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British Journal of Pharmacology
RESEARCH PAPER
In vitro and in vivo pharmacological characterization of nociceptin/orphanin FQ tetrabranched derivatives
DOI:10.1111/bph.12799 www.brjpharmacol.org
Correspondence Girolamo Calo, Department of Medical Sciences, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 19, 44121 Ferrara, Italy. E-mail: [email protected] ----------------------------------------------------------------
Received 1 October 2013
Revised 6 May 2014
Accepted 14 May 2014
A Rizzi1, D Malfacini1, M C Cerlesi1, C Ruzza1, E Marzola2, M F Bird3, D J Rowbotham3, S Salvadori2, R Guerrini2, D G Lambert3 and G Calo1 1
Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, and 2Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy, and 3Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, University of Leicester, Leicester, UK
BACKGROUND AND PURPOSE An innovative chemical approach, named peptide welding technology (PWT), allows the synthesis of multibranched peptides with extraordinary high yield, purity and reproducibility. With this approach, three different tetrabranched derivatives of nociceptin/orphanin FQ (N/OFQ) have been synthesized and named PWT1-N/OFQ, PWT2-N/OFQ and PWT3-N/OFQ. In the present study we investigated the in vitro and in vivo pharmacological profile of PWT N/OFQ derivatives and compared their actions with those of the naturally occurring peptide.
EXPERIMENTAL APPROACH The following in vitro assays were used: receptor and [35S]-GTPγS binding, calcium mobilization in cells expressing the human N/OFQ peptide (NOP) receptor, or classical opioid receptors and chimeric G proteins, electrically stimulated mouse vas deferens bioassay. In vivo experiments were performed; locomotor activity was measured in normal mice and in animals with the NOP receptor gene knocked out [NOP(−/−)].
KEY RESULTS In vitro PWT derivatives of N/OFQ behaved as high affinity potent and rather selective full agonists at human recombinant and animal native NOP receptors. In vivo PWT derivatives mimicked the inhibitory effects exerted by the natural peptide on locomotor activity showing 40-fold higher potency and extremely longer lasting action. The effects of PWT2-N/OFQ were no longer evident in NOP(−/−) mice.
CONCLUSIONS AND IMPLICATIONS The results showed that the PWT can be successfully applied to the peptide sequence of N/OFQ to generate tetrabranched derivatives characterized by a pharmacological profile similar to the native peptide and associated with a higher potency and marked prolongation of action in vivo. 4138
British Journal of Pharmacology (2014) 171 4138–4153
© 2014 The British Pharmacological Society
N/OFQ tetrabranched derivatives as NOP ligands
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Abbreviations DOP, δ opioid peptide receptor; KOP, κ opioid peptide receptor; MOP, μ opioid peptide receptor; mVD, mouse vas deferens; N/OFQ, nociceptin/orphanin FQ; NOP, N/OFQ peptide receptor; NOP(−/−), mice knockout for the NOP receptor gene; NOP(+/+), wild-type mice; PWT, peptide welding technology; LA, locomotor activity
Table of Links TARGETS
LIGANDS
δ opioid receptor
Dynorphin A
κ opioid receptor
[3H]-diprenorphine
μ opioid receptor
DPDPE
NOP receptor
GTPγS Naloxone Norbinaltorphimine Probenecid SB-612111
This Table lists key protein targets and ligands in this document, which are hyperlinked to corresponding entries in http:// www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).
Introduction Most of the currently marketed drugs are small molecules characterized by oral bioavailability and suitable pharmacokinetic properties. However, at least in general, relatively low selectivity for the target is often the cause of their side effects. In contrast, peptides are characterized by an extraordinary selectivity of action. However, the pharmacokinetic properties of peptides have been disappointing due to their low bioavailability, poor ability to cross barriers and high susceptibility to enzymatic degradation. To improve the pharmacokinetic properties of peptides, several different strategies have been used, including cyclization, substitutions with unnatural amino acids, modifications of the peptide bond, chemical protections at the N- and C-terminal, and use of multibranched compounds. The advantage of a multibranched peptide is its reduced susceptibility to enzymatic degradation, while the biological effects and the high selectivity of action of the peptide sequence are maintained. In the peptide receptor field, multibranched molecules have been synthesized and evaluated using different peptide sequences including neurotensin (Falciani et al., 2007a), melanocytestimulating hormone (Brabez et al., 2011), enkephalins (Falciani et al., 2007b) and nociceptin/orphanin FQ (N/OFQ) (Bracci et al., 2003). In these examples, the biological activity of the peptide sequences was maintained and their metabolic stability was prolonged by several fold. However, the synthesis of these molecules is rather challenging and is in general characterized by low yield and purity of the final product. We have recently developed an innovative chemical approach, named peptide welding technology (PWT), that allows the synthesis of multibranched peptides with an
extraordinary high yield, purity and reproducibility. Details of the PWT chemical strategy have been recently published (Guerrini et al., 2014). With this approach, three different tetrabranched derivatives of N/OFQ have been synthesized and named PWT1-N/OFQ, PWT2-N/OFQ and PWT3-N/OFQ. These compounds differ exclusively in the core used: (Lys)2Lys-NH2 for PWT1-N/OFQ, Cyclam for PWT2-N/OFQ and (Lys)2-ethylendiamine for PWT3-N/OFQ. The chemical structures of the PWT derivatives of N/OFQ are shown in Figure 1. N/OFQ (FGGFTGARKSARKLANQ) was identified as the endogenous ligand of a previously described orphan receptor (Meunier et al., 1995; Reinscheid et al., 1995), now referred to as the N/OFQ peptide (NOP) receptor. NOP is a GPCR whose activation produces inhibition of cAMP levels, stimulation of potassium currents and inhibition of calcium channels. The peptide and its receptor are widely distributed in the nervous system where, via inhibitory mechanisms, they modulate several different biological functions (Lambert, 2008). In the present study, we investigated the in vitro and in vivo pharmacological profile of PWT N/OFQ derivatives and compared their actions with those of the naturally occurring peptide.
