Karel Guillemyn

Joanna Starnowska

Camille Lagard

Jolanta Dyniewicz

Ewelina Rojewska

Joanna Mika

Nga N Chung

Valerie Utard

Piotr Kosson

Andrzej W Lipkowski

Joanna Sarnowska

Valérie Utard #

Lucie Chevillard

Pol Arranz-Gibert

Meritxell Teixidó

Bruno Megarbane

Dirk Tourwé

Frédéric Simonin #

Barbara Przewlocka
email: barbara.przewlocka@gmail.com.

Peter W Schiller

Steven Ballet
email: sballet@vub.ac.be.

Bifunctional Peptide-Based Opioid Agonist-Nociceptin Antagonist Ligands for Dual Treatment of Acute and Neuropathic Pain

come

INTRODUCTION

The efficient treatment of pain remains of paramount importance to our society. Not only does it influence the individual's quality of life, it also significantly impacts healthcare systems around the world. Pain affects almost one in five adults across Europe (ca. 160 million people) and in the U.S., and one-third of all European households are affected by chronic pain. [START_REF] Breivik | Survey of Chronic Pain in Europe: Prevalence, Impact on Daily Life, and Treatment[END_REF] To overcome pain, potent opioids like morphine, oxycodone, buprenorphine (1), and fentanyl are widely prescribed. However, these drugs are not effective in all patients and may even be not well-tolerated as a consequence of deleterious effects such as constipation, [START_REF] Kalso | Opioids in Chronic Non-cancer Pain: Systematic Review of Efficacy and Safety[END_REF] nausea and vomiting, [START_REF] Porreca | Nausea and Vomiting Side Effects with Opioid Analgesics during Treatment of Chronic Pain: Mechanisms, Implications, and Management Options[END_REF] physical dependence, [START_REF] Wikler | Hindlimb Reflexes of Chronic Spinal Dogs during Cycles of Addiction to Morphine and Methadon[END_REF][START_REF] Martin | A Comparison between Acute an Chronic Physical Dependence in the Chronic Spinal Dog[END_REF] tolerance, [START_REF] Cochin | Development and Loss of Tolerance to Morphine in the Rat after Single and Multiple Injections[END_REF] and respiratory depression. [START_REF] Pattinson | Opioids and the Control of Respiration[END_REF] During the last 10 years, increasing prescriptions, availability, and misuse resulted in a rising number of fatalities due to respiratory depression in relation to unintentional opioid overdoses. [START_REF] Dart | Trends in Opioid Analgesic Abuse and Mortality in the United States[END_REF] Opioids represent about 8.6% of all drug-induced fatalities reported by the American Association of Poison Control Centers. [START_REF] Mowry | Annual report of the American Association of Poison Control Centers' National Poison Data System (NPDS): 31st Annual Report[END_REF] To eliminate or reduce opioid-related side effects, other receptors involved in pain can be targeted. Examples of this strategy include the use of neurokinin-1 antagonists, [START_REF] Powell | Inhibition of Neurokinin-1-Substance P Receptor and Prostanoid Activity Prevents and Reverses the Development of Morphine Tolerance in vivo and the Morphine-induced Increase in CGRP Expression in Cultured Dorsal Root Ganglion Neurons[END_REF] neurotensin agonists, [START_REF] Boules | Analgesic Synergy of Neurotensin Receptor Subtype 2 Agonist NT79 and Morphine[END_REF][START_REF] Yano | Simultaneous Activation of Two Different Receptor Systems by Enkephalin/Neurotensin Conjugates Having Spacer Chains of Various Lengths[END_REF] or nociceptin (ant)agonists [START_REF] Tian | Bidirectional Modulatory Effect of Orphanin FQ on Morphine-induced Analgesia: Antagonism in Brain and Potentiation in Spinal Cord of the Rat[END_REF] in combination with opioids. The nociceptin receptor (NOP), previously called the opioid-receptor like-1 (ORL-1) receptor, is a G protein-coupled receptor that was discovered in 1994. [START_REF] Mollereau | ORL1, a Novel Member of the Opioid Receptor Family. Cloning, Functional Expression and Localization[END_REF][START_REF] Cox | Opioid Receptors[END_REF] The endogenous ligand of this receptor is the heptadecapeptide nociceptin/orphanin FQ (N/OFQ, H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-OH). Activation of the nociceptin receptor induces several biological responses including hypotension and immunodepression, and it strongly modulates nociceptive transmission and potentiates the effect of morphine in neuropathic pain. [START_REF] Lambert | The Nociceptin/Orphanin FQ Receptor: a Target with Broad Therapeutic Potential[END_REF][START_REF] Mogil | The Molecular and Behavioral Pharmacology of the Orphanin FQ/ Nociceptin Peptide and Receptor Family[END_REF] The nociceptin/orphanin FQ system seems to be involved in the modulation of acute nociceptive stimulation, as well as in chronic pain processes, for example, in inflammation [START_REF] Andoh | Nociceptin Gene Expression in Rat Dorsal Root Ganglia Induced by Peripheral Inflammation[END_REF][START_REF] Jia | Nociceptin/Orphanin FQ Binding Increases in Superficial Laminae of the Rat Spinal Cord during Persistent Peripheral Inflammation[END_REF] and neuropathic pain. [START_REF] Briscini | Upregulation of ORL-1 Receptors in Spinal Tissue of Allodynic Rats after Sciatic Nerve Injury[END_REF][START_REF] Mika | Morphine and Endomorphin-1 Differently Influence Pronociceptin/Orphanin