JPET #99986 Functional Characterization of Opioid Receptor

Home , Morphiceptin

JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 JPET FastThis Forward.article has not Published been copyedited on and February formatted. The23, final 2006 version as DOI:10.1124/jpet.105.099986may differ from this version. JPET #99986

Functional Characterization of Opioid Receptor Ligands by Aequorin Luminescence-

based Calcium Assay

Jakub Fichna, Katarzyna Gach, Mariola Piestrzeniewicz, Emmanuel Burgeon, Jeroen Poels,

Jozef Vanden Broeck, and Anna Janecka

Laboratory of Biomolecular Chemistry, Medical University of Lodz, Lodz, Poland (J.F., K.G., Downloaded from M.P., A.J.);

Euroscreen s.a., Gosselies, Belgium (E.B.);

Laboratory for Developmental Physiology, Genomics and Proteomics, Zoological Institute, jpet.aspetjournals.org

Catholic University, Leuven, Belgium (J.P., J.V.B.). at ASPET Journals on September 26, 2021

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Functional Characterization of the Opioid Receptor Ligands

Corresponding author:

Dr. Anna Janecka

Laboratory of Biomolecular Chemistry

Medical University of Lodz

Mazowiecka 6/8 Downloaded from 92-215 Lodz, Poland

Phone/fax: (4842)6784277

E-mail: [email protected] jpet.aspetjournals.org

No. of text pages: 16

No. of tables: 2 at ASPET Journals on September 26, 2021

No. of figures: 4

No. of references: 27

No. of words in the Abstract: 187

No. of words in the Introduction: 615

No. of words in the Discussion: 489

List of abbreviations:

Aeq, Apoaequorin; BSA, Bovine serum albumin; CHO, Chinese hamster ovary; DOR, Delta opioid receptor; FAB, Fast atom bombardment; Fmoc, 9-fluorenylmethoxycarbonyl; GPCR,

G protein-coupled receptor; GPI, guinea pig ileum; MVD, mouse vas deferens; MOR, Mu opioid receptor; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

2 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Abstract

A functional assay, based on aequorin-derived luminescence triggered by receptor- mediated changes in intracellular calcium levels, was used to examine relative potency and efficacy of the µ-opioid agonists, endomorphin-1, endomorphin-2, morphiceptin and their position 3-substituted analogs, as well as the δ-agonist, deltorphin-II. The results of the aequorin assay, performed on recombinant cell lines, were compared with those obtained in Downloaded from the functional assay on isolated tissue preparations (guinea pig ileum and mouse vas deferens). A range of nine opioid peptide ligands produced a similar rank order of potency for

µ δ µ

the - and -opioid receptor agonists in both functional assays. The highest potency at the - jpet.aspetjournals.org receptor was observed for endomorphin-1, endomorphin-2, and [D-1-Nal3]morphiceptin, while deltorphin-II was the most potent δ-receptor agonist. In the aequorin assay, the µ- and

-agonist-triggered luminescence was inhibited by opioid antagonists, naloxone and at ASPET Journals on September 26, 2021 naltrindole, respectively.

We can conclude that the use the aequorin assay for new µ- and δ-receptor selective opioid analogs gives pharmacologically relevant data and allows high-throughput compound screening, which does not involve radioactivity or animal tissues. This is the first study that validates the application of this assay in screening of opioid analogs.

3 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Introduction

Current methods for the identification and characterization of new ligands at the three main opioid receptor types (µ, δ, κ) typically employ binding assays, where a radioligand with high affinity to one receptor type is displaced by a tested analog. Apart from binding studies, different functional assays can also be used to determine relative potency and efficacy of new analogs at the opioid receptors. The most popular functional assay used for opioid activity Downloaded from determination in vitro is performed on isolated tissue preparations. This assay is based on inhibition of electrically evoked contractions of guinea pig ileum (GPI) and mouse vas

deferens (MVD). The opioid effect in the GPI preparations is mainly mediated by the - jpet.aspetjournals.org receptors, whereas the predominant receptors of the MVD are of the δ-type. The GPI/MVD assay makes it possible to determine the µ- and δ-receptor interactions, which is the main

focus in standard tests for opioid activity. at ASPET Journals on September 26, 2021

In the present study we employed an alternative functional assay, which does not involve radioactivity or animal tissues, and allows high-throughput screening of opioid peptide analogs. This assay is based on the recombinant mammalian cell line expressing an opioid receptor together with a luminescent reporter protein.