Methods Cell culture and membrane preparation CHO cells stably expressing human classical opioid receptors (MOP, KOP and DOP receptors) were maintained in Nutrient F12 containing 10% FBS; CHO cells expressing human NOP receptors were maintained in DMEM/Nutrient F12 (50/50) British Journal of Pharmacology (2014) 171 4138–4153
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H2N
NH2
O
HN FGGFTGARKSARKLANQ
O
S
O
O
N
H FGGFTGARKSARKLANQ N
O S
H2N
O
O
O HN
O
O
NH
PWT1-N/OFQ
NH2
HN O
O
O
H2N
N
NH
S
O HN
O
H2N FGGFTGARKSARKLANQ
N H
S
O
O
O O N
FGGFTGARKSARKLANQ
S
S
N O
O
S
O
N QNALKRASKRAGTFGGF H
O
O
O
N
O
FGGFTGARKSARKLANQ N H
O O
H N QNALKRASKRAGTFGGF
O
NH2
N
PWT2-N/OFQ
H2N
O
N
N
H2N
O
H2N
N
S O
O
O N
O
O
S
H N
QNALKRASKRAGTFGGF
QNALKRASKRAGTFGGF HN NH2
O
N
NH
O
N
N H
O
H N O
O
O
NH
O O
H2N O
Chemical structures of PWT derivatives of N/OFQ. British Journal of Pharmacology (2014) 171 4138–4153
N O
S H2N
O
N QNALKRASKRAGTFGGF H
N S
Figure 1
O
H N
N H
PWT3-N/OFQ
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NH QNALKRASKRAGTFGGF
N
NH
O
N
O
O
HN
O
S
O N QNALKRASKRAGTFGGF H
N/OFQ tetrabranched derivatives as NOP ligands
with 5% FBS; all media were further supplemented with penicillin (100 IU·mL−1), streptomycin (100 μg·mL−1) and fungizone (2.5 μg·mL−1). Stock cultures additionally contained geneticin (G418) (200 μg·mL−1) for CHOMOP/KOP/DOP cells, or G418 (200 μg·mL−1) and hygromycin B (200 μg·mL−1) for CHONOP cells. Cultures were sustained at 37°C with 5% carbon dioxide humidified air and subcultured twice weekly. Membranes were prepared from freshly harvested cells. Cells were suspended in homogenizing buffer consisting of either Tris (50 mM) for [3H]-diprenorphine displacement binding, Tris (50 mM), MgSO4 (5 mM) for [3H]-UFP-101 displacement binding or Tris (50 mM), EGTA (0.2 mM) for [35S]-GTPγS assays. Cell suspensions were homogenized and membranes were collected via centrifugation at 20 374× g for 10 min at 4°C. This process was repeated three times and protein concentrations were determined (Lowry et al., 1951).
Receptor binding
CHONOP membranes (40 μg) were incubated in 0.5 mL of buffer consisting of Tris (50 mM), BSA (0.5%), ∼0.8 nM [3H]UFP-101 and varying concentrations (10 μM–1 pM) of ligands. Non-specific binding was determined in the presence of 1 μM N/OFQ. In addition, 50 μg of membranes from CHO cells expressing classical opioid receptors was incubated in 0.5 mL buffer consisting of Tris (50 mM), BSA (0.5%), ∼0.8 nM [3H]-DPN and a range of concentrations of ligands. Non-specific binding was determined in the presence of 10 μM naloxone. For both displacement assays, reactions were incubated at room temperature for 1 h and terminated by vacuum filtration through polyethylenimine (0.5%)-soaked Whatman GF/B filters (Fisher Scientific, Loughborough, UK), using a Brandel harvester. Radioactivity was determined following an 8 h extraction of filters in ScintiSafe Gel using liquid scintillation spectroscopy.