FQ System in Neuropathic Rats[END_REF][START_REF] Popiolek-Barczyk | Minocycline Enhances the Effectiveness of Nociceptin/Orphanin FQ during Neuropathic Pain[END_REF][START_REF] Yamamoto | Effects of Intrathecally Administered Nociceptin, an Opioid Receptor-like1 (ORL1) Receptor Agonist, on the Thermal Hyperalgesia Induced by Unilateral Constriction Injury to the Sciatic Nerve in the Rat[END_REF] It is well established that N/OFQ may perform both pro-and antinociceptive actions, depending on the administration route [START_REF] Mustazza | Development of Nociceptin Receptor (NOP) Agonists and Antagonists[END_REF] or dosage, [START_REF] Mika | The Role of Nociceptin and Dynorphin in Chronic Pain: Implications of Neuro-glial Interaction[END_REF] and both agonists and antagonists of NOP appear to be useful. [START_REF] Lambert | The Nociceptin/Orphanin FQ Receptor: a Target with Broad Therapeutic Potential[END_REF][START_REF] Mustazza | Development of Nociceptin Receptor (NOP) Agonists and Antagonists[END_REF][START_REF] Corradini | The Putative OP 4 Antagonist, [Nphe 1 ]Nociceptin(1-13)NH 2 , Prevents the Effects of Nociceptin in Neuropathic Rats[END_REF] NOP antagonists on their own do not demonstrate antinociceptive potency, neither in acute [START_REF] Khroyan | Nociceptin/Orphanin FQ Receptor Activation Attenuates Antinociception Induced by Mixed Nociceptin/Orphanin FQ/mu-Opioid Receptor Agonists[END_REF] nor in neuropathic pain models, although they are reported to potentiate the antiallodynic effect of morphine under neuropathic conditions when coadministered iv, [START_REF] Khroyan | Differential Effects of Nociceptin/Orphanin FQ (NOP) Receptor Agonists in Acute Versus Chronic Pain: Studies with Bifunctional NOP/μ Receptor Agonists in the Sciatic Nerve Ligation Chronic Pain Model in Mice[END_REF] and to enhance DAMGO ([D-Ala, [START_REF] Kalso | Opioids in Chronic Non-cancer Pain: Systematic Review of Efficacy and Safety[END_REF] NMePhe, [START_REF] Wikler | Hindlimb Reflexes of Chronic Spinal Dogs during Cycles of Addiction to Morphine and Methadon[END_REF] Gly-ol]-enkephalin, MOP agonist) induced analgesia after microinjection to the ventrolateral periaqueductal gray, being the main output pathway involved in descending pain control. [START_REF] Scoto | Blockade of the Nociceptin/Orphanin FQ/NOP Receptor System in the Rat Ventrolateral Periaqueductal Gray Potentiates DAMGO Analgesia[END_REF] Natural N/OFQ administered intrathecally, but not supraspinally, induces naloxone insensitive antinociceptive actions in rhesus monkeys. [START_REF] Ko | Effects of Intrathecally Administered Nociceptin/ Orphanin FQ in Monkeys: Behavioral and Mass Spectrometric Studies[END_REF] NOP agonists in contrast may demonstrate powerful antinociceptive potency in acute pain but only in low doses (0.001-1 nmol it vs morphine >1 nmol it), as was demonstrated by Micheli and co-workers in rats. [START_REF] Micheli | Acute and Subchronic Antinociceptive Effects of Nociceptin/Orphanin FQ Receptor Agonists Infused by Intrathecal Route in Rats[END_REF] In the range of 0.001-1 nmol, a dose of 0.3 nmol it administered NOP agonist [pF-Phe, [START_REF] Wikler | Hindlimb Reflexes of Chronic Spinal Dogs during Cycles of Addiction to Morphine and Methadon[END_REF] Aib, [START_REF] Pattinson | Opioids and the Control of Respiration[END_REF] Arg, [START_REF] Mollereau | ORL1, a Novel Member of the Opioid Receptor Family. Cloning, Functional Expression and Localization[END_REF] Lys 15 ]N/OFQ-NH 2 (UFP-112) afforded the highest antinociceptive effect. In addition, NOP agonists have not only been reported to be effective in acute pain but also not to lose their potency in neuropathic pain, as opioids do. [START_REF] Obara | Local Peripheral Opioid Effects and Expression of Opioid Genes in the Spinal Cord and Dorsal Root Ganglia in Neuropathic and Inflammatory Pain[END_REF][START_REF] Khroyan | Antinociceptive and Anti-allodynic Effects of a High Affinity NOP Hexapeptide [Ac-RY(3-Cl)YRWR-NH 2 ] (Syn 1020) in Rodents[END_REF] Interestingly, morphine's effect in acute pain was significantly greater in naïve than in NOP(-/-) animals which suggests that the endogenous NOP system contributes to acute pain processing, at least in rats. Other authors report that this effect does not occur in mice models. [START_REF] Mamiya | Morphine Tolerance and Dependence in the Nociceptin Receptor Knockout Mice[END_REF] NOP agonists proved to be especially potent in nonhuman primates, which suggests a promising outlook for human patients therapies. [START_REF] Lin | The Therapeutic Potential of Nociceptin/Orphanin FQ Receptor Agonists as Analgesics without Abuse Liability[END_REF] There is also evidence substantiating the use of NOP/MOP agonist hybrids in pain: intrathecal administration of a combination of inactive doses of UFP-112 and morphine attenuated hyperalgesia in rhesus monkeys. [START_REF] Hu | Long-lasting Antinociceptive Spinal Effects in Primates of the Novel Nociceptin/Orphanin FQ Receptor Agonist UFP-112[END_REF] Consequently, targeting two distinct pathways to produce analgesia may potentiate the overall analgesic effect, while ameliorating side effects, for example by requiring lower doses.