Opioid receptors belong to a large family of the G protein-coupled receptors (GPCRs) which upon stimulation by a ligand induce the activation of the phospholipase C, with the subsequent generation of the primary second messenger metabolites: diacylglycerol and inositol 1,4,5-trisphosphate, followed by a release of calcium from intracellular stores. These agonist-induced receptor-mediated changes in calcium levels can be quantitatively assessed and used to determine agonist potency and efficacy.

A number of groups (Stables et al., 1997; Ungrin et al., 1999; George et al., 2000;

Torfs et al., 2002a; Niedernberg et al., 2003) have reported the use of the photoprotein

4 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986 aequorin (Shimomura et al., 1962) as a sensitive detector of increases in Ca2+ levels.

Apoaequorin is a 21 kDa photoprotein, isolated from a jellyfish Aequorea victoria, which forms a bioluminescent complex when linked to the chromophore cofactor coelenterazine

(Shimomura, 1995). The binding of Ca2+ to this complex results in an oxidation reaction of coelenterazine, followed by the production of apoaequorin, coelenteramide, CO2, and light with a λmax of 469 nm, which can be detected by conventional luminometry (Stables et al.,

1997). Since its discovery in 1960s aequorin has been used as an intracellular calcium Downloaded from indicator. The first applications of aequorin involved its microinjections into cells. The cloning of its gene in 1985 opened the way to the stable expression of apoaequorin in

different cell lines (Inouye et al., 1985). In a cell line expressing recombinant apoaequorin, jpet.aspetjournals.org reconstitution of aequorin can be simply obtained by addition of coelenterazine to the medium

(Brini et al., 1995).

Methods have been developed to use aequorin-based functional assay for agonist and at ASPET Journals on September 26, 2021 antagonist screening of GPCRs, ion channels and tyrosine kinase receptors (Dupriez et al.,

2002; Le Poul et al., 2002; Liu et al., 2003). For example, this assay was extensively used for characterization of human, as well as insect neurokinin receptors (Torfs et al., 2002b; Torfs et al., 2002c; Poels et al., 2004; Janecka et al., 2005), a histamine receptor (Brini et al., 1995) and mouse α-4, β-2 nicotinic acetylcholine receptors (Karadsheh et al., 2004).

The aim of the present study was to characterize the relative agonist potency and efficacy of three structurally related µ-selective opioid peptides, endomorphin-1, endomorphin-2 and morphiceptin and their position 3-substituted analogs, as well as a δ- agonist, deltorphin-II in the aequorin assay, performed on genetically engineered Chinese hamster ovary cell lines, CHO-MOR-Aeq and CHO-DOR-Aeq, co-expressing, respectively,

µ- or δ-opioid receptors and apoaequorin. A comparison with the data obtained previously

5 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986 from conventional opioid receptor binding and functional GPI/MVD assays was also performed.

Methods

Peptide synthesis; Peptides were synthesized by standard solid-phase procedures as described before (Fichna et al., 2004), using techniques for 9-fluorenylmethoxycarbonyl Downloaded from (Fmoc)-protected amino acids on MBHA Rink peptide resin (100-200 mesh, 0.8 mM/g,

Novabiochem). 20% Piperidine in dimethylformamide was used for deprotection of Fmoc-

groups and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) jpet.aspetjournals.org was employed to facilitate coupling. Simultaneous deprotection and cleavage from the resin was accomplished by treatment with trifluoroacetic acid (TFA) : triisopropylsilane : water (95

: 2.5 : 2.5) for 3 h at room temperature. Crude peptides were purified by RP HPLC on a at ASPET Journals on September 26, 2021

Vydac C18 column (1 x 25 cm) using the solvent system of 0.1% TFA in water (A)/80% acetonitrile in water containing 0.1% TFA (B) and a linear gradient. Calculated values for protonated molecular ions were in agreement with those obtained using FAB mass spectrometry.