Stimulation of [35S]-GTPγS binding
CHONOP membranes, 40 μg, were incubated in 0.5 mL buffer containing Tris (50 mM), EGTA (0.2 mM), MgCl2 (1 mM), NaCl (100 mM), BSA (0.1%), bacitracin (0.15 mM), GDP (100 mM) and ∼150 pM [35S]-GTPγS. NOP ligands were included in varying concentrations, and non-specific binding was determined in the presence of 10 μM GTPγS. Reactions were incubated for 1 h at 30°C with gentle shaking and terminated by vacuum filtration through dry Whatman GF/B filters. Radioactivity was determined following an 8 h extraction of filters in ScintiSafe Gel using liquid scintillation spectroscopy.
Calcium mobilization CHO cells stably co-expressing the human NOP, KOP or MOP receptor and the C-terminally modified Gαqi5 and CHO cells expressing the DOP receptor and the GαqG66Di5 protein were generated as previously described (Camarda et al., 2009; Camarda and Calo, 2013). Cells were maintained in culture medium consisting of DMEM/HAM’S F-12 (50/50) supplemented with 10% FCS, penicillin (100 IU·mL−1), streptomycin (100 μg·mL−1), fungizone (2.5 μg·mL−1), geneticin (G418; 200 μg·mL−1) and hygromycin B (200 mg·mL−1). Cell cultures were kept at 37°C in 5% CO2 humidified air. Experimental cultures were free from selection agents (hygromycin B,
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G418). When confluence was reached (3–4 days), cells were subcultured as required using trypsin/EDTA and used for experimentation. Cells were seeded at a density of 50 000 cells per well into 96-well black, clear-bottom plates. After 24 h of incubation, cells were loaded with medium supplemented with 2.5 mM probenecid, 3 μM of the calciumsensitive fluorescent dye Fluo-4 AM and 0.01% pluronic acid for 30 min at 37°C. Afterwards, the loading solution was aspirated and 100 μL per well of assay buffer: HBSS supplemented with 20 mM HEPES, 2.5 mM probenecid and 500 μM Brilliant Black was added. Stock solutions (1 mM) of peptides and PWT compounds were made in distilled water and stored at −20°C. SB-612111 was solubilized (10 mM) in DMSO. Serial dilutions of ligands for experimental use were made in HBSS/ HEPES (20 mM) buffer (containing 0.03% BSA fraction V). After the cell culture and compound plates had been placed in the FlexStation II (Molecular Device, Union City, CA USA), fluorescence changes were measured. Online additions were carried out in a volume of 50 μL per well. To facilitate drug diffusion into the wells in antagonist-type experiments, the present studies were performed at 37°C and three cycles of mixing (25 μL from each well) were performed immediately after antagonist injection.
Electrically stimulated mouse vas deferens (mVD) Tissues were taken from NOP(+/+) or NOP(−/−) male mice. The mVD tissues were prepared as previously described (Calo et al., 1996). Tissues were suspended in 5 mL of organ baths containing heated Krebs solution (composition in mM: NaCl 118.5, KCl 4.7, KH2PO4 1.2, NaHCO3 25, glucose 10 and CaCl2 2.5) oxygenated with 95% O2 and 5% CO2. The bath temperature was set at 33°C. Tissues were continuously stimulated through two platinum ring electrodes with supramaximal rectangular pulses of 1 ms duration and 0.05 Hz frequency. A resting tension of 0.3× g was applied to the vas deferens. The electrically-evoked contractions (twitches) were measured isotonically with a strain gauge transducer (Basile 7006; Ugo Basile s.r.l., Varese, Italy) and recorded with the PC-based acquisition system Power Lab (ADInstrument, Colorado Springs, CO, USA). Following an equilibration period of 60 min, the contractions induced by electrical field stimulation were stable. At this time, cumulative concentration–response curves to N/OFQ, its PWT derivatives or DPDPE were performed.