Activation or blockage of distinct targets can be achieved by combination therapy (i.e., using drug cocktails) or by the use of designed multiple ligands (DMLs). [START_REF] Morphy | Designed Multiple Ligands. An Emerging Drug Discovery Paradigm[END_REF] The latter are single chemical entities able to bind two or more well-chosen receptor types. Although both strategies proved to be very useful, major advantages of DMLs consist of an early stage, thus less expensive optimization in the drug discovery process, and less complex pharmacokinetics. [START_REF] Morphy | Designed Multiple Ligands. An Emerging Drug Discovery Paradigm[END_REF] Several small molecules with nanomolar binding affinities at both the nociceptin and the opioid systems have been reported (Figure 1). [START_REF] Khroyan | Differential Effects of Nociceptin/Orphanin FQ (NOP) Receptor Agonists in Acute Versus Chronic Pain: Studies with Bifunctional NOP/μ Receptor Agonists in the Sciatic Nerve Ligation Chronic Pain Model in Mice[END_REF][START_REF] Johnson | Buprenorphine: Considerations for Pain Management[END_REF][START_REF] Khroyan | The First Universal Opioid Ligand, (2S)-2-[(5R,6R,7R,14S)-N-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3hydroxy-6-meth oxymorphinan-7-yl]-3,3-dimethylpentan-2-ol (BU08028): Characterization of the in vitro Profile and in vivo Behavioral Effects in Mouse Models of Acute Pain and Cocaineinduced Reward[END_REF][START_REF] Zaveri | A Novel Series of Piperidin-4-yl-1,3-Dihydroindol-2-ones as Agonist and Antagonist Ligands at the Nociceptin Receptor[END_REF][START_REF] Zaveri | Designing Bifunctional NOP Receptor-mu Opioid Receptor Ligands from NOP Receptor-selective Scaffolds. Part I[END_REF][START_REF] Blair Journigan | Designing Bifunctional NOP Receptor-mu Opioid Receptor Ligands from NOP-Receptor Selective Scaffolds. Part II[END_REF][START_REF] Khroyan | SR 16435 [1-(1-(bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a Novel Mixed Nociceptin/Orphanin FQ/mu-opioid Receptor Partial Agonist: Analgesic and Rewarding Properties in Mice[END_REF][START_REF] Schunk | Discovery of a Potent Analgesic NOP and Opioid Receptor Agonist: Cebranopadol[END_REF] Depending on their structural architectures, they behave as agonists, partial agonists, or antagonists on one or both of these systems. Several of these small molecule hybrids demonstrated interesting analgesic properties when tested in vivo. The high affinity, nonselective NOP/MOP partial agonist SR16435 (2) produced an antiallodynic response in the rat chronic constriction injury (CCI) model of neuropathic pain after intraperitoneal administration. [START_REF] Khroyan | Activity of New NOP Receptor Ligands in a Rat Peripheral Mononeuropathy Model: Potentiation of Morphine Antiallodynic Activity by NOP Receptor Antagonists[END_REF] In a thermal antinociception test, the development of analgesic tolerance was reduced compared with morphine. [START_REF] Khroyan | SR 16435 [1-(1-(bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a Novel Mixed Nociceptin/Orphanin FQ/mu-opioid Receptor Partial Agonist: Analgesic and Rewarding Properties in Mice[END_REF] Co-administration of a NOP antagonist potentiated the antiallodynic effect of 2 and of morphine. This led the authors to suggest that NOP/MOP ligands with a dual profile of NOP antagonism and MOP agonism may be particularly useful for the treatment of chronic pain. [START_REF] Khroyan | Activity of New NOP Receptor Ligands in a Rat Peripheral Mononeuropathy Model: Potentiation of Morphine Antiallodynic Activity by NOP Receptor Antagonists[END_REF] Compound 3, a full NOP agonist and weak partial (<20%) MOP agonist, did not have an effect in an acute pain model, but after subcutaneous administration to neuropathic sciatic nerve ligation (SNL) mice, it proved to possess an antiallodynic activity which could be suppressed by co-administration of a NOP, but not MOP, antagonist. [START_REF] Khroyan | SR 16435 [1-(1-(bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a Novel Mixed Nociceptin/Orphanin FQ/mu-opioid Receptor Partial Agonist: Analgesic and Rewarding Properties in Mice[END_REF] However, compound 3 was less potent than morphine in the SNL model. This may implicate the need to find a proper balance between activation of MOP and NOP receptors in allodynia alleviation. [START_REF] Khroyan | Differential Effects of Nociceptin/Orphanin FQ (NOP) Receptor Agonists in Acute Versus Chronic Pain: Studies with Bifunctional NOP/μ Receptor Agonists in the Sciatic Nerve Ligation Chronic Pain Model in Mice[END_REF] Interestingly, cebranopadol 4, a highly potent agonist at MOP, DOP, KOP, and NOP, produced potent antinociception in rat models of acute and chronic pain and presented a clearly delayed development of tolerance. [START_REF] Schunk | Discovery of a Potent Analgesic NOP and Opioid Receptor Agonist: Cebranopadol[END_REF][START_REF] Linz | Cebranopadol: A Novel Potent Analgesic Nociceptin/Orphanin FQ Peptide and Opioid Receptor Agonist[END_REF] Also peptide and peptidomimetic bifunctional compounds have been reported. Kawano 50 linked the opioid agonist dermorphin sequence to the Ac-RYYRIK-NH 2 sequence that was shown to have high NOP affinity and to behave as an agonist, partial agonist, or antagonist depending on the assay. [START_REF] Khroyan | Antinociceptive and Anti-allodynic Effects of a High Affinity NOP Hexapeptide [Ac-RY(3-Cl)YRWR-NH 2 ] (Syn 1020) in Rodents[END_REF][START_REF] Judd | Structure-activity Studies on High Affinity NOP-active Hexapeptides[END_REF][START_REF] Berger | Antagonism by Acetyl-RYYRIK-NH2 of G Protein Activation in Rat Brain Preparations and of Chronotropic Effect on Rat Cardiomyocytes Evoked by Nociceptin/Orphanin FQ[END_REF] A synergistic and improved affinity for both the MOP and NOP was observed, leading to subnanomolar affinities when a long spacer -Gly-Gly-Gly-Lys(Gly-Gly-) was incorporated between the pharmacophores (5). In the mouse tail flick test, hybrid 5 showed an antinociceptive effect comparable to that of the isolated dermorphin sequence after it administration but less potent after icv administration. The antinociception was lower for the hybrid than for the coadministered opioid and nociceptin monomeric components despite the higher receptor affinities of the hybrid. No studies in neuropathic pain models were reported for hybrid 5. [START_REF] Kawano | Receptor Binding Properties and Antinociceptive Effects of Chimeric Peptides Consisting of a μ-Opioid Receptor Agonist and an ORL1 Receptor Antagonist[END_REF] Becker 52 screened a library of peptides, all incorporating β-turn inducing motifs for their binding at the opioid and nociceptin receptors and identified peptide III-BTD 6 with nanomolar binding affinities, and agonist properties at the opioid receptors and antagonist properties for NOP. [START_REF] Becker | Ligands for κ-Opioid and ORL1 Receptors Identified from a Conformationally Constrained Peptide Combinatorial Library[END_REF][START_REF] Bigoni | In vitro Pharmacological Profile of Peptide III-BTD: A Novel Ligand for Nociceptin/Orphanin FQ and Opioid Receptors[END_REF] On the basis of the potential of dual opioid-nociceptin ligands for the treatment of chronic and neuropathic pain, which remain an unmet medical need, we now report the design and synthesis of new DMLs. [START_REF] Morphy | Designed Multiple Ligands. An Emerging Drug Discovery Paradigm[END_REF] In contrast to the work of Kawano, the opioid part is directly connected to the nociceptin pharmacophore. [START_REF] Kawano | Synthesis and Receptor Binding Properties of Chimeric Peptides Containing a μ-Opioid Receptor Ligand and Nociceptin/Orphanin FQ Receptor Ligand Ac-RYYRIK-amide[END_REF] The opioid agonist part consists of the earlier reported and optimized H-Dmt-D-Arg-Aba-β-Ala-NH 2 tetrapeptide 7, [START_REF] Guillemyn | Synthesis and Biological Evaluation of Compact, Conformationally Constrained Bifunctional Opioid Agonist -Neurokinin-1 Antagonist Peptidomimetics[END_REF] which is characterized by subnanomolar affinity and agonist activity for the opioid receptors. The nociceptin part is based on hexapeptides that were discovered by Dooley [START_REF] Dooley | Binding and In Vitro Activities of Peptides with High Affinity for the Nociceptin/Orphanin FQ Receptor, ORL1[END_REF] (i.e., Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 [START_REF] Dart | Trends in Opioid Analgesic Abuse and Mortality in the United States[END_REF] and analogues). Substitution of the N-terminal Ile-Lys dipeptide led to the Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH 2 sequence which has a K i of 0.1 nM for the human NOP and also behaves as a partial agonist in the [ 35 S]GTPγS assay. [START_REF] Judd | Structure-activity Studies on High Affinity NOP-active Hexapeptides[END_REF] The hexapeptides behave as partial agonists in cells expressing NOP, [START_REF] Judd | Structure-activity Studies on High Affinity NOP-active Hexapeptides[END_REF][START_REF] Dooley | Binding and In Vitro Activities of Peptides with High Affinity for the Nociceptin/Orphanin FQ Receptor, ORL1[END_REF] but antagonism has been observed in rat brain preparations and in acute and chronic pain models in mice and rats. 33,55 Replacement of the Tyr 3 residue in this hexapeptide with a para-fluorophenylalanine (pF-Phe) results in an even higher affinity (K i = 0.05 nM). [START_REF] Judd | Structure-activity Studies on High Affinity NOP-active Hexapeptides[END_REF] The latter product behaves as a partial agonist (70% stimulation) similar to the parent compound. [START_REF] Judd | Structure-activity Studies on High Affinity NOP-active Hexapeptides[END_REF] Introduction of a 2′,6′-dimethyl-L-tyrosine (Dmt) residue at position 3 also gives a high affinity (K i = 0.04 nM) partial agonist. [START_REF] Judd | Structure-activity Studies on High Affinity NOP-active Hexapeptides[END_REF] The N-terminus of these four peptides was linked to the C-terminus of our opioid pharmacophore in order to obtain bifunctional peptides. These new bifunctional peptide ligands were evaluated in vitro for their affinity and activity on both the opioid and the nociceptin receptors, and additionally these compounds were tested in vivo for acute and neuropathic pain alleviation. One of the hybrids was examined for effects on respiratory depression.

