Accepted Manuscript Structure activity studies of nociceptin/orphanin FQ(1-13)-NH2 derivatives modified in position 5 Remo Guerrini, Erika Marzola, Claudio Trapella, Salvatore Pacifico, Maria Camilla Cerlesi, Davide Malfacini, Federica Ferrari, Mark Francis Bird, David George Lambert, Severo Salvadori, Girolamo Calo PII: DOI: Reference:
S0968-0896(15)00092-9 http://dx.doi.org/10.1016/j.bmc.2015.02.008 BMC 12071
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
12 January 2015 27 January 2015 4 February 2015
Please cite this article as: Guerrini, R., Marzola, E., Trapella, C., Pacifico, S., Cerlesi, M.C., Malfacini, D., Ferrari, F., Bird, M.F., Lambert, D.G., Salvadori, S., Calo, G., Structure activity studies of nociceptin/orphanin FQ(1-13)NH2 derivatives modified in position 5, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/ j.bmc.2015.02.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structure activity studies of nociceptin/orphanin FQ(1-13)-NH2 derivatives modified in position 5
Remo Guerrini1,2*, Erika Marzola1,2, Claudio Trapella1,2, Salvatore Pacifico 1, Maria Camilla Cerlesi3, Davide Malfacini3, Federica Ferrari3, Mark Francis Bird 4, David George Lambert4, Severo Salvadori1,2 and Girolamo Calo’3
1
Department of Chemical and Pharmaceutical Sciences, 2Laboratorio per le tecnologie delle terapie
avanzate (LTTA), 3Department of Medical Science, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, 44121 Ferrara, Italy. 4Department of Cardiovascular Sciences, University of Leicester, Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE2 7LX. UK.
*Corresponding author: Remo Guerrini, PhD tel: +39 0532 455 988 e.mail: [email protected]
Keywords: nociceptin/orphanin FQ; peptide synthesis, NOP receptor, [35S]GTPγS binding assay, calcium mobilization assay.
1
Abstract
Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide acting as the endogenous ligand of the N/OFQ peptide receptor (NOP). N/OFQ(1-13)-NH2 is the shortest N/OFQ sequence maintaining the same potency and efficacy as the natural peptide. Thus N/OFQ(1-13)-NH2 was used as chemical template for investigating the structure activity relationship of threonine in position 5. 28 [X5]N/OFQ(1-13)-NH2 derivatives, in which Thr was substituted with natural and unnatural residues, were synthesized and characterized pharmacologically for their effects at the human NOP receptor. Two different functional assays were used: agonist stimulated [35S]GTPγS binding in cell membranes and calcium mobilization in whole cells co-expressing chimeric G proteins. All [X5]N/OFQ(1-13)-NH2 derivatives behaved as full NOP agonists showing large differences in their potency. There was an excellent correlation between the results obtained in the two assays. The results of this study suggest that: position 5 does not play a pivotal role in receptor activation; the secondary alcoholic function of Thr is not important for receptor binding; side chain size, lipo / hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. In conclusion this study demonstrates that a simple ethyl side chain as in compound 23 is sufficient in N/OFQ position 5 for maintaining bioactivity.
2
1. Introduction The heptadecapeptide nociceptin/orphanin FQ1, 2 (N/OFQ) is the endogenous ligand of a previously orphan G-protein coupled receptor now named NOP. The N/OFQ-NOP receptor system modulates several physiological functions both at central and peripheral levels including nociception, locomotor activity, anxiety and depression, learning and memory, drug abuse, cardiovascular and gastrointestinal functions, immunity, and some reflexes such as cough and micturition. 3 Thus the NOP receptor represents an attractive target for the identification and development of innovative drugs to treat a large variety of pathologies. Structure activity relationship (SAR) studies indicated N/OFQ(1-13)-NH2 as the minimal bioactive sequence.
4, 5
This peptide has been used as chemical template for a series of SAR studies. In
particular a large series of N/OFQ(1-13)-NH2 derivatives were generated for investigating the chemical requirements of the N terminal tetrapeptide Phe-Gly-Gly-Phe5, the Phe1-Gly2 peptide bond 6, 7, the importance of Phe1 for selectivity over classical opioid receptors8, the crucial role of Phe1 for ligand efficacy9, the structure activity relationship of Nphe1
10
and Phe4
11
. These SAR
studies were instrumental in describing the chemical features of N/OFQ essential for NOP receptor occupation and activation and led to the discovery of NOP ligands encompassing full ([(pF)Phe4]N/OFQ(1-13)-NH2, NH2,
12, 13
) and partial agonist ([Phe1ψ(CH2-NH)Gly2 ] N/OFQ(1-13)-
6, 14
) as well as pure antagonist ([Nphe1]N/OFQ(1-13)-NH2,
15
) activity. These NOP ligands
have been extensively investigated and characterized in several in vitro and in vivo assays.