Mouse locomotor activity All experimental procedures adopted for in vivo studies were as humane as possible and complied with the ARRIVE guidelines (Kilkenny et al., 2010) and the standards of the European Communities Council directives (86/609/EEC) and national regulations (D.L. 116/92). Male CD-1 mice (30–38 g, Harlan, San Pietro in Natisone, Udine, Italy) and male NOP(+/+) or NOP(−/−) mice were used. The total number of mice used for this study was 360. They were housed in Plexiglas® cages (Tecniplast, Buguggiate, Varese, Italy), under standard conditions (22°C, 55% humidity, 12 h light–dark cycle, lights on 07:00 h) with food and water available ad libitum for at least 5 days before experiments were begun. Experiments were performed according to the procedures British Journal of Pharmacology (2014) 171 4138–4153
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described previously (Guerrini et al., 2009). For these experiments, the ANY-maze video tracking system was used (Ugo Basile, Varese, Italy, application version 4.52c Beta). Mice were positioned in a square plastic cage (40 × 40 cm), 1 mouse per cage. Four mice were monitored in parallel. Mouse horizontal activity was monitored by a camera, whereas vertical activity was measured by an infrared beam array. The parameters measured were cumulative distance travelled (total distance in metres that the animal travelled during the test), immobility time (the animal is considered immobile when 90% of it remains in the same place for a minimum of 2.5 s) and the number of rearing (the number of beam breaks due to vertical movements; this input is triggered when the beam is interrupted for a minimum of 200 ms). N/OFQ, PWT1-N/ OFQ, PWT2-N/OFQ and PWT3-N/OFQ were given i.c.v. (2 μL per mouse). Free hand i.c.v. injections were given, under isoflurane anaesthesia (just sufficient to produce a loss of the righting reflex), in the left ventricle according to literature procedures (Laursen and Belknap, 1986).
Data analysis and statistical procedures
In vitro data were expressed as mean ± SEM of at least three separate experiments. The concentration of drug producing 50% displacement of specific binding (IC50) was corrected for the competing mass of radioligand according to Cheng and Prusoff (1973) to yield Ki using KD. KD values of [3H]-UFP-101 at NOP and [3H]-diprenorphine at classical opioid receptors were taken from previous studies (Kitayama et al., 2003; Ibba et al., 2008). [35S]-GTPγS data are expressed as stimulation factor (agonist specific binding/basal specific binding). In calcium mobilization experiments, maximum change in fluorescence, expressed as percentage over the baseline fluorescence, was used to determine agonist response. Non-linear regression analysis using GraphPad Prism software (5.0; GraphPad Software, San Diego, CA, USA) allowed logistic iterative fitting of the resultant responses and the calculation of agonist potencies and maximal effects. Agonist potencies were given as pEC50 (the negative logarithm to base 10 of the molar concentration of an agonist that produces 50% of the maximal possible effect). In vivo data are expressed as mean ± SEM of n animals. Data were analysed using one-way ANOVA followed by Dunnett’s post hoc test or with Student’s t-test. Differences were considered statistically significant when P < 0.05.
Results Receptor binding In CHONOP cell membranes, N/OFQ displaced [3H]-UFP-101 binding in a concentration-dependent manner showing high affinity (pKi 9.42). Similar results were obtained with PWT derivatives of N/OFQ; however, they displayed increased affinity by 5 (PWT3-N/OFQ) up to 15 (PWT1-N/OFQ) fold (Table 1). To investigate selectivity over classical opioid receptors, similar experiments were performed using membranes obtained from CHO cells expressing the MOP, KOP and DOP receptors and [3H]-diprenorphine as radioligand. N/OFQ up to 1 μM did not bind to KOP and DOP receptors while it displayed micromolar affinity at MOP receptors (pKi 6.06). All PWT derivatives showed higher affinity than N/OFQ at classical opioid receptors. However, their selectivity for the NOP receptor (ranging from 2570- to 14 125-fold) was never inferior to that displayed by the natural peptide (2291-fold). In parallel experiments, standard ligands for classical opioid receptors displayed the expected high affinity (morphine, pKi 8.57 for MOP receptors; Dmt-Tic, pKi 8.87 for DOP receptors; and norbinaltorphimine, pKi 10.69 for KOP receptors).
Stimulation of [35S]-GTPγS binding In CHONOP cell membranes, N/OFQ stimulated [35S]-GTPγS binding in a concentration-dependent manner with maximal effects of 3.5 ± 0.2 and pEC50 of 8.84 (Figure 2, top panel). PWT derivatives of N/OFQ mimicked the stimulatory effect of the natural peptide showing similar maximal effects but higher potency (pEC50 9.71–10.12; Figure 2, top panel).