Cell lines; The Chinese hamster ovary (CHO) cell lines, CHO-MOR-Aeq and CHO-

DOR-Aeq, permanently expressing both, mitochondria-targeted apoaequorin and the mammalian µ and δ receptor, respectively, were obtained from Euroscreen (Gosselies,

o Belgium). The cells were cultured at 37 C with 5% CO2 in the Ham’s F12 medium

(BioWhittaker, Belgium) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin,

100 µg/ml streptomycin, 400 µg/ml G 418 and 250 µg/ml Zeocin (all cell medium components were purchased from Invitrogen). The cells were grown as monolayers and were trypsinized every 3 days.

6 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Aequorin charging protocol; The CHO cells were grown until 90% confluent monolayers were obtained. Cells in mid-log phase were detached from culture flasks by flushing with PBS-EDTA (5 mM EDTA), centrifuged (5 min, 150 g) and resuspended in

‘BSA medium’ (D-MEM/F-12 with HEPES and 0.1 % BSA, without phenol red) at a concentration of 5 x 106 cells/ml. Cells were incubated at room temperature for 4 h in “BSA medium” supplemented with 5 µM coelenterazine h (Molecular Probes, The Netherlands).

After coelenterazine-loading, the cells were diluted 10 times in the same medium and Downloaded from incubated for 1h.

Aequorin assay; Test substances were dissolved in 50 µl ‘BSA medium’ and pipetted

into the wells of the white 96-well plates. Light emission was recorded (EG&G Microplate jpet.aspetjournals.org

Luminometer LB96V, Berthold, Germany) for 30 s immediately after injection of 50 µl cell suspension (i.e. 25000 cells) into each well. Cells were then lysed by a second injection of 50

µl 0.3% Triton X-100, followed by a 15 s monitoring period. Luminescence data (peak at ASPET Journals on September 26, 2021 integration) were calculated using WinglowTM software (Perkin-Elmer), which was linked to the Microsoft Excel program. Results are expressed as the fractional luminescence, i.e. the ratio of the agonist generated signal and the total luminescence (agonist + lysed cells) thereby correcting for potential well-to-well variation in the number of injected cells (Knight et al.,

2003).

Data analysis; Statistical and curve-fitting analyses were performed using Prism 4.0

(GraphPad Software Inc.). Three independent experiments for each assay were carried out in duplicate. All values are expressed as mean ± SEM.

Results

7 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Calcium measurements and concentration-response curves. Recombinant mammalian cell-lines, CHO-MOR-Aeq and CHO-DOR-Aeq, expressing, respectively, the µ- or δ-opioid receptors and apoaequorin, were used to study agonist-induced bioluminescent responses. Three structurally related µ-selective opioid peptides, endomorphin-1, endomorphin-2 and morphiceptin and their position 3-substituted analogs, as well as a δ- agonist, deltorphin-II were chosen for the study. The concentration-response curves for the

2+ Ca responses in the µ-receptor expressing cells are depicted in Fig. 1. Calculated EC50 Downloaded from values and maximal Ca2+ rises are listed in Table 1. A maximal increase in Ca2+ concentration at the µ-opioid receptor was obtained with endomorphin-1, endomorphin-2, morphiceptin and

analogs 4 and 5. For analogs 6-8 the maximal Ca stimulation reached only about 75% of the jpet.aspetjournals.org level found with peptides 1-5, indicating that these analogs behaved as partial agonists at the

µ-opioid receptor. Despite the lower maximal responses, activity of 6-8 was detectable at

-11 -12 2+

concentrations as low as 10 -10 M. Concentration-response curves for the Ca response at ASPET Journals on September 26, 2021 induced in the δ-opioid receptor expressing cells are shown in Fig. 2. Deltorphin-II behaved

2+ as a potent δ-agonist, with the EC50 value of 0.0017 ± 0.0001 nM and a maximal Ca stimulation of 86 % (100% = total luminescence as determined after cell lysis). All other tested peptides behaved as partial agonists, reaching 51 to 75 % of the total luminescence level. Their EC50 values were at least 2-3 orders of magnitude higher than the value obtained for deltorphin-II.