RESULTS AND DISCUSSION

Synthesis

The targeted bifunctional peptides all contain a tetrahydro-4-amino-2-benzazepinone (Aba) subunit which serves as a conformationally constrained Phe analogue. Incorporation of this building block, which was previously developed by us, [START_REF] Tourwe | Dermorphin Sequence with High Delta-affinity by Fixing the Phe Side-chain to Trans at Alpha-1[END_REF][START_REF] Tourwe | Conformational Restriction of Tyr and Phe Side-chain in Opioid Peptides: Information about Preferred and Bioactive Side-chain Topology[END_REF] into several bioactive peptide sequences has successfully led to more stable and GPCR-subtype selective ligands and has provided highly potent opioid agonists. [START_REF] Ballet | Synthesis and Biological Evaluation of Constrained Analogues of the Opioid Peptide H-Tyr-D-Ala-Phe-Gly-NH 2 Using 4-Amino-2-Benzazepin-3-one Scaffold[END_REF][START_REF] Vandormael | Superpotent [Dmt 1 ]Dermorphin Tetrapeptides Containing the 4-Aminotetrahydro-2-Benzazepin-3-one Scaffold with Mixed μ/δ Opioid Receptor Agonistic Properties[END_REF][START_REF] Ballet | Novel Selective Human Melanocortin-3 Receptor Ligands: Use of the 4-Amino-1,2,4,5-Tetrahydro-2-Benzazepin-3-one (Aba) Scaffold[END_REF][START_REF] Ballet | Bradykinin Analogs Containing the 4-Amino-2-Benzazepin-3-one Scaffold at the C-terminus[END_REF] In the current work, its insertion into the desired opioid-nociceptin hybrids was realized in two ways (indicated as pathways A and B in Scheme 1).