Calcium mobilization In CHONOP cells stably expressing the Gαqi5 chimeric protein, N/OFQ and its PWT derivatives evoked a concentrationdependent stimulation of calcium release (Figure 2, bottom panel). N/OFQ displayed high potency (pEC50 9.39) and maximal effects (237 ± 15%). PWT derivatives of N/OFQ mimicked the peptide stimulatory effects showing similar maximal effects but slightly lower potency (pEC50 8.75–9.16; Figure 2, bottom panel). Inhibition experiments were performed by testing increasing concentrations of the NOP receptor antagonist SB-612111 against a fixed concentration of agonist approximately corresponding to its EC80 (10 nM for
Table 1 Affinities of N/OFQ and its PWT derivatives in membranes of CHO cells expressing the human NOP or classical opioid receptors
N/OFQ
NOP
MOP
KOP
DOP
pKi (CL95%)
pKi (CL95%)
pKi (CL95%)
pKi (CL95%)
British Journal of Pharmacology
RESEARCH PAPER
In vitro and in vivo pharmacological characterization of nociceptin/orphanin FQ tetrabranched derivatives
DOI:10.1111/bph.12799 www.brjpharmacol.org
Correspondence Girolamo Calo, Department of Medical Sciences, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 19, 44121 Ferrara, Italy. E-mail: [email protected] ----------------------------------------------------------------
Received 1 October 2013
Revised 6 May 2014
Accepted 14 May 2014
A Rizzi1, D Malfacini1, M C Cerlesi1, C Ruzza1, E Marzola2, M F Bird3, D J Rowbotham3, S Salvadori2, R Guerrini2, D G Lambert3 and G Calo1 1
Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, and 2Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy, and 3Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, University of Leicester, Leicester, UK
BACKGROUND AND PURPOSE An innovative chemical approach, named peptide welding technology (PWT), allows the synthesis of multibranched peptides with extraordinary high yield, purity and reproducibility. With this approach, three different tetrabranched derivatives of nociceptin/orphanin FQ (N/OFQ) have been synthesized and named PWT1-N/OFQ, PWT2-N/OFQ and PWT3-N/OFQ. In the present study we investigated the in vitro and in vivo pharmacological profile of PWT N/OFQ derivatives and compared their actions with those of the naturally occurring peptide.
EXPERIMENTAL APPROACH The following in vitro assays were used: receptor and [35S]-GTPγS binding, calcium mobilization in cells expressing the human N/OFQ peptide (NOP) receptor, or classical opioid receptors and chimeric G proteins, electrically stimulated mouse vas deferens bioassay. In vivo experiments were performed; locomotor activity was measured in normal mice and in animals with the NOP receptor gene knocked out [NOP(−/−)].
KEY RESULTS In vitro PWT derivatives of N/OFQ behaved as high affinity potent and rather selective full agonists at human recombinant and animal native NOP receptors. In vivo PWT derivatives mimicked the inhibitory effects exerted by the natural peptide on locomotor activity showing 40-fold higher potency and extremely longer lasting action. The effects of PWT2-N/OFQ were no longer evident in NOP(−/−) mice.
CONCLUSIONS AND IMPLICATIONS The results showed that the PWT can be successfully applied to the peptide sequence of N/OFQ to generate tetrabranched derivatives characterized by a pharmacological profile similar to the native peptide and associated with a higher potency and marked prolongation of action in vivo. 4138
British Journal of Pharmacology (2014) 171 4138–4153
© 2014 The British Pharmacological Society
N/OFQ tetrabranched derivatives as NOP ligands
BJP
Abbreviations DOP, δ opioid peptide receptor; KOP, κ opioid peptide receptor; MOP, μ opioid peptide receptor; mVD, mouse vas deferens; N/OFQ, nociceptin/orphanin FQ; NOP, N/OFQ peptide receptor; NOP(−/−), mice knockout for the NOP receptor gene; NOP(+/+), wild-type mice; PWT, peptide welding technology; LA, locomotor activity
Table of Links TARGETS
LIGANDS
δ opioid receptor
Dynorphin A
κ opioid receptor
[3H]-diprenorphine
μ opioid receptor
DPDPE
NOP receptor
GTPγS Naloxone Norbinaltorphimine Probenecid SB-612111
This Table lists key protein targets and ligands in this document, which are hyperlinked to corresponding entries in http:// www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).
Introduction Most of the currently marketed drugs are small molecules characterized by oral bioavailability and suitable pharmacokinetic properties. However, at least in general, relatively low selectivity for the target is often the cause of their side effects. In contrast, peptides are characterized by an extraordinary selectivity of action. However, the pharmacokinetic properties of peptides have been disappointing due to their low bioavailability, poor ability to cross barriers and high susceptibility to enzymatic degradation. To improve the pharmacokinetic properties of peptides, several different strategies have been used, including cyclization, substitutions with unnatural amino acids, modifications of the peptide bond, chemical protections at the N- and C-terminal, and use of multibranched compounds. The advantage of a multibranched peptide is its reduced susceptibility to enzymatic degradation, while the biological effects and the high selectivity of action of the peptide sequence are maintained. In the peptide receptor field, multibranched molecules have been synthesized and evaluated using different peptide sequences including neurotensin (Falciani et al., 2007a), melanocytestimulating hormone (Brabez et al., 2011), enkephalins (Falciani et al., 2007b) and nociceptin/orphanin FQ (N/OFQ) (Bracci et al., 2003). In these examples, the biological activity of the peptide sequences was maintained and their metabolic stability was prolonged by several fold. However, the synthesis of these molecules is rather challenging and is in general characterized by low yield and purity of the final product. We have recently developed an innovative chemical approach, named peptide welding technology (PWT), that allows the synthesis of multibranched peptides with an
extraordinary high yield, purity and reproducibility. Details of the PWT chemical strategy have been recently published (Guerrini et al., 2014). With this approach, three different tetrabranched derivatives of N/OFQ have been synthesized and named PWT1-N/OFQ, PWT2-N/OFQ and PWT3-N/OFQ. These compounds differ exclusively in the core used: (Lys)2Lys-NH2 for PWT1-N/OFQ, Cyclam for PWT2-N/OFQ and (Lys)2-ethylendiamine for PWT3-N/OFQ. The chemical structures of the PWT derivatives of N/OFQ are shown in Figure 1. N/OFQ (FGGFTGARKSARKLANQ) was identified as the endogenous ligand of a previously described orphan receptor (Meunier et al., 1995; Reinscheid et al., 1995), now referred to as the N/OFQ peptide (NOP) receptor. NOP is a GPCR whose activation produces inhibition of cAMP levels, stimulation of potassium currents and inhibition of calcium channels. The peptide and its receptor are widely distributed in the nervous system where, via inhibitory mechanisms, they modulate several different biological functions (Lambert, 2008). In the present study, we investigated the in vitro and in vivo pharmacological profile of PWT N/OFQ derivatives and compared their actions with those of the naturally occurring peptide.