16
In
parallel, the replacement of position 14 and 15 with a couple of Arg-Lys residues in the natural peptide sequence or in the NOP antagonist peptide [Nphe1]N/OFQ-NH2 generated highly potent NOP ligands acting as agonist17, 18 and antagonist19, respectively. This suggests that the C-terminal part of the peptide is important for NOP receptor occupation. N/OFQ and Dynorphin (Dyn) share some similarities, in particular the same length, the N-terminal part with two aromatic residues linked by a Gly-Gly spacer and a C-terminal part rich in basic residues. Despite these structural homologies, N/OFQ is highly selective for the NOP and Dyn for the KOP receptor. The systematic replacement in the C-terminus of the Dyn sequence with residues of the C-terminus of N/OFQ slightly increased affinity but did not affect potency at the NOP receptor. In contrast, further replacement of position 6 (Arg/Gly) and 5 (Lue/Thr) substantially increased affinity and produced a dramatic enhancement of NOP potency.
20
An important role of
N/OFQ Thr5 for NOP occupation/activation is also suggested by classical Ala scan studies that demonstrated a substantial loss of affinity and an even greater loss of potency of [Ala5 ]N/OFQ.4, 21 3
Thus, the aim of this study was the investigation of the structure activity relationship of the Thr residue in position 5 of the N/OFQ sequence. To this aim N/OFQ(1-13)-NH2 was used as chemical template and 28 [X5]N/OFQ(1-13)-NH2 derivatives, in which Thr was substituted with natural and unnatural residues (Figure 1), were synthesized, purified and characterized for their pharmacological action at the NOP receptor.
2. Results 2.1 [35S]GTPγS binding studies Data obtained in [35S]GTPγS binding experiments with membranes of CHO cells stably expressing the human recombinant NOP receptor were reported in Table 1 In line with previous findings22, N/OFQ stimulated [35S]GTPγS binding to CHOhNOP membranes in a concentration dependent manner showing a maximal effect of 210 ± 17 % over the basal value and a pEC50 of 8.45. This stimulatory effect of N/OFQ was mimicked by N/OFQ(1-13)-NH2 that produced a superimposable concentration response curve (Emax 199 ± 16 %, pEC50 8.57). Under the same experimental conditions, [X5]N/OFQ(1–13)-NH2 analogues in which Thr was substituted with natural amino acids (compounds 1-13) stimulated [35S]GTPγS binding in a concentration dependent manner showing maximal effects similar to those evoked by the reference compound N/OFQ(1–13)-NH2 (Table 1). As far as potency is concerned, compound 5 and 10 displayed potency similar to that of N/OFQ(1–13)-NH2, while all the other analogues showed lower potency. In particular compounds 4, 8, 9 and 12 had pEC50 values from 3 to 10 fold lower than the reference peptide, compounds 1, 2, 6, 7, 11 and 13 had pEC50 values from 10 to 100 fold lower than N/OFQ(1–13)-NH2, while compound 3 was 117 fold less potent than the standard. In a second series of experiments the effects of [X5]N/OFQ(1–13)-NH2 analogues in which Thr was substituted with unnatural amino acids (compounds 14-28) were investigated. All compounds stimulated [35S]GTPγS binding in a concentration dependent with maximal effects similar to those evoked by N/OFQ(1–13)-NH2 (Table 1). As far as potency is concerned, compounds 16 and 23 displayed similar potency to that of N/OFQ(1–13)-NH2, while all the other analogues displayed lower potency. In particular compounds 15, 19-22, and 26 showed pEC50 values from 3 to 10 fold lower than the reference peptide, compounds 18, 24, 25, 27, and 28 had pEC50 values from 10 to 100 fold lower than N/OFQ(1–13)-NH2, while compounds 14 and 17 were more than 100 fold less potent than the standard. 4