Effect of opioid antagonists on agonist-evoked responses in CHO-MOR-Aeq and

CHO-DOR-Aeq cells. Opioid antagonists, naloxone and naltrindole, were used to assess their influence on the agonist-induced bioluminescent responses in the µ- and δ-opioid receptor expressing cells, respectively. Naloxone produced a concentration-dependent rightward shift of concentration-response curves for the µ-selective peptides in CHO-MOR-Aeq cells. The exemplary results for endomorphin-1 and the most potent µ-agonist analog 5 are shown in

8 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Fig. 3. The effect produced by deltorphin-II was blocked by pretreatment with δ-antagonist naltrindole (Fig. 4).

Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

9 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Discussion

Endogenous ligands with high affinity and selectivity towards the µ-opioid receptor, endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin–2 (Tyr-Pro-Phe-Phe-NH2) were isolated from mammalian brain in 1997 (Zadina et al., 1997). These tetrapeptide amides are structurally related to another µ-selective ligand, morphiceptin (Tyr-Pro-Phe-Pro-NH2), obtained from the enzymatic digest of bovine casein (Chang et al., 1981, 1985). Downloaded from Here we have selected endomorphins and morphiceptin, as well as their position 3- modified analogs to study their relative potency and efficacy at the µ- and δ-opioid receptors,

δ D expressed in CHO cells. For comparison, a highly -selective peptide, deltorphin-II (Tyr- - jpet.aspetjournals.org

Ala-Phe-Glu-Val-Val-Gly-NH2), originating from amphibian skin (Kreil et al., 1989) has also been studied. We have employed a functional assay based on agonist-evoked changes of

2+ µ δ

intracellular Ca levels in CHO cells stably transfected with the opioid receptor ( or ) and at ASPET Journals on September 26, 2021 apoaequorin cDNA. The light emission responses obtained in the aequorin-based assay for all tested peptides were concentration-dependent, and they were inhibited by opioid antagonists.

The results from this study were compared with our previous data obtained in conventional functional assay (GPI/MVD) and with a traditional binding assay on rat brain membrane preparations (Table 2). In both functional assays, the most potent compounds at the

µ-receptor were endogenous ligands, endomorphin-1, endomorphin-2, and morphiceptin analog 5 (Tyr-Pro-D-1-Nal-Pro-NH2). These three peptides were almost equipotent in the

GPI/MVD assay, but showed a significant difference in potency in the calcium assay.

Endomorphin-1 was the most potent and selective peptide of the series. Analog 5 was 7-times less potent, while the second endogenous ligand, endomorphin-2, was 180-times less potent than endomorphin-1. Morphiceptin was the only peptide showing much higher selectivity in

10 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

the GPI/MVD assay than in the calcium assay. Analog 7 (Tyr-Pro- D-ClPhe-Pro-NH2) was in both tests the only analog with higher potency for the δ-receptor over the µ-receptor.

Although the rank order of potencies of tested peptides was comparable in both functional assays, the EC50 values observed in the bioluminescent calcium assay were several orders of magnitude lower than the IC50 values observed in the GPI/MVD assay and in the radioligand binding assay on rat brain membrane preparations. This clearly illustrates the extraordinary sensitivity of the cell-based aequorin assay, which therefore can differentiate Downloaded from the potency of opioid analogs much better than the GPI/MVD assay. This may be due to the fact that cell clones expressing a specific receptor type assure much higher selectivity of this

assay system then the traditional bioassays on complex tissue preparations. jpet.aspetjournals.org

In conclusion, the selected group of peptides including well-known ligands of high µ- affinity (endomorphin-1 and endomorphin-2), moderate µ-affinity (morphiceptin), high δ-

affinity (deltorphin-II), and position 3-substituted analogs of endomorphins and morphiceptin, at ASPET Journals on September 26, 2021 which in the binding experiments exhibited different degree of µ- or δ-affinity, were tested on the cell lines expressing µ- or δ-opioid receptor and a reporter protein, apoaequorin. The obtained results allowed to differentiate all tested peptides in terms of their potency in a much greater degree than it was possible using a functional assay on tissue preparations.

