministry of Education of China and Gansu province Natural Science Foundation of China (YS031-A21-003).

References [1] Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 1995;377:532 – 5. [2] Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, et al. Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science 1995;270:792 – 4. [3] Cox BM, Chavkin C, Christie MJ, Civelli O, Evans C, Hamon MD, et al. Opioid receptors, in the IUPHAR compendium of receptor characterization and classification. London’ IUPHAR Media Ltd.; 2000. p. 321 – 33. [4] Chen Y, Fan Y, Liu J, Mestek A, Tian M, Kozak CA, et al. Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family. FEBS Lett 1994;347:279 – 83. [5] Mollereau C, Parmentier M, Mailleux P, Butour JL, Moisand C, Chalon P, et al. ORL1, a novel member of the opioid receptor family. Cloning, functional expression and localization. FEBS Lett 1994;341: 33 – 8. [6] Wang JB, Johnson PS, Imai Y, Persico AM, Ozenberger BA, Eppler CM, et al. cDNA cloning of an orphan opiate receptor gene family member and its splice variant. FEBS Lett 1994;348:75 – 9. [7] Calo G, Guerrini R, Rizzi A, Salvadori S, Regoli D. Pharmacology of nociceptin and its receptor: a novel therapeutic target. Br J Pharmacol 2000;129:1261 – 83. [8] Mogil JS, Pasternak GW. The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family. Pharmacol Rev 2001;53:381 – 415. [9] Guerrini R, Calo G, Bigoni R, Rizzi A, Varani K, Toth G, et al. Further studies on nociceptin-related peptides: discovery of a new chemical template with antagonist activity on the nociceptin receptor. J Med Chem 2000;43:2805 – 13. [10] Calo G, Guerrini R, Bigoni R, Rizzi A, Marzola G, Okawa H, et al. Characterization of [Nphe1]nociceptin(1 – 13)NH2, a new selective nociceptin receptor antagonist. Br J Pharmacol 2000;129:1183 – 93. [11] Dooley CT, Houghten RA. Orphanin FQ: receptor binding and analog structure activity relationships in rat brain. Life Sci 1996;59:L23 – 9. [12] Okada K, Sujaku T, Chuman Y, Nakashima R, Nose T, Costa T, et al. Highly potent nociceptin analog containing the Arg – Lys triple repeat. Biochem Biophys Res Commun 2000;278:493 – 8. [13] Rizzi D, Rizzi A, Bigoni R, Camarda V, Marzola G, Guerrini R, et al. [Arg14, Lys15]nociceptin, a highly potent agonist of the nociceptin/or-

122

[14]

[15]

[16]

[17]

[18]

[19]

[20] [21]

[22]

[23]

[24]

[25] [26]

M. Chang et al. / Regulatory Peptides 130 (2005) 116 – 122 phanin FQ receptor: in vitro and in vivo studies. J Pharmacol Exp Ther 2002;300:57 – 63. Guerrini R, Calo G, Bigoni R, Rizzi D, Rizzi A, Varani K, et al. Structure – activity studies of the Phe4 residue of nociceptin(1 – 13)NH2: identification of highly potent agonists of the nociceptin/orphanin FQ receptor. J Med Chem 2001;44:3956 – 64. Bigoni R, Rizzi D, Rizzi A, Camarda V, Guerrini R, Lambert DG, et al. Pharmacological characterisation of [(pX)Phe4]nociceptin(1 – 13)amide analogues: 1. In vitro studies. Naunyn Schmiedebergs Arch Pharmacol 2002;365(6):442 – 9. Rizzi A, Salis MB, Ciccocioppo R, Marzola G, Bigoni R, Guerrini R, et al. Pharmacological characterisation of [(pX)Phe4]nociceptin(1 – 13)amide analogues: 2. In vivo studies. Naunyn Schmiedebergs Arch Pharmacol 2002;365(6):450 – 6. Zhang CW, Miller W, Valenzano KJ, Kyle DJ. Novel, potent ORL-1 receptor agonist peptides containing a-helix-promoting conformational constraints. J Med Chem 2002;45:5280 – 6. Calo G, Rizzi A, Rizzi D, Bigoni R, Guerrini R, Marzola G, et al. [Nphe1,Arg14,Lys15] Nociceptin-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor. Br J Pharmacol 2002;136:303 – 11. Guerrini R, Calo G, Lambert DG, Carra G, Arduin M, Barnes TA, et al. N- and C-terminal modifications of nociceptin/orphanin FQ generate highly potent NOP receptor ligands. J Med Chem 2005;48:1421 – 7. Atherton E, Sheppard RC. Solid-phase peptide synthesis: a practical approach. Oxford, England’ IRL Press; 1989. Varani K, Calo G, Rizzi A, Merighi S, Toth G, Guerrini R, et al. Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships. Br J Pharmacol 1998;125:1485 – 90. Calo G, Guerrini R, Bigoni R, Rizzi A, Bianchi C, Regoli D, et al. Structure-activity study of the nociceptin-(1 – 13)-NH2 N-terminal tetrapeptide and discovery of a nociceptin receptor antagonist. J Med Chem 1998;4:3360 – 6. Hughes J, Kosterlitz HW, Leslie FM. Effect of morphine on adrenergic transmission in the mouse vas deferens. Assessment of agonist and antagonist potencies of narcotic analgesics. Br J Pharmacol 1975; 53:371 – 81. Jenkinson DH, Barnard EA, Hoyer D, Humphrey PPA, Leff P, et al. International Union of Pharmacology Committee on receptor nomenclature and drug classification: XI. Recommendations on terms and symbols in quantitative pharmacology. Pharmacol Rev 1995;47:255 – 66. Hruby VJ. Designing peptide receptor agonists and antagonists. Nat Rev Drug Discov 2002;1:847 – 58. Schwyzer R. ACTH: a short introductory review. Ann N Y Acad Sci 1977;297:3 – 25.