2.2 Calcium mobilization studies Data obtained in calcium mobilization experiments in CHO cells stably co-expressing the human recombinant NOP receptor and the Gαqi5 chimeric protein are reported in Table 2. In line with previous findings23, N/OFQ stimulated calcium mobilization in a concentration dependent manner with a maximal effect of 238 ± 17 % over the basal values and a pEC50 of 8.74. The stimulatory effect of N/OFQ was mimicked by N/OFQ(1-13)-NH2 that produced a superimposable concentration response curve (Emax of 232 ± 13, pEC50 9.07). Under the same experimental conditions, the effects of [X5]N/OFQ(1–13)-NH2 analogues in which Thr was substituted with natural amino acids were evaluated. All compounds stimulated calcium mobilization with maximal effects similar to those evoked by the reference compound N/OFQ(1– 13)-NH2 (Table 2). As far as potency is concerned, compound 5 and 10 displayed potency similar to that of N/OFQ(1–13)-NH2, while all the other analogues showed lower potency. In particular compounds 9 and 12 had pEC50 values from 3 to 10 fold lower than the reference peptide, compounds 1, 4, 6-8, and 11 had pEC50 values from 10 to 100 fold lower than N/OFQ(1–13)-NH2, while compound 2 and 13 were 159 and 760 fold less potent than the standard, respectively. Finally the potency of compound 3 could not be estimated because the compound produced an incomplete concentration response curve. In the last series of experiments the effects of [X5]N/OFQ(1–13)-NH2 analogues in which Thr was substituted with unnatural amino acids (compounds 14-28) were investigated. All compounds stimulated calcium mobilization in a concentration dependent manner showing maximal effects similar to those evoked by N/OFQ(1–13)-NH2 (Table 2). As far as potency is concerned, compounds 21-23 displayed potency similar to that of N/OFQ(1–13)-NH2, while all the other analogues showed lower potency. In particular, compound 16 was 4 fold less potent than the reference peptide, compounds 15, 19, 20, and 26-28 had pEC50 values from 10 to 100 fold lower than N/OFQ(1–13)-NH2, while compounds 14, 17, 18, 24 and 25 were more than 100 fold less potent than the standard. The results obtained in the [35S]GTPγS binding and in the calcium mobilization assay have been compared in figure 2: a determination coefficient (r2) of 0.82 and a value of p 25 displayed by their linear analogs 18 > 3. Collectively these findings demonstrated that both the presence of the basic amino function and its distance from the peptide backbone play a very important role in disrupting peptide receptor binding. Finally modulation of the distance from the peptide backbone of the nitrogen atom obtained with aromatic derivatives as in compounds 26-28 does not substantially affect ligand potency. This result is similar to that obtained with the natural aromatic residues Phe, Tyr and His. Thus, these findings corroborate the proposal that NOP receptor binding is sensitive to the spatial disposition of the positive charge of the basic nitrogen atom of the X5 residue. 7
4. Conclusions In conclusion this study investigated the structure activity relationship of the position 5 of N/OFQ sequence. The major findings of this investigation can be summarized as follows: i) position 5 does not play a pivotal role in receptor activation; ii) the secondary alcoholic function of Thr is not important for receptor binding; iii) side chain size, lipo / hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; iv) an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. This study in addition to extending our knowledge on the structure activity relationship of N/OFQ allowed the identification of N/OFQ(1-13)-NH2 derivatives (i.e. compound 23) as potent as the natural sequence. The presence of unnatural residues e.g. Abu in the peptide sequence may at least partially prevent enzymatic degradation thus generating longer lasting effects in vivo. Further studies are needed to validate this proposal.
4. Experimental Section 4.1. General Amino acid derivatives, reagents and solvents were purchased from Sigma Aldrich (Steinheim, Germany) or Bachem (Bubendorf, Switzerland). The purity of the tested compounds has been assessed by RP-HPLC. All compounds showed >95% purity. MS analyses were performed on an ESI-Micromass ZMD 2000.