Acknowledgments

The authors wish to thank Dr. M. Detheux (Euroscreen, Belgium) for advocating the use of the cell lines and Jozef Cieslak and Sofie Van Soest for their excellent technical assistance.

11 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

References

Brini M, Marsault R, Bastianutto C, Alvarez J, Pozzan T, Rizzuto R (1995) Transfected

aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical

evaluation. J Biol Chem 270:9896-9903.

Chang KJ, Lillian A, Hazum E, Cuatrecasas P, Chang J-K (1981) Morphiceptin (NH4-Tyr-

Pro-Phe-Pro-CONH2): a potent and specific agonist for morphine (mu) receptors. Science Downloaded from

212:75-77.

Chang KJ, Su YF, Brent DA, Chang JK (1985) Isolation of a specific mu-opiate receptor jpet.aspetjournals.org

peptide, morphiceptin, from an enzymatic digest of milk proteins. J Biol Chem 260:9706-

9712.

Dupriez VJ, Maes K, Le Poul E, Burgeon E, Detheux M (2002) Aequorin-based functional at ASPET Journals on September 26, 2021

assays for G-protein-coupled receptors, ion channels, and tyrosine kinase receptors.

Receptors Channels 8:319-330.

Fichna J, Do-Rego JC, Costentin J, Chung NN, Schiller PW, Kosson P, Janecka A (2004)

Opioid receptor binding and in vivo antinociceptive activity of position 3-substituted

morphiceptin analogs. Biochem Biophys Res Commun 320, 531-536.

George SE, Schaeffer MT, Cully D, Beer MS, McAllister G (2000) A high-throughput glow-

type aequorin assay for measuring receptor-mediated changes in intracellular calcium

levels. Anal Biochem 286:231-237.

Inouye S, Noguchi M, Sakaki Y, Tagaki Y, Miyata T, Iwanaga S, Miyata T, Tsuji S (1985)

Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc Natl

Acad Sci USA 82:3154-3158.

12 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Janecka A, Poels J, Fichna J, Studzian K, Vanden Broeck J (2005) Comparison of antagonist

activity of spantide family at human neurokinin receptors measured by aequorin

luminescence-based functional calcium assay. Regul Pept 131:23-28.

Karadsheh MS, Shah MS, Tang X, Macdonald RL, Stitzel JA (2004) Functional

characterization of mouse alpha4beta2 nicotinic acetylcholine receptors stably expressed

in HEK293T cells. J Neurochem 91:1138-1150. Downloaded from Knight PJK, Pfeifer TA, Grigliatti TA (2003) A functional assay for G-protein-coupled

receptors using stably transformed insect tissue culture cell lines. Anal Biochem 320:88–

103. jpet.aspetjournals.org

Kreil G, Barra D, Simmaco M, Erspamer V, Falconieri-Erspamer G, Negri L, Severini C,

Corsi R, Melchiorri P (1989) Deltorphin, a novel amphibian skin peptide with high

selectivity and affinity for delta opioid receptors. Eur J Pharmacol 162:123-128. at ASPET Journals on September 26, 2021

Kruszynski R, Fichna J, do-Rego J-C, Chung NN, Schiller PW, Kosson P, Costentin J,

Janecka A (2005) Novel endomorphin-2 analogs with µ-opioid receptor antagonist

activity. J Pept Res 66:125-131.

Le Poul E, Hisada S, Mizuguchi Y, Dupriez VJ, Burgeon E, Detheux M (2002) Adaptation of

aequorin functional assay to high throughput screening. J Biomol Screen 7:57-65.