Methods Cell culture and membrane preparation CHO cells stably expressing human classical opioid receptors (MOP, KOP and DOP receptors) were maintained in Nutrient F12 containing 10% FBS; CHO cells expressing human NOP receptors were maintained in DMEM/Nutrient F12 (50/50) British Journal of Pharmacology (2014) 171 4138–4153
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H2N
NH2
O
HN FGGFTGARKSARKLANQ
O
S
O
O
N
H FGGFTGARKSARKLANQ N
O S
H2N
O
O
O HN
O
O
NH
PWT1-N/OFQ
NH2
HN O
O
O
H2N
N
NH
S
O HN
O
H2N FGGFTGARKSARKLANQ
N H
S
O
O
O O N
FGGFTGARKSARKLANQ
S
S
N O
O
S
O
N QNALKRASKRAGTFGGF H
O
O
O
N
O
FGGFTGARKSARKLANQ N H
O O
H N QNALKRASKRAGTFGGF
O
NH2
N
PWT2-N/OFQ
H2N
O
N
N
H2N
O
H2N
N
S O
O
O N
O
O
S
H N
QNALKRASKRAGTFGGF
QNALKRASKRAGTFGGF HN NH2
O
N
NH
O
N
N H
O
H N O
O
O
NH
O O
H2N O
Chemical structures of PWT derivatives of N/OFQ. British Journal of Pharmacology (2014) 171 4138–4153
N O
S H2N
O
N QNALKRASKRAGTFGGF H
N S
Figure 1
O
H N
N H
PWT3-N/OFQ
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NH QNALKRASKRAGTFGGF
N
NH
O
N
O
O
HN
O
S
O N QNALKRASKRAGTFGGF H
N/OFQ tetrabranched derivatives as NOP ligands
with 5% FBS; all media were further supplemented with penicillin (100 IU·mL−1), streptomycin (100 μg·mL−1) and fungizone (2.5 μg·mL−1). Stock cultures additionally contained geneticin (G418) (200 μg·mL−1) for CHOMOP/KOP/DOP cells, or G418 (200 μg·mL−1) and hygromycin B (200 μg·mL−1) for CHONOP cells. Cultures were sustained at 37°C with 5% carbon dioxide humidified air and subcultured twice weekly. Membranes were prepared from freshly harvested cells. Cells were suspended in homogenizing buffer consisting of either Tris (50 mM) for [3H]-diprenorphine displacement binding, Tris (50 mM), MgSO4 (5 mM) for [3H]-UFP-101 displacement binding or Tris (50 mM), EGTA (0.2 mM) for [35S]-GTPγS assays. Cell suspensions were homogenized and membranes were collected via centrifugation at 20 374× g for 10 min at 4°C. This process was repeated three times and protein concentrations were determined (Lowry et al., 1951).
Receptor binding
CHONOP membranes (40 μg) were incubated in 0.5 mL of buffer consisting of Tris (50 mM), BSA (0.5%), ∼0.8 nM [3H]UFP-101 and varying concentrations (10 μM–1 pM) of ligands. Non-specific binding was determined in the presence of 1 μM N/OFQ. In addition, 50 μg of membranes from CHO cells expressing classical opioid receptors was incubated in 0.5 mL buffer consisting of Tris (50 mM), BSA (0.5%), ∼0.8 nM [3H]-DPN and a range of concentrations of ligands. Non-specific binding was determined in the presence of 10 μM naloxone. For both displacement assays, reactions were incubated at room temperature for 1 h and terminated by vacuum filtration through polyethylenimine (0.5%)-soaked Whatman GF/B filters (Fisher Scientific, Loughborough, UK), using a Brandel harvester. Radioactivity was determined following an 8 h extraction of filters in ScintiSafe Gel using liquid scintillation spectroscopy.