4.2. General procedures for the solid phase peptide synthesis All peptides were synthesized following the procedures previously reported5 using Fmoc/t-butyl chemistry with a Syro XP multiple peptide synthesizer (MultiSynTech GmbH, Witten Germany). Rink amide MBHA resin (loading 0.51 mmol/g) was used as a solid support. Final peptides were side-chain deprotected and simultaneously cleaved from the resin by using the mixture TFA/TIS/water (90/5/5, v/v/v) for 3 h at room temperature. After filtration of the exhausted resin, precipitation from ice-cold diethyl ether and recovery by centrifugation provided the crude products. 8
4.3. Peptide purification and analytical determinations
Crude peptides were purified by preparative reversed-phase HPLC using aWaters Delta Prep 3000 system with a Jupiter column C18 (250 x 30 mm, 300 A, 15 µ spherical particle size). The column was perfused at a flow rate of 20 mL/min with a mobile phase containing solvent A (10%, v/v, acetonitrile in 0.1% TFA), and a linear gradient from 0% to 40% of solvent B (60%, v/v, acetonitrile in 0.1% TFA) over 25 min for the elution of peptides. Analytical HPLC analyses were performed on a Beckman 116 liquid chromatograph equipped with a Beckman 166 diode array detector. Analytical purity of the peptides were determined using a Luna C18 column (4.6 x 100 mm, 3 µ particle size) with the above solvent system (solvents A and B) programmed at a flow rate of 0.5 mL/min with a linear gradient from 0% to 65% B over 25 min. All analogues showed >95% purity when monitored at 220 nm. Molecular weights of final compounds were determined by a mass spectrometer ESI Micromass ZMD-2000.
4.4. Calcium mobilization assay
Chinese Hamster Ovary (CHO) cells stably co-expressing human recombinant NOP receptors and the C-terminally modified Gαqi5, were generated as previously described.23, 26 Cells were cultured in Dulbecco’s MEM/HAM’S F12 (1:1) culture medium supplemented with 10% fetal calf serum, penicillin (100 IU/ml), streptomycin (100 mg/ml), geneticin (G418; 200 g/ml) and hygromycin B (100 g/ml). Cell cultures were kept at 37◦C in 5% CO2/humidified air. When confluence was reached (3–4 days), cells were subcultured using trypsin/EDTA and used for the assay. Cells were seeded at a density of 50,000 cells/well into 96-wellblack, clear-bottom plates. After 24 h incubation, the cells were treated with the loading solution of the culture medium supplemented with 2.5 mM probenecid, 3 µM of the calcium sensitive fluorescent dye Fluo-4 AM and 0.01% pluronic acid and HEPES 1M for 30 min at 37◦C. The loading solution was aspirated and a 100/well of the assay buffer (Hank’s Balanced Salt Solution (HBSS) supplemented with 20 mM HEPES, 2.5 mM probenecid, and 500 µM Brilliant Black) was added. The tested peptides were dissolved in the bi-distilled water to the final concentration of 1 mM. The successive dilutions were made in the HBSS/HEPES (20 mM) buffer (containing 0.02% BSA fraction V).After placing both plates (cell culture and compound plate) into the FlexStation II, the on-line additions were carried out in a volume of 50/well and fluorescence changes were measured at 37◦C.
9
4.5. Stimulation of [35S]-GTPγS binding assay CHO cells stably expressing human NOP receptors were maintained in DMEM/Nutrient F12 (50/50) with 5% FBS; 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) and hygromycin B (200 µg·mL−1) and used experimentally when confluent. Cultures were sustained at 37°C with 5% carbon dioxide humidified air and sub-cultured using trypsin. Membranes were prepared from freshly harvested cells. Cells were centrifuged at 423 g for 3 minutes before being suspended in homogenizing buffer consisting of Tris (50 mM) and EGTA (0.2 mM). Cell suspensions were homogenized and membranes were collected via centrifugation at 10,000 g for 10 min at 4°C. This process was repeated twice and protein concentrations were determined. 27 CHONOP membranes, 40 µg, were incubated in 0.5 mL of assay buffer containing Tris-HCl (50 mM), EGTA (0.2 mM), NaCl (100 mM), MgCl2 (1 mM), bacitracin (0.15 mM), BSA (0.15 mM), GDP (33 µM) and ∼150 pM [35S]GTPγS. The ligands were added in serial concentrations (10µM – 1 pM), 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.