Liu AMF, Ho MKC, Wong CSS, Chan JHP, Pau AHM, Wong YH (2003) Gα16/z chimeras

efficiently link a wide range of G protein-coupled receptors to calcium mobilization. J

Biomol Screen 8:39-49.

Niedernberg A, Tunaru S, Blaukat A, Harris B, Kostenis E (2003) Comparative analysis of

functional assays for characterization of agonist ligands at G protein-coupled receptors. J

Biomol Screen 8:500-510.

13 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Paterlini MG, Avitabile F, Ostrowski BG, Ferguson DM, Protoghese PS (2000)

Stereochemical requirements for receptor recognition of the mu-opioid peptide

endomorphin-1. Biophys J 78:590-599.

Poels J, Van Loy T, Franssens V, Detheux M, Nachman RJ, Oonk HB, Ackerman KE,

Vassart G, Parmentier M, De Loof A, Torfs H, Vanden Broeck J (2004) Substitution of

conserved glycine residue by alanine in natural and synthetic neuropeptide ligands causes

partial agonism at the stomoxytachykinin receptor. J Neurochem 90:472-478. Downloaded from

Sasaki Y, Sasaki A, Ariizumi T, Igari Y, Sato K, Kohara H, Niizuma H, Ambo A (2004)

2’,6’-Dimethylphenylalanine (Dmp) can mimic the N-Terminal Tyr in opioid peptides. jpet.aspetjournals.org Biol Pharm Bull 27:244-247.

Shimomura O (1995) Luminescence of aequorin is triggered by the binding of two calcium

ions. Biochem Biophys Res Commun 211:359-363. at ASPET Journals on September 26, 2021

Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of

aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell

Comp Physiol 59:223-239.

Stables J, Green A, Marshall F, Fraser N, Knight E, Sautel M, Milligan G, Lee M, Rees S

(1997) A bioluminescent assay for agonist activity at potentially any G-protein-coupled

receptor. Anal Biochem 252:115-126.

Torfs H, Poels J, Detheux M, Dupriez V, Van Loy T, Vercammen L, Vassart G, Parmentier

M, Vanden Broeck J (2002a) Recombinant aequorin as a reporter for receptor-mediated

changes of intracellular Ca2+ -levels in Drosophila S2 cells. Invert Neurosci 4:119-124.

14 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Torfs H, Detheux M, Oonk HB, Akerman KE, Poels J, Van Loy T, De Loof A, Vassart G,

Parmentier M, Vanden Broeck J (2002b) Analysis of C-terminally substituted tachykinin-

like peptide agonists by means of aequorin-based luminescent assays for human and insect

neurokinin receptors. Biochem Pharmacol 63:1675-1682.

Torfs H, Akerman KE, Nachman RJ, Oonk HB, Detheux M, Poels J, Van Loy T, De Loof A,

Meloen RH, Vassart G, Parmentier M, Vanden Broeck J (2002c) Functional analysis of

synthetic insectatachykinin analogs on recombinant neurokinin receptor expressing cell Downloaded from

lines. Peptides 23:1999-2005.

Ungrin MD, Singh LM, Stocco R, Sas DE, Abramovitz M (1999) An automated aequorin jpet.aspetjournals.org

luminescence-based functional calcium assay for G-protein-coupled receptors. Anal

Biochem 272:34-42.

Yaksh TL, Malmberg AB, Ro S, Schiller PW, Goodman M (1995) Characterization of the at ASPET Journals on September 26, 2021

spinal antinociceptive activity of constrained peptidomimetic opioids. J Pharmacol Exp

Ther 275:63-72.

Zadina JE, Hackler L, Ge LJ, Kastin AJ (1997) A potent and selective endogenous agonist for

the mu-opiate receptor. Nature 386:499-502.

15 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Footnote:

This work was supported by a grant for ‘Bilateral Scientific Cooperation’ between Flanders and Poland (BIL03/18). In addition, the authors gratefully acknowledge the Belgian

‘Interuniversity Attraction Poles Programme’ (IUAP/PAI P5/30, Belgian Science Policy) and the ‘FWO’ for financial support. J. Poels is supported by the ‘FWO’ as a postdoctoral research associate.