Both strategies start with the synthesis of the resin-bound hexapeptides 10 by use of standard Fmoc-solid phase peptide synthesis (SPPS) on Rink amide AM resin. In pathway A, a segment condensation was performed between resin-bound 10 (with Xxx, Tyr(OtBu); Yyy, Ile; and Zzz, Lys(Boc)) and a preassembled protected tetrapeptide analogue Boc-Dmt-D-Arg(Pbf)-Aba-β-Ala-OH (11), which was prepared on 2-chlorotrityl resin, to obtain hybrid 12. The Aba-β-Ala component was formed directly on the solid support as previously reported. [START_REF] Guillemyn | Synthesis and Biological Evaluation of Compact, Conformationally Constrained Bifunctional Opioid Agonist -Neurokinin-1 Antagonist Peptidomimetics[END_REF] After full protecting group removal of 12 and simultaneous cleavage from the resin, the product was purified by means of preparative RP-HPLC to obtain the pure compound 13a. In route B, dipeptide Fmoc-Aba-β-Ala-OH (14), was prepared by solution synthesis (Scheme 2) and coupled to the hexapeptides 10 to give 15. Fmoc-Aba-β-Ala-OH (14) was obtained, starting from phthaloyl protected phenylalanine 16 (Scheme 2), which was coupled to β-Ala-OEt•HCl using TBTU as coupling reagent and triethylamine as base. After crystallization of the resulting dipeptide 17, formation of the desired azepinone ring was achieved by use of an adapted literature procedure. [START_REF] Robl | Processes and Intermediates for Preparing Benzo-Fused Azepinone and Piperidinone Compounds Useful in the Inhibition of ACE and NEP[END_REF] Dipeptide 17 was mixed with trioxane, AcOH, and H 3 PO 4 in benzene at reflux in a Dean-Stark apparatus. P 2 O 5 was added to avoid hydrolysis of the ester in the starting material due to the presence of traces of water. After refluxing for 4 h, ring closure proved to be complete. Following workup and flash chromatography purification of the crude mixture, the ester in 18 was hydrolyzed with a 1 N HCl in water/acetone (1:1) mixture at 90 °C for 16 h to isolate 19. The phthaloyl protecting group was then removed by hydrazinolysis. Subsequently, the free dipeptide was N-protected with a Fmoc group and the pure and SPPS-compatible building block 14 was isolated after final purification with flash chromatography in 22% yield (over four steps). After coupling of dipeptide mimetic 14 onto resin-bound 10 (Scheme 1), further peptide elongation via SPPS, full deprotection, and cleavage from the resin, the final peptides of type 13 were obtained in high purity (>95%) after preparative HPLC purification. The overall yield of pathway B was slightly higher and this pathway was easier to perform on larger scale (0.2 mmol). Only the first sequence 13a was synthesized via both methods; the rest of the series (i.e., 13b-13d, Table 1) was synthesized by use of pathway B. To allow comparison with the parent nociceptin receptor ligands, compounds 8 (Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 ) and 20 (H-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 ) were also prepared via SPPS and evaluated in vitro and in vivo.

In Vitro Biological Evaluation

In vitro binding and functional activity at the MOP, DOP, and KOP as well as at the nociceptin receptor were established for the parent (7, 8, 20) and hybrid compounds 13a-d (Table 2). In contrast to the parent structures, all hybrids maintained affinities within the moderate to low nanomolar range at both the opioid receptors and NOP. As expected, the parent structures possessed good binding only at the opioid or NOP receptors.

Hybrid 13a combines the opioid ligand 7 with the peptide Ac-RYYRIK-NH 2 8. This fusion led to a decrease in nociceptin and opioid receptor binding compared to the parent compounds, which is in contrast to the synergistic effect reported for hybrid 5. [START_REF] Kawano | Receptor Binding Properties and Antinociceptive Effects of Chimeric Peptides Consisting of a μ-Opioid Receptor Agonist and an ORL1 Receptor Antagonist[END_REF] In comparison to NOP, the effect was less pronounced at the opioid receptors. Changing the last two amino acids (Ile-Lys to Trp-Arg, see 13d) did not have a beneficial influence on affinity in contrast to the improvement observed in the parent compound (going from 1.5 to 0.6 nM). [START_REF] Judd | Structure-activity Studies on High Affinity NOP-active Hexapeptides[END_REF][START_REF] Dooley | Binding and In Vitro Activities of Peptides with High Affinity for the Nociceptin/Orphanin FQ Receptor, ORL1[END_REF] Substitution of Tyr with para-fluoro phenylalanine gave a small decrease in opioid affinity for 13b, but the NOP binding improved almost 2-fold (47 to 27 nM). Introduction of a 2′,6′-dimethyltyrosine (Dmt) at this position (13c) improved DOP and KOP binding compared to 13b but with a slight drop in NOP affinity. With respect to nociceptin receptor binding, no significant differences were observed between the four investigated hybrids because the affinity varied maximally with a factor of 2.

The in vitro opioid functional activity of the hybrids was verified by means of the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, representative of µ-and δ-opioid receptor activity, respectively. In agreement with the affinity data of these compounds, low nanomolar activities were determined for all analogues, 13b being the most potent of the series. Some discrepancies between DOP receptor binding data and agonist potencies in the MVD assay were observed. For example, compound 13b showed quite low DOP binding affinity (K i δ = 194 nM), but turned out to be a potent agonist in the MVD assay (IC 50 = 1.4 nM). There is evidence to indicate that in the vas preparation a cooperative effect between MOP and DOP receptors may be produced by some compounds, [START_REF] Maldonado | Activity of mu-and delta-Opioid Agonists in Vas Deferens from Mice Deficient in MOR Gene[END_REF] and such cooperativity may be the cause for the unexpected high agonist potency of compound 13b. An alternative explanation would be that this compound may interact with another unknown receptor in the vas, as was also suggested to be the case with the NOP agonist/MOP partial agonist SR16476. [START_REF] Spagnolo | Activities of Mixed NOP and μ-opioid Receptor Ligands[END_REF] The activity of nociceptin parent NOP ligands 8 and 20, as well as hybrid molecules 13a-13d, was then assessed in the forskolin (FSK)-stimulated 3′,5′-cyclic adenosine monophosphate (cAMP) assay in HEK293 cells stably expressing the human NOP as described in the experimental section. As expected, nociceptin efficiently inhibited FSKstimulated cAMP production (Figure 2) with an EC 50 of 0.23 ± 0.02 nM. Compound 8 displayed partial agonist activity at NOP (70 ± 1% of nociceptin maximum activity) with an EC 50 of 29 ± 1 nM, while 20 displayed neither agonist activity up to 10 µM nor antagonist activity up to 20 µM (Table 2). Hybrids 13a-13d did not display agonist activity at NOP up to 10 µM. Moreover, 20 µM of each compound significantly shifted the dose-response curve of nociceptin to the right (Figure 2), indicating that they displayed antagonist properties at this receptor. We then determined pA 2 values for each hybrid (Table 2). Our results show that 13a was the less potent NOP antagonist (pA 2 = 5.39), while the performed structural modifications led to improved antagonism for 13b-d, with pA 2 values of 6.00, 6.25 and 6.01, respectively.