4.6 Data analysis Agonist potencies were given as pEC50 representing a negative logarithm of the molar concentration of an agonist that produces 50% of the maximal possible effect. Concentration response curves were fitted with the four parameter logistic nonlinear regression model. Curve fittings were performed using GraphPad PRISM 5.0 (GraphPad Software Inc., San Diego, USA) Ligand efficacy was expressed as intrinsic activity (α) calculated as the ratio between the Emax of the ligand and that of the standard N/OFQ(1-13)-NH2. At least five independent experiments for each assay were carried out in duplicate. Data have been statistically analyzed with one way ANOVA followed by the Dunnett’s test for multiple comparisons; p values
S0968-0896(15)00092-9 http://dx.doi.org/10.1016/j.bmc.2015.02.008 BMC 12071
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
12 January 2015 27 January 2015 4 February 2015
Please cite this article as: Guerrini, R., Marzola, E., Trapella, C., Pacifico, S., Cerlesi, M.C., Malfacini, D., Ferrari, F., Bird, M.F., Lambert, D.G., Salvadori, S., Calo, G., Structure activity studies of nociceptin/orphanin FQ(1-13)NH2 derivatives modified in position 5, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/ j.bmc.2015.02.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structure activity studies of nociceptin/orphanin FQ(1-13)-NH2 derivatives modified in position 5
Remo Guerrini1,2*, Erika Marzola1,2, Claudio Trapella1,2, Salvatore Pacifico 1, Maria Camilla Cerlesi3, Davide Malfacini3, Federica Ferrari3, Mark Francis Bird 4, David George Lambert4, Severo Salvadori1,2 and Girolamo Calo’3
1
Department of Chemical and Pharmaceutical Sciences, 2Laboratorio per le tecnologie delle terapie
avanzate (LTTA), 3Department of Medical Science, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, 44121 Ferrara, Italy. 4Department of Cardiovascular Sciences, University of Leicester, Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE2 7LX. UK.
*Corresponding author: Remo Guerrini, PhD tel: +39 0532 455 988 e.mail: [email protected]
Keywords: nociceptin/orphanin FQ; peptide synthesis, NOP receptor, [35S]GTPγS binding assay, calcium mobilization assay.
1
Abstract
Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide acting as the endogenous ligand of the N/OFQ peptide receptor (NOP). N/OFQ(1-13)-NH2 is the shortest N/OFQ sequence maintaining the same potency and efficacy as the natural peptide. Thus N/OFQ(1-13)-NH2 was used as chemical template for investigating the structure activity relationship of threonine in position 5. 28 [X5]N/OFQ(1-13)-NH2 derivatives, in which Thr was substituted with natural and unnatural residues, were synthesized and characterized pharmacologically for their effects at the human NOP receptor. Two different functional assays were used: agonist stimulated [35S]GTPγS binding in cell membranes and calcium mobilization in whole cells co-expressing chimeric G proteins. All [X5]N/OFQ(1-13)-NH2 derivatives behaved as full NOP agonists showing large differences in their potency. There was an excellent correlation between the results obtained in the two assays. The results of this study suggest that: position 5 does not play a pivotal role in receptor activation; the secondary alcoholic function of Thr is not important for receptor binding; side chain size, lipo / hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. In conclusion this study demonstrates that a simple ethyl side chain as in compound 23 is sufficient in N/OFQ position 5 for maintaining bioactivity.
2
1. Introduction The heptadecapeptide nociceptin/orphanin FQ1, 2 (N/OFQ) is the endogenous ligand of a previously orphan G-protein coupled receptor now named NOP. The N/OFQ-NOP receptor system modulates several physiological functions both at central and peripheral levels including nociception, locomotor activity, anxiety and depression, learning and memory, drug abuse, cardiovascular and gastrointestinal functions, immunity, and some reflexes such as cough and micturition. 3 Thus the NOP receptor represents an attractive target for the identification and development of innovative drugs to treat a large variety of pathologies. Structure activity relationship (SAR) studies indicated N/OFQ(1-13)-NH2 as the minimal bioactive sequence.
4, 5
This peptide has been used as chemical template for a series of SAR studies. In
particular a large series of N/OFQ(1-13)-NH2 derivatives were generated for investigating the chemical requirements of the N terminal tetrapeptide Phe-Gly-Gly-Phe5, the Phe1-Gly2 peptide bond 6, 7, the importance of Phe1 for selectivity over classical opioid receptors8, the crucial role of Phe1 for ligand efficacy9, the structure activity relationship of Nphe1
10
and Phe4
11
. These SAR
studies were instrumental in describing the chemical features of N/OFQ essential for NOP receptor occupation and activation and led to the discovery of NOP ligands encompassing full ([(pF)Phe4]N/OFQ(1-13)-NH2, NH2,
12, 13
) and partial agonist ([Phe1ψ(CH2-NH)Gly2 ] N/OFQ(1-13)-
6, 14
) as well as pure antagonist ([Nphe1]N/OFQ(1-13)-NH2,
15
) activity. These NOP ligands
have been extensively investigated and characterized in several in vitro and in vivo assays.