Downloaded from

Corrresponding author and person to receive reprint requests:

Dr. Anna Janecka jpet.aspetjournals.org

Laboratory of Biomolecular Chemistry

Medical University of Lodz

Mazowiecka 6/8 at ASPET Journals on September 26, 2021

92-215 Lodz, Poland

Phone/fax: (4842)6784277

E-mail: [email protected]

16 JPET Fast Forward. Published on February 23, 2006 as DOI: 10.1124/jpet.105.099986 This article has not been copyedited and formatted. The final version may differ from this version. JPET #99986

Legends for Figures

Fig. 1.

Concentration-response curves for the calcium increase induced by µ-and δ-selective opioid peptides in the CHO-MOR-Aeq cells. The data represent mean ± SEM of three independent experiments carried out in duplicate.

Fig. 2. Downloaded from Concentration-response curves for the calcium increase induced by µ-and δ-selective opioid peptides in the CHO-DOR-Aeq cells. The data represent mean ± SEM of three independent

experiments carried out in duplicate. jpet.aspetjournals.org

Fig. 3.

Concentration-dependent effect of naloxone on the concentration-response curves for the at ASPET Journals on September 26, 2021 calcium rise induced by endomorphin-1 1 (A) and [D-1-Nal3]morphiceptin 5 in the CHO-

MOR-Aeq cells. The data represent mean ± SEM of three independent experiments carried out in duplicate.

Fig. 4.

Concentration-dependent effect of naltrindole on the concentration-response curves for the calcium rise induced by deltorphin-II 9 (A) and [D-ClPhe3]morphiceptin 7 in the CHO-DOR-

Aeq cells. The data represent mean ± SEM of three independent experiments carried out in duplicate.

17 JPET #99986

Table 1. Comparison of EC50 values and maximal calcium rise for the µ- and δ-mediated intracellular calcium response induced by µ-and δ- selective opioid peptides. This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. No. Peptide CHO-MOR-Aeq CHO-DOR-Aeq JPET FastForward.PublishedonFebruary23,2006asDOI:10.1124/jpet.105.099986 2+ 2+ EC50± SEM Max Ca -rise EC50± SEM Max Ca -rise [nM] [%] [nM] [%]

1 Tyr-Pro-Trp-Phe-NH2 (endomorphin-1) 0.00050 ± 0.00003 76.2 ± 3.0 0.47 ± 0.05 65.1 ± 2.5

2 Tyr-Pro-Phe-Phe-NH2 (endomorphin-2) 0.090 ± 0.005 78.1 ± 4.7 1.11 ± 0.15 68.3 ± 3.1

3 Tyr-Pro-Phe-Pro-NH2 (morphiceptin) 4.63 ± 0.22 78.2 ± 5.2 6.47 ± 0.44 71.5 ± 3.3

4 Tyr-Pro-D-1-Nal-Phe-NH2 0.26 ± 0.02 84.3 ± 6.0 4.41 ± 0.27 54.3 ± 2.5

5 Tyr-Pro-D-1-Nal-Pro-NH2 0.0033 ± 0.0001 79.0 ± 2.2 0.28 ± 0.01 75.2 ± 3.1

6 Tyr-Pro-D-Phe-Pro-NH2 4.85 ± 0.18 68.1 ± 4.0 3.42 ± 0.22 51.3 ± 2.0

7 Tyr-Pro-D-ClPhe-Pro-NH2 5.72 ± 0.31 53.2 ± 2.7 1.20 ± 0.09 53.5 ± 2.0

8 Tyr-Pro-D-Cl2Phe-Pro-NH2 0.95 ± 0.04 58.1 ± 2.9 6.59 ± 0.48 57.3 ± 2.4

9 Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 5.30 ± 0.31 57.5 ± 3.3 0.0017 ± 0.0001 86.6 ± 5.5 (deltorphin-II)

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at JPET #99986

Table 2. Competitive binding and GPI/MVD functional assay data obtained with µ- and δ-selective opioid peptides.