In Vitro BBB Permeation Assays

To validate BBB permeation, a selected hybrid (13a) and its parent opioid (7) and NOP parent structures (8 and 20) were assayed in a PAMPA assay using porcine brain polar lipids as a model to study their passive diffusion transport through the blood-brain barrier (BBB) [START_REF] Di | High Throughput Artificial Membrane Permeability Assay for Blood-Brain Barrier[END_REF] at a concentration of 500 µM in the donor compartment. Propranolol, a β-adrenergic receptor blocker with high brain penetration, was used as a positive control. Permeability (P e ) is considered excellent if values >4.0 × 10 -6 cm/s, uncertain between 2.0 × 10 -6 and 4.0 × 10 -6 cm/s, and poor with values below 2.0 × 10 -6 cm/s. [START_REF] Di | High Throughput Artificial Membrane Permeability Assay for Blood-Brain Barrier[END_REF] Thus, looking at the results depicted in Table 3, none of the peptide analogues show a significant passive diffusion transport, as all of them had permeabilities below 2.0 × 10 -6 cm/s (see Table 3).

Next, to shed light on potential transport of these compounds through the BBB and study whether they are able to cross by active transport, an in vitro cell-based model of the BBB was used (Figure 3). [START_REF] Cecchelli | A Stable and Reproducible Human Blood-Brain Barrier Model Derived from Hematopoietic Stem Cells[END_REF] This human in vitro model of the BBB uses brain-like endothelial cells, generated from human cord blood-derived hematopoietic stem cells cocultured with perycites. Peptide quantification and integrity were analyzed by UPLC and MALDI-TOF, respectively. In this model, Lucifer Yellow lithium salt was used as internal control, whose permeability (P app ) should stay below 17 × 10 -6 cm/s. In all cases, the integrity of the model was preserved.

Excellent transports were obtained in all cases (ranging from 7.0 to 13.7 × 10 -6 cm/s), except for the peptide 20 (2.7 × 10 -6 cm/s). This compound, in spite of its high degree of similarity with 8 (the N-terminal acetylated analogue of 20), showed a 3-fold reduction in transport, likely to be caused by the action of aminopeptidases (half the peptide was hydrolyzed after 2 h, see mass balance in Table 4 and Supporting Information). Compound stability was preserved in compound 13a, which also contains moiety 20 at the C-erminus.

Thus, the in vitro cell-based human model of the BBB, together with the BBB-PAMPA assay, enabled us to determine that all these compounds are transported by active mechanisms and not through passive diffusion. Moreover, as it is known, the BBB is also an enzymatic barrier, [START_REF] Aigner | Transport and Detoxication: Principles, Approaches, and Perspectives for Research on the Blood -Brain Barrier[END_REF][START_REF] Hitchcock | Structure-Brain Exposure Relationships[END_REF] and in this regard we observed an increased resistance to proteases for compounds 8 and 13a thanks to the N-terminal capping either with an acetyl or the opioid pharmacophore, respectively.

Behavioral Studies in Mice and Rats

Acute Pain in Mice (Tail-Flick Test)-Evaluation of the antinociceptive potency of hybrid 13a by the tail-flick test in mice after iv administration revealed a maximal effect at 60 min post administration while the maximal effect of morphine was typically reached after 15-30 min (Figure 4). Compared to morphine, an equipotent effect was measured at much lower doses (6, 31, 61 nmol vs morphine in 390 nmol dose). The %MPE of morphine started to decline from 60 min postadministration, while the nociceptive effect of 13a maintained high %MPE values for longer periods of time. Longer lasting and maximal effects were measured at 180 and 240 min for both the 31 and 61 nmol doses (100 %MPE for both doses at both time points and 79.8 %MPE for 6 nmol dose at 180 min after administration (data not recorded for morphine at these time-points)). The above results suggested that potent CNS-mediated effects can be obtained by iv injection of hybrid 13a and, hence, BBB permeation did not seem to represent a limitation for this compound as indicated above (cf., in vitro BBB permeability assay). This peptide was modified at the Nand C-terminus and internally by the Aba lactam, which should increase its resistance to enzymatic degradation. The stability of 13a in plasma was determined and, to our satisfaction, revealed a half-life time of 585 min at 37 °C in human plasma (see Supporting Information).

When hybrid 13a was administered orally to mice at doses of 61, 122, and 196 nmol, no analgesic effects were observed (see Supporting Information, no %MPE > 5% was observed), even at such high doses.