16
In
parallel, the replacement of position 14 and 15 with a couple of Arg-Lys residues in the natural peptide sequence or in the NOP antagonist peptide [Nphe1]N/OFQ-NH2 generated highly potent NOP ligands acting as agonist17, 18 and antagonist19, respectively. This suggests that the C-terminal part of the peptide is important for NOP receptor occupation. N/OFQ and Dynorphin (Dyn) share some similarities, in particular the same length, the N-terminal part with two aromatic residues linked by a Gly-Gly spacer and a C-terminal part rich in basic residues. Despite these structural homologies, N/OFQ is highly selective for the NOP and Dyn for the KOP receptor. The systematic replacement in the C-terminus of the Dyn sequence with residues of the C-terminus of N/OFQ slightly increased affinity but did not affect potency at the NOP receptor. In contrast, further replacement of position 6 (Arg/Gly) and 5 (Lue/Thr) substantially increased affinity and produced a dramatic enhancement of NOP potency.
20
An important role of
N/OFQ Thr5 for NOP occupation/activation is also suggested by classical Ala scan studies that demonstrated a substantial loss of affinity and an even greater loss of potency of [Ala5 ]N/OFQ.4, 21 3
Thus, the aim of this study was the investigation of the structure activity relationship of the Thr residue in position 5 of the N/OFQ sequence. To this aim N/OFQ(1-13)-NH2 was used as chemical template and 28 [X5]N/OFQ(1-13)-NH2 derivatives, in which Thr was substituted with natural and unnatural residues (Figure 1), were synthesized, purified and characterized for their pharmacological action at the NOP receptor.
2. Results 2.1 [35S]GTPγS binding studies Data obtained in [35S]GTPγS binding experiments with membranes of CHO cells stably expressing the human recombinant NOP receptor were reported in Table 1 In line with previous findings22, N/OFQ stimulated [35S]GTPγS binding to CHOhNOP membranes in a concentration dependent manner showing a maximal effect of 210 ± 17 % over the basal value and a pEC50 of 8.45. This stimulatory effect of N/OFQ was mimicked by N/OFQ(1-13)-NH2 that produced a superimposable concentration response curve (Emax 199 ± 16 %, pEC50 8.57). Under the same experimental conditions, [X5]N/OFQ(1–13)-NH2 analogues in which Thr was substituted with natural amino acids (compounds 1-13) stimulated [35S]GTPγS binding in a concentration dependent manner showing maximal effects similar to those evoked by the reference compound N/OFQ(1–13)-NH2 (Table 1). As far as potency is concerned, compound 5 and 10 displayed potency similar to that of N/OFQ(1–13)-NH2, while all the other analogues showed lower potency. In particular compounds 4, 8, 9 and 12 had pEC50 values from 3 to 10 fold lower than the reference peptide, compounds 1, 2, 6, 7, 11 and 13 had pEC50 values from 10 to 100 fold lower than N/OFQ(1–13)-NH2, while compound 3 was 117 fold less potent than the standard. In a second series of experiments the effects of [X5]N/OFQ(1–13)-NH2 analogues in which Thr was substituted with unnatural amino acids (compounds 14-28) were investigated. All compounds stimulated [35S]GTPγS binding in a concentration dependent with maximal effects similar to those evoked by N/OFQ(1–13)-NH2 (Table 1). As far as potency is concerned, compounds 16 and 23 displayed similar potency to that of N/OFQ(1–13)-NH2, while all the other analogues displayed lower potency. In particular compounds 15, 19-22, and 26 showed pEC50 values from 3 to 10 fold lower than the reference peptide, compounds 18, 24, 25, 27, and 28 had pEC50 values from 10 to 100 fold lower than N/OFQ(1–13)-NH2, while compounds 14 and 17 were more than 100 fold less potent than the standard. 4