No. Peptide IC50 ± SEM [nM] This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

µa δb GPI MVD JPET FastForward.PublishedonFebruary23,2006asDOI:10.1124/jpet.105.099986

c 1 Tyr-Pro-Trp-Phe-NH2 (endomorphin-1) 0.97 ± 0.22 ND 6.70 ± 0.20 ND

d d 2 Tyr-Pro-Phe-Phe-NH2 (endomorphin-2) 3.90 ± 0.20 >1000 7.71 ± 1.47 15.3 ± 1.8

e e 3 Tyr-Pro-Phe-Pro-NH2 (morphiceptin) 79.4 ± 3.4 >1000 318 ± 26 4800 ± 330

4 Tyr-Pro-D-1-Nal-Phe-NH2 89.1 ± 3.8 >1000 ND ND

f f 5 Tyr-Pro-D-1-Nal-Pro-NH2 1.90 ± 0.20 >1000 9.57 ± 1.01 35.4 ± 2.3

f f 6 Tyr-Pro-D-Phe-Pro-NH2 50.1 ± 3.1 >1000 149 ± 23 410 ± 69

f f 7 Tyr-Pro-D-ClPhe-Pro-NH2 >1000 250 ± 24 1320 ± 230 314 ± 40

f f 8 Tyr-Pro-D-Cl2Phe-Pro-NH2 20.0 ± 2.8 >1000 576 ± 165 1360 ± 230

g g 9 Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 314 ± 53 0.68 ± 0.11 >1000 0.55 ± 0.03 (deltorphin-II) a Displacement of [3H]naloxone. b Displacement of [3H][Ile5,6]deltorphin-II. c Data from Paterlini et al., 2000 d Data from Kruszynski et al., 2005 e Data from Yaksh et al., 1995 f Data from Fichna et al., 2004 g Data from Sasaki et al., 2004

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at endomorphin-1 1 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

endomorphin-2 2 JPET FastForward.PublishedonFebruary23,2006asDOI:10.1124/jpet.105.099986 morphiceptin 3 3 [D-1-Nal ]endomorphin 4 3 [D-1-Nal ]morphiceptin 5 3 [D-Phe ]morphiceptin 6 3 [D-ClPhe ]morphiceptin 7 1.0 3 [D-Cl2Phe ]morphiceptin 8 deltorphin-II 9 0.8

0.6

A/(A+L) 0.4

0.2

0.0 -16 -14 -12 -10 -8 -6 -4 logC

Figure 1

0.6

A/(A+L) 0.4

0.2

0.0 -16 -14 -12 -10 -8 -6 -4 logC

Figure 2

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonFebruary23,2006asDOI:10.1124/jpet.105.099986

endomorphin-1 1 endomorphin-1 1 + naloxone 10-5 M endomorphin-1 1 + naloxone 10-6 M endomorphin-1 1 + naloxone 10-7 M endomorphin-1 1 + naloxone 10-8 M 1.0

0.8

0.6

0.4 A/(A+L)

0.2

0.0 -16 -14 -12 -10 -8 -6 -4 logC

Figure 3A

3 [D-1-Nal ]morphiceptin 5 3 -5 [D-1-Nal ]morphiceptin 5 + naloxone 10 M 3 -6 [D-1-Nal ]morphiceptin 5 + naloxone 10 M 3 -7 [D-1-Nal ]morphiceptin 5 + naloxone 10 M 3 -8 [D-1-Nal ]morphiceptin 5 + naloxone 10 M 1.0

0.8

0.6

0.4 A/(A+L)

0.2

0.0 -16 -14 -12 -10 -8 -6 -4 logC

Figure 3B

deltorphin-2 9 deltorphin-2 9 + naltrindole 10-5 M deltorphin-2 9 + naltrindole 10-6 M deltorphin-2 9 + naltrindole 10-7 M deltorphin-2 9 + naltrindole 10-8 M 1.0

0.8

0.6

0.4 A/(A+L)

0.2

0.0 -16 -14 -12 -10 -8 -6 -4 logC

Figure 4

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at