Neuropathic Pain Tests in Mice-All bifunctional compounds (13a-13d), the parent NOP sequence (20), as well as its N-acetylated form (8), were tested in neuropathic pain models in mice. The ligands were all examined as potential suppressors of allodynia and hyperalgesia, phenotypes which were caused by chronic constriction injury to the sciatic nerve (CCI) or by a streptozocin (STZ)-induced type 1 diabetes. [START_REF] Mika | Differential Activation of Spinal Microglial and Astroglial Cells in a Mouse Model of Peripheral Neuropathic Pain[END_REF][START_REF] Zychowska | Mnocycline Influences the Anti-inflammatory Interleukins and Enhances the Effectiveness of Morphine Under Mice Diabetic Neuropathy[END_REF] The results of these tests are presented in Figure 5 (von Frey test, allodynia) and Figure 6 (cold plate test, hyperalgesia). The calculated ED 50 values for the parent compounds and all four hybrids at one time point (30 min) are presented in Table 3. The results for the parent opioid sequence 7 in mice and rats were obtained during an earlier study. [START_REF] Guillemyn | Synthesis and Biological Evaluation of Compact, Conformationally Constrained Bifunctional Opioid Agonist -Neurokinin-1 Antagonist Peptidomimetics[END_REF][START_REF] Guillemyn | Neuropathic Pain: A Maladaptive Response of the Nervous System to Damage[END_REF] As can be noticed, all bifunctional compounds proved to be significantly more effective than morphine, both in terms of antiallodynic and antihyperalgesic effects. Interestingly and with exception of hybrid 13b, the hybrids performed better than the nociceptin parent compounds corresponding to the NOP pharmacophore of 13a (Figures 5 and6). The hybrids were highly effective at low doses, especially when compared to morphine [e.g., ED 50 (13a) = 0.003 nmol vs ED 50 (morphine) = 7 nmol, allodynia; ED 50 (13a) = 0.004 nmol vs ED 50 (morphine) = 7.46 nmol, hyperalgesia]. When compared to the opioid parent compound 7 with ED 50 values of 0.85 nmol (von Frey) and 1.60 nmol (cold plate), the hybrids were also much more potent. [START_REF] Guillemyn | Neuropathic Pain: A Maladaptive Response of the Nervous System to Damage[END_REF] These observations support the hypothesis that more potent effects can be induced by the presence of both pharmacophores in 13a-13d when compared to the effect of a single opioid or nociceptin unit. The effect of opioid-nociceptin hybrids, in spite of their low NOP antagonist potency, is profound because it originates from the simultaneous modulation of the transmitted signal in the same part of the nociceptive pathway and the influence concerns both endogenous opioid and nociceptin systems which are very important for nociceptive transmission. Moreover, the endogenous nociceptin/ orphanin FQ system, apart from its analgesic action, is known to exhibit anti-opioid activity and may contribute to lower responsiveness to morphine in neuropathic pain. [START_REF] Mika | Morphine and Endomorphin-1 Differently Influence Pronociceptin/Orphanin FQ System in Neuropathic Rats[END_REF] [START_REF] Mogil | Heritability of Nociception II. 'Types' of Nociception Revealed by Genetic Correlation Analysis[END_REF] but also by different afferent fibers types. [START_REF] Shir | A-fibersMediate Mechanical Hyperesthesia and Allodynia and C-fibers Mediate Thermal Hyperalgesia in a New Model of Causalgiform Pain Disorders in Rats[END_REF] Hybrid 13a seems to be the best of all investigated hybrids, parent peptides, and morphine. The antiallodynic and antihyperalgesic effects of 13a were more prolonged than those produced by a classic analgesic like morphine and stayed extremely high even 180 min after it administration and injection of very low doses (e.g., 0.0005 nmol). The effect of the parent compound 20 is transient, while the acetylated parent 8 is efficient at higher doses only (0.8 vs 0.05). Hybrid 13a is clearly superior (even at very low doses) to the parents in the cold plate test as well as two neuropathy models. Hence, in contrast to common opioids such as morphine, the tested opioid-NOP DMLs exhibit high efficacy in neuropathic pain models. In mice, morphine performs dose-dependently in acute pain, with a dose of 10 µg/animal giving 63.8 ± 15.9 %MPE in the tail-flick test. The effect reaches 100 %MPE upon increased dosing. [START_REF] Guillemyn | Neuropathic Pain: A Maladaptive Response of the Nervous System to Damage[END_REF] On the contrary, under neuropathic conditions, morphine provides 36.5 ± 9.5 %MPE in von Frey test and 30.15 ± 8.2 %MPE in cold plate test at the respective dose of 10 µg/animal. The effect never reaches maximum values; it sustains at about 50 %MPE no matter how high the dose is. [START_REF] Guillemyn | Neuropathic Pain: A Maladaptive Response of the Nervous System to Damage[END_REF] Of all hybrids, compound 13b was the least effective [ED 50 of 0.18 nmol (von Frey) and 0.14 nmol (cold plate)] and it only showed a maximal effect, comparable to the other hybrids, at a high dose of 0.5 nmol (Figure 5A and 6A). Hybrids 13c-13d seemed also to be promising compounds in the investigated neuropathic pain models. Taking the results depicted in Figures 5 and6 into consideration, these analogues presented the most potent and long lasting effects, especially with regard to antiallodynic (von Frey) efficacy.

Altogether, hybrid 13a and 13d seemed to be the most promising compounds in the investigated neuropathic pain models. Taking the results depicted in Figure 5 and 6 into consideration, these analogues presented the most potent and long lasting effects, especially with regard to antiallodynic (von Frey) efficacy (Table 5).

The observations made in the injury-induced neuropathy models also applied to the diabetic neuropathy, although in the latter model the antiallodynic action of the compounds showed a maximum at 30 min which slightly decreased at 180 min (Figure 4B). This decrease was also present in the antihyperalgesic action (Figure 5B) except for 13a. This may result from intermodel differences in mechanisms underlying the neuropathic symptoms occurrence and maintenance, although it was still promising that 13a was able to alleviate the painful symptoms of both hyperalgesia and allodynia in distinct models. As the discovery of a multifunctional drug alleviating assorted symptoms of neuropathy remains of high interest, the above results provide a worthy outlook toward other novel therapies.

All compounds were also evaluated in the Rota-Rod test to examine motor function of mice. Only the 0.5 nmol dose of 13b gave way to impaired motor function. At the used doses, all other compounds did not impair motor functions (data not shown).

Respiratory Depression in Rats-Respiratory effects in rats were determined by plethysmography after iv administration of equipotent antinociceptive doses (in tail-flick test) of 13a (0.5 mg/kg, 6 nmol/kg) and morphine (5 mg/kg, 390 nmol/kg). On the basis of the measurement of the ventilation minute, administration of 13a did not result in a significant respiratory depression in comparison to saline, a result that stands in contrast to the deleterious effect of morphine (p < 0.01; Figure 7F). Interestingly, morphine induced a significant increase in inspiratory time in comparison to saline and 13a (p < 0.05; Figure 7B), while 13a induced a significant decrease in expiratory time in comparison to morphine (p < 0.05; Figure 7E). Combining both effects, morphine induced a decrease in respiratory frequency (p < 0.001, Figure 7D), while 13a slightly increased this frequency. The tidal volume was significantly reduced with 13a in comparison to saline and morphine (p < 0.05; Figure 7C).

An increase in inspiratory time is a marker of opioid-related effects on the respiratory control centers in the brainstem. [START_REF] Chevillard | Characteristics and Comparative Severity of Respiratory Response to Toxic Doses of Fentanyl, Methadone, Morphine, and Buprenorphine in Rats[END_REF] Remarkably, 13a did not result in any significant increase in inspiratory time at the studied 0.5 mg/kg dose. Moreover, in comparison to morphine, 13a significantly reduced the expiratory time. Both effects show the limited respiratory effects related to 13a in comparison to the deleterious ones of morphine, studied only at a single therapeutic dose. A more elaborate study will be carried out to compare the ratio of the ED 50 % of the respiratory-to-analgesic effects of 13a versus morphine.