2.2 Calcium mobilization studies Data obtained in calcium mobilization experiments in CHO cells stably co-expressing the human recombinant NOP receptor and the Gαqi5 chimeric protein are reported in Table 2. In line with previous findings23, N/OFQ stimulated calcium mobilization in a concentration dependent manner with a maximal effect of 238 ± 17 % over the basal values and a pEC50 of 8.74. The stimulatory effect of N/OFQ was mimicked by N/OFQ(1-13)-NH2 that produced a superimposable concentration response curve (Emax of 232 ± 13, pEC50 9.07). Under the same experimental conditions, the effects of [X5]N/OFQ(1–13)-NH2 analogues in which Thr was substituted with natural amino acids were evaluated. All compounds stimulated calcium mobilization with maximal effects similar to those evoked by the reference compound N/OFQ(1– 13)-NH2 (Table 2). As far as potency is concerned, compound 5 and 10 displayed potency similar to that of N/OFQ(1–13)-NH2, while all the other analogues showed lower potency. In particular compounds 9 and 12 had pEC50 values from 3 to 10 fold lower than the reference peptide, compounds 1, 4, 6-8, and 11 had pEC50 values from 10 to 100 fold lower than N/OFQ(1–13)-NH2, while compound 2 and 13 were 159 and 760 fold less potent than the standard, respectively. Finally the potency of compound 3 could not be estimated because the compound produced an incomplete concentration response curve. In the last series of experiments the effects of [X5]N/OFQ(1–13)-NH2 analogues in which Thr was substituted with unnatural amino acids (compounds 14-28) were investigated. All compounds stimulated calcium mobilization in a concentration dependent manner showing maximal effects similar to those evoked by N/OFQ(1–13)-NH2 (Table 2). As far as potency is concerned, compounds 21-23 displayed potency similar to that of N/OFQ(1–13)-NH2, while all the other analogues showed lower potency. In particular, compound 16 was 4 fold less potent than the reference peptide, compounds 15, 19, 20, and 26-28 had pEC50 values from 10 to 100 fold lower than N/OFQ(1–13)-NH2, while compounds 14, 17, 18, 24 and 25 were more than 100 fold less potent than the standard. The results obtained in the [35S]GTPγS binding and in the calcium mobilization assay have been compared in figure 2: a determination coefficient (r2) of 0.82 and a value of p 25 displayed by their linear analogs 18 > 3. Collectively these findings demonstrated that both the presence of the basic amino function and its distance from the peptide backbone play a very important role in disrupting peptide receptor binding. Finally modulation of the distance from the peptide backbone of the nitrogen atom obtained with aromatic derivatives as in compounds 26-28 does not substantially affect ligand potency. This result is similar to that obtained with the natural aromatic residues Phe, Tyr and His. Thus, these findings corroborate the proposal that NOP receptor binding is sensitive to the spatial disposition of the positive charge of the basic nitrogen atom of the X5 residue. 7
4. Conclusions In conclusion this study investigated the structure activity relationship of the position 5 of N/OFQ sequence. The major findings of this investigation can be summarized as follows: i) position 5 does not play a pivotal role in receptor activation; ii) the secondary alcoholic function of Thr is not important for receptor binding; iii) side chain size, lipo / hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; iv) an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. This study in addition to extending our knowledge on the structure activity relationship of N/OFQ allowed the identification of N/OFQ(1-13)-NH2 derivatives (i.e. compound 23) as potent as the natural sequence. The presence of unnatural residues e.g. Abu in the peptide sequence may at least partially prevent enzymatic degradation thus generating longer lasting effects in vivo. Further studies are needed to validate this proposal.
4. Experimental Section 4.1. General Amino acid derivatives, reagents and solvents were purchased from Sigma Aldrich (Steinheim, Germany) or Bachem (Bubendorf, Switzerland). The purity of the tested compounds has been assessed by RP-HPLC. All compounds showed >95% purity. MS analyses were performed on an ESI-Micromass ZMD 2000.