Stimulation of [35S]-GTPγS binding
CHONOP membranes, 40 μg, were incubated in 0.5 mL buffer containing Tris (50 mM), EGTA (0.2 mM), MgCl2 (1 mM), NaCl (100 mM), BSA (0.1%), bacitracin (0.15 mM), GDP (100 mM) and ∼150 pM [35S]-GTPγS. NOP ligands were included in varying concentrations, and non-specific binding was determined in the presence of 10 μM GTPγS. Reactions were incubated for 1 h at 30°C with gentle shaking and terminated by vacuum filtration through dry Whatman GF/B filters. Radioactivity was determined following an 8 h extraction of filters in ScintiSafe Gel using liquid scintillation spectroscopy.
Calcium mobilization CHO cells stably co-expressing the human NOP, KOP or MOP receptor and the C-terminally modified Gαqi5 and CHO cells expressing the DOP receptor and the GαqG66Di5 protein were generated as previously described (Camarda et al., 2009; Camarda and Calo, 2013). Cells were maintained in culture medium consisting of DMEM/HAM’S F-12 (50/50) supplemented with 10% FCS, penicillin (100 IU·mL−1), streptomycin (100 μg·mL−1), fungizone (2.5 μg·mL−1), geneticin (G418; 200 μg·mL−1) and hygromycin B (200 mg·mL−1). Cell cultures were kept at 37°C in 5% CO2 humidified air. Experimental cultures were free from selection agents (hygromycin B,
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G418). When confluence was reached (3–4 days), cells were subcultured as required using trypsin/EDTA and used for experimentation. Cells were seeded at a density of 50 000 cells per well into 96-well black, clear-bottom plates. After 24 h of incubation, cells were loaded with medium supplemented with 2.5 mM probenecid, 3 μM of the calciumsensitive fluorescent dye Fluo-4 AM and 0.01% pluronic acid for 30 min at 37°C. Afterwards, the loading solution was aspirated and 100 μL per well of assay buffer: HBSS supplemented with 20 mM HEPES, 2.5 mM probenecid and 500 μM Brilliant Black was added. Stock solutions (1 mM) of peptides and PWT compounds were made in distilled water and stored at −20°C. SB-612111 was solubilized (10 mM) in DMSO. Serial dilutions of ligands for experimental use were made in HBSS/ HEPES (20 mM) buffer (containing 0.03% BSA fraction V). After the cell culture and compound plates had been placed in the FlexStation II (Molecular Device, Union City, CA USA), fluorescence changes were measured. Online additions were carried out in a volume of 50 μL per well. To facilitate drug diffusion into the wells in antagonist-type experiments, the present studies were performed at 37°C and three cycles of mixing (25 μL from each well) were performed immediately after antagonist injection.
Electrically stimulated mouse vas deferens (mVD) Tissues were taken from NOP(+/+) or NOP(−/−) male mice. The mVD tissues were prepared as previously described (Calo et al., 1996). Tissues were suspended in 5 mL of organ baths containing heated Krebs solution (composition in mM: NaCl 118.5, KCl 4.7, KH2PO4 1.2, NaHCO3 25, glucose 10 and CaCl2 2.5) oxygenated with 95% O2 and 5% CO2. The bath temperature was set at 33°C. Tissues were continuously stimulated through two platinum ring electrodes with supramaximal rectangular pulses of 1 ms duration and 0.05 Hz frequency. A resting tension of 0.3× g was applied to the vas deferens. The electrically-evoked contractions (twitches) were measured isotonically with a strain gauge transducer (Basile 7006; Ugo Basile s.r.l., Varese, Italy) and recorded with the PC-based acquisition system Power Lab (ADInstrument, Colorado Springs, CO, USA). Following an equilibration period of 60 min, the contractions induced by electrical field stimulation were stable. At this time, cumulative concentration–response curves to N/OFQ, its PWT derivatives or DPDPE were performed.