[27] Cowell SM, Balse-Srinivasan PM, Ahn JM, Hruby VJ. Design and synthesis of peptide antagonists and inverse agonists for G proteincoupled receptors. Methods Enzymol 2002;343:49 – 72. [28] Guerrini R, Calo G, Rizzi A, Bianchi C, Lazarus LH, Salvadori S, et al. Address and message sequences for the nociceptin receptor: a structure – activity study of nociceptin-(1 – 13)-peptide amide. J Med Chem 1997;40:1789 – 93. [29] Klaudel L, Legowska A, Brzozowski K, Silberring J, Wojcik J, Rolka K. Solution conformational study of nociceptin and its 1 – 13 and 1 – 11 fragments using circular dichroism and two-dimensional NMR in conjunction with theoretical conformational analysis. J Pept Sci 2004;10(11):678 – 90. [30] Orsini MJ, Nesmelova I, Young HC, Hargittai B, Beavers MP, Liu J, et al. The nociceptin pharmacophore site for opioid receptor binding derived from the NMR structure and bioactivity relationships. J Biol Chem 2005;280:8134 – 42. [31] Salvadori S, Guerrini R, Calo’ G, Regoli D. Structure activity studies on nociceptin/orphanin FQ: from full agonist, to partial agonist, to pure antagonist. Il Famaco 1999;54:810 – 25. [32] Carra G, Rizzi A, Guerrini R, Barnes TA, Mc Donald J, Hebbes CP, et al. [(pF)Phe4,Arg14, Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/orphanin FQ receptor. J Pharmacol Exp Ther 2005;312:1114 – 23. [33] Reinscheid RK, Ardati A, Monsma Jr FJ, Civelli O. Structure – activity relationship studies on the novel neuropeptide orphanin FQ. J Biol Chem 1996;271:14163 – 8. [34] Chen Z, Miller WS, Shan S, Valenzano KJ. Design and parallel synthesis of piperidine libraries targeting the nociceptin (N/OFQ) receptor. Bioorg Med Chem Lett 2003;19:3247 – 52. [35] Guerrini R, Carra G, Calo G, Trapella C, Marzola E, Rizzi D, et al. Nonpeptide/peptide chimeric ligands for the nociceptin/orphanin FQ receptor: design, synthesis and in vitro pharmacological activity. J Pept Res 2004;63:477 – 84. [36] Topham CM, Mouledous L, Poda G, Maigret B, Meunier JC. Molecular modeling of the ORL1 receptor and its complex with nociceptin. Protein Eng 1998;11:1163 – 79. [37] Mouledous L, Topham CM, Moisand C, Mollereau C, Meunier JC. Functional inactivation of the nociceptin receptor by alanine substitution of glutamine 286 at the C terminus of transmembrane segment VI: evidence from a site-directed mutagenesis study of the ORL1 receptor transmembrane-binding domain. Mol Pharmacol 2000;57:495 – 502. [38] Pogozheva ID, Lomize AL, Mosberg HI. Opioid receptor threedimensional structures from distance geometry calculations with hydrogen bonding constraints. Biophys J 1998;75:612 – 34.