4.2. General procedures for the solid phase peptide synthesis All peptides were synthesized following the procedures previously reported5 using Fmoc/t-butyl chemistry with a Syro XP multiple peptide synthesizer (MultiSynTech GmbH, Witten Germany). Rink amide MBHA resin (loading 0.51 mmol/g) was used as a solid support. Final peptides were side-chain deprotected and simultaneously cleaved from the resin by using the mixture TFA/TIS/water (90/5/5, v/v/v) for 3 h at room temperature. After filtration of the exhausted resin, precipitation from ice-cold diethyl ether and recovery by centrifugation provided the crude products. 8
4.3. Peptide purification and analytical determinations
Crude peptides were purified by preparative reversed-phase HPLC using aWaters Delta Prep 3000 system with a Jupiter column C18 (250 x 30 mm, 300 A, 15 µ spherical particle size). The column was perfused at a flow rate of 20 mL/min with a mobile phase containing solvent A (10%, v/v, acetonitrile in 0.1% TFA), and a linear gradient from 0% to 40% of solvent B (60%, v/v, acetonitrile in 0.1% TFA) over 25 min for the elution of peptides. Analytical HPLC analyses were performed on a Beckman 116 liquid chromatograph equipped with a Beckman 166 diode array detector. Analytical purity of the peptides were determined using a Luna C18 column (4.6 x 100 mm, 3 µ particle size) with the above solvent system (solvents A and B) programmed at a flow rate of 0.5 mL/min with a linear gradient from 0% to 65% B over 25 min. All analogues showed >95% purity when monitored at 220 nm. Molecular weights of final compounds were determined by a mass spectrometer ESI Micromass ZMD-2000.
4.4. Calcium mobilization assay
Chinese Hamster Ovary (CHO) cells stably co-expressing human recombinant NOP receptors and the C-terminally modified Gαqi5, were generated as previously described.23, 26 Cells were cultured in Dulbecco’s MEM/HAM’S F12 (1:1) culture medium supplemented with 10% fetal calf serum, penicillin (100 IU/ml), streptomycin (100 mg/ml), geneticin (G418; 200 g/ml) and hygromycin B (100 g/ml). Cell cultures were kept at 37◦C in 5% CO2/humidified air. When confluence was reached (3–4 days), cells were subcultured using trypsin/EDTA and used for the assay. Cells were seeded at a density of 50,000 cells/well into 96-wellblack, clear-bottom plates. After 24 h incubation, the cells were treated with the loading solution of the culture medium supplemented with 2.5 mM probenecid, 3 µM of the calcium sensitive fluorescent dye Fluo-4 AM and 0.01% pluronic acid and HEPES 1M for 30 min at 37◦C. The loading solution was aspirated and a 100/well of the assay buffer (Hank’s Balanced Salt Solution (HBSS) supplemented with 20 mM HEPES, 2.5 mM probenecid, and 500 µM Brilliant Black) was added. The tested peptides were dissolved in the bi-distilled water to the final concentration of 1 mM. The successive dilutions were made in the HBSS/HEPES (20 mM) buffer (containing 0.02% BSA fraction V).After placing both plates (cell culture and compound plate) into the FlexStation II, the on-line additions were carried out in a volume of 50/well and fluorescence changes were measured at 37◦C.
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4.5. Stimulation of [35S]-GTPγS binding assay CHO cells stably expressing human NOP receptors were maintained in DMEM/Nutrient F12 (50/50) with 5% FBS; 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) and hygromycin B (200 µg·mL−1) and used experimentally when confluent. Cultures were sustained at 37°C with 5% carbon dioxide humidified air and sub-cultured using trypsin. Membranes were prepared from freshly harvested cells. Cells were centrifuged at 423 g for 3 minutes before being suspended in homogenizing buffer consisting of Tris (50 mM) and EGTA (0.2 mM). Cell suspensions were homogenized and membranes were collected via centrifugation at 10,000 g for 10 min at 4°C. This process was repeated twice and protein concentrations were determined. 27 CHONOP membranes, 40 µg, were incubated in 0.5 mL of assay buffer containing Tris-HCl (50 mM), EGTA (0.2 mM), NaCl (100 mM), MgCl2 (1 mM), bacitracin (0.15 mM), BSA (0.15 mM), GDP (33 µM) and ∼150 pM [35S]GTPγS. The ligands were added in serial concentrations (10µM – 1 pM), 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.
4.6 Data analysis Agonist potencies were given as pEC50 representing a negative logarithm of the molar concentration of an agonist that produces 50% of the maximal possible effect. Concentration response curves were fitted with the four parameter logistic nonlinear regression model. Curve fittings were performed using GraphPad PRISM 5.0 (GraphPad Software Inc., San Diego, USA) Ligand efficacy was expressed as intrinsic activity (α) calculated as the ratio between the Emax of the ligand and that of the standard N/OFQ(1-13)-NH2. At least five independent experiments for each assay were carried out in duplicate. Data have been statistically analyzed with one way ANOVA followed by the Dunnett’s test for multiple comparisons; p values