Mouse locomotor activity All experimental procedures adopted for in vivo studies were as humane as possible and complied with the ARRIVE guidelines (Kilkenny et al., 2010) and the standards of the European Communities Council directives (86/609/EEC) and national regulations (D.L. 116/92). Male CD-1 mice (30–38 g, Harlan, San Pietro in Natisone, Udine, Italy) and male NOP(+/+) or NOP(−/−) mice were used. The total number of mice used for this study was 360. They were housed in Plexiglas® cages (Tecniplast, Buguggiate, Varese, Italy), under standard conditions (22°C, 55% humidity, 12 h light–dark cycle, lights on 07:00 h) with food and water available ad libitum for at least 5 days before experiments were begun. Experiments were performed according to the procedures British Journal of Pharmacology (2014) 171 4138–4153
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described previously (Guerrini et al., 2009). For these experiments, the ANY-maze video tracking system was used (Ugo Basile, Varese, Italy, application version 4.52c Beta). Mice were positioned in a square plastic cage (40 × 40 cm), 1 mouse per cage. Four mice were monitored in parallel. Mouse horizontal activity was monitored by a camera, whereas vertical activity was measured by an infrared beam array. The parameters measured were cumulative distance travelled (total distance in metres that the animal travelled during the test), immobility time (the animal is considered immobile when 90% of it remains in the same place for a minimum of 2.5 s) and the number of rearing (the number of beam breaks due to vertical movements; this input is triggered when the beam is interrupted for a minimum of 200 ms). N/OFQ, PWT1-N/ OFQ, PWT2-N/OFQ and PWT3-N/OFQ were given i.c.v. (2 μL per mouse). Free hand i.c.v. injections were given, under isoflurane anaesthesia (just sufficient to produce a loss of the righting reflex), in the left ventricle according to literature procedures (Laursen and Belknap, 1986).
Data analysis and statistical procedures
In vitro data were expressed as mean ± SEM of at least three separate experiments. The concentration of drug producing 50% displacement of specific binding (IC50) was corrected for the competing mass of radioligand according to Cheng and Prusoff (1973) to yield Ki using KD. KD values of [3H]-UFP-101 at NOP and [3H]-diprenorphine at classical opioid receptors were taken from previous studies (Kitayama et al., 2003; Ibba et al., 2008). [35S]-GTPγS data are expressed as stimulation factor (agonist specific binding/basal specific binding). In calcium mobilization experiments, maximum change in fluorescence, expressed as percentage over the baseline fluorescence, was used to determine agonist response. Non-linear regression analysis using GraphPad Prism software (5.0; GraphPad Software, San Diego, CA, USA) allowed logistic iterative fitting of the resultant responses and the calculation of agonist potencies and maximal effects. Agonist potencies were given as pEC50 (the negative logarithm to base 10 of the molar concentration of an agonist that produces 50% of the maximal possible effect). In vivo data are expressed as mean ± SEM of n animals. Data were analysed using one-way ANOVA followed by Dunnett’s post hoc test or with Student’s t-test. Differences were considered statistically significant when P < 0.05.
Results Receptor binding In CHONOP cell membranes, N/OFQ displaced [3H]-UFP-101 binding in a concentration-dependent manner showing high affinity (pKi 9.42). Similar results were obtained with PWT derivatives of N/OFQ; however, they displayed increased affinity by 5 (PWT3-N/OFQ) up to 15 (PWT1-N/OFQ) fold (Table 1). To investigate selectivity over classical opioid receptors, similar experiments were performed using membranes obtained from CHO cells expressing the MOP, KOP and DOP receptors and [3H]-diprenorphine as radioligand. N/OFQ up to 1 μM did not bind to KOP and DOP receptors while it displayed micromolar affinity at MOP receptors (pKi 6.06). All PWT derivatives showed higher affinity than N/OFQ at classical opioid receptors. However, their selectivity for the NOP receptor (ranging from 2570- to 14 125-fold) was never inferior to that displayed by the natural peptide (2291-fold). In parallel experiments, standard ligands for classical opioid receptors displayed the expected high affinity (morphine, pKi 8.57 for MOP receptors; Dmt-Tic, pKi 8.87 for DOP receptors; and norbinaltorphimine, pKi 10.69 for KOP receptors).
Stimulation of [35S]-GTPγS binding In CHONOP cell membranes, N/OFQ stimulated [35S]-GTPγS binding in a concentration-dependent manner with maximal effects of 3.5 ± 0.2 and pEC50 of 8.84 (Figure 2, top panel). PWT derivatives of N/OFQ mimicked the stimulatory effect of the natural peptide showing similar maximal effects but higher potency (pEC50 9.71–10.12; Figure 2, top panel).
Calcium mobilization In CHONOP cells stably expressing the Gαqi5 chimeric protein, N/OFQ and its PWT derivatives evoked a concentrationdependent stimulation of calcium release (Figure 2, bottom panel). N/OFQ displayed high potency (pEC50 9.39) and maximal effects (237 ± 15%). PWT derivatives of N/OFQ mimicked the peptide stimulatory effects showing similar maximal effects but slightly lower potency (pEC50 8.75–9.16; Figure 2, bottom panel). Inhibition experiments were performed by testing increasing concentrations of the NOP receptor antagonist SB-612111 against a fixed concentration of agonist approximately corresponding to its EC80 (10 nM for
Table 1 Affinities of N/OFQ and its PWT derivatives in membranes of CHO cells expressing the human NOP or classical opioid receptors
N/OFQ
NOP
MOP
KOP
DOP
pKi (CL95%)
pKi (CL95%)
pKi (CL95%)
pKi (CL95%)