CONCLUSIONS

In vitro, the opioid-nociceptin hybrids behaved as potent opioid agonists and weak nociceptin antagonists. Although fusion of the two peptide fragments led to a slight loss in opioid and nociceptin receptor binding, low nanomolar affinities were maintained. On the basis of the in vitro evaluation, compound 13a was selected for further in vivo tests. After intravenous administration to naïve mice, it was more active than morphine and showed a prolonged analgesic action (>3 h) in an acute pain model. This result indicated that hybrid 13a was transported through the BBB, a hypothesis which was further supported by a cellbased in vitro BBB model. In addition, all hybrid compounds were administered intrathecally to CCI mice and were proven to be more effective than morphine and the parent NOP ligands in both antiallodynic and antihyperalgesic effects, with compounds 13a and 13d exhibiting the best in vivo profile. Compared to morphine, the effects of 13a were much more prolonged and extremely high at low doses even at 180 min postadministration.

Because 13a proved to be very potent in both acute and neuropathic pain models, potential respiratory depression effects linked to the use of this product were evaluated. Inspiratory and expiratory times show that the administration of 13a did not result in a significant respiratory depression. Altogether, the opioid-NOP hybrids are highly effective analgesics in the investigated neuropathic pain models. The best compounds have ED 50 values about 1000 times lower than those of the opioid agonist/NK1 antagonist hybrid H-Dmt-D-Arg-Abaβ-Ala-NMe-Bn(CF 3 ) 2 in the von Frey test and about 20 times lower in the cold plate test. [START_REF] Guillemyn | Synthesis and Biological Evaluation of Compact, Conformationally Constrained Bifunctional Opioid Agonist -Neurokinin-1 Antagonist Peptidomimetics[END_REF][START_REF] Guillemyn | Neuropathic Pain: A Maladaptive Response of the Nervous System to Damage[END_REF] This shows that opioid-nociceptin hybrids are highly promising compounds as analgesics for the dual treatment of acute and neuropathic pain, devoid of respiratory depression.

MATERIALS AND METHODS

General

Thin-layer chromatography (TLC) was performed on glass plates precoated with silica gel 60F 254 (Merck, Darmstadt, Germany) using the mentioned solvent systems. Purification of organic molecules was done with flash chromatography (Davisil LC60A, 40-63 µm). Mass spectrometry (MS) was performed on a Micromass Q-Tof Micro spectrometer with electrospray ionization (ESI). Data collection and spectrum analysis was done with Masslynx software. Analytical RP-HPLC was performed using a Waters 717plus autosampler, a Waters 1525 binary HPLC pump, and a Waters 2487 dual absorbance wavelength detector (Milford, MA) on a Grace (Deerfield, IL) Vydac RP C18 column (25 cm × 4.6 mm × 5 µm) using UV detection at 215 nm. The mobile phase was a mixture of water and acetonitrile both containing 0.1% TFA. The used gradient runs from 3 to 100% acetonitrile in 20 min at a flow rate of 1 mL/min. Preparative RP-HPLC purification was done on a Gilson (Middleton, WI) HPLC system with Gilson 322 pumps, controlled by the software package Unipoint and a reversed phase C18 column (DiscoveryBIO SUPELCO Wide Pore C18 column, 25 cm × 2.21 cm, 5 µm) using a gradient that increased by 1%/min of acetonitrile in water (both containing 0.1% TFA) until the product eluted. After purification, the purity of all compounds was evaluated as being more than 95% by analytical RP-HPLC. All fractions were lyophilized using a Flexy-Dry lyophilizer (FTS Systems, Warminster, PA). 1 H and [START_REF] Tian | Bidirectional Modulatory Effect of Orphanin FQ on Morphine-induced Analgesia: Antagonism in Brain and Potentiation in Spinal Cord of the Rat[END_REF] C NMR spectra were recorded at 500 and 125 MHz on a Bruker Avance II 500 (Bruker Corp, Billerica, MA). Tetramethylsilane (TMS) or residual solvent signals were used as internal standard. The solvent used is mentioned in all cases, and the abbreviations used are as follows: s (singlet), d (doublet), dd (double doublet), t (triplet), q (quadruplet), and m (multiplet).

Synthesis

General Peptide Synthesis-All peptides were synthesized manually by Fmoc-based solid phase peptide synthesis (SPPS) on Rink amide AM resin. For standard couplings, a 3fold excess of the Fmoc-protected amino acids and 3-fold excess of coupling reagent (HCTU) in 0.4 NMM in DMF was used for 1.5 h. For the coupling of Fmoc-Aba-β-Ala-OH, 1.5-fold excess for both the dipeptide and the coupling reagent was used and coupling was left for 3 h. Fmoc deprotection was carried out by treatment of the resin with 20% 4methylpiperidine in DMF for 5 and 15 min. After every reaction step, the resin was washed with DMF (3 × 1 min), iPrOH (3 × 1 min), and CH 2 Cl 2 (3 × 1 min).

When the sequence was complete, the peptide was cleaved from the resin and completely deprotected with TFA/TES/H 2 O (95:2.5:2.5) for 3 h. The resin was filtered, and the filtrate was concentrated and added to cold ether. The precipitated peptide was then dissolved in acetonitrile/H 2 O and lyophilized to get the crude peptides as a powder.

The crude peptides were dissolved in H 2 O, and acetonitrile was added until complete dissolution was observed. The solution was injected on a Gilson preparative RP-HPLC.

Fractions containing the pure peptide were collected, combined, and lyophilized. The peptides were obtained as white powders with a purity of >95% as determined by analytical HPLC. The structures were confirmed by high-resolution electrospray mass spectrometry.

Coupling of the Protected Peptide (11) to the Sixmer (10) (Pathway A)-The protected peptide 11 (1.5 equiv) was dissolved in DMF, and DIC (1.5 equiv) and HOBt (1.5 equiv) were added to the solution. This coupling solution was then added to the resin and shaken for 3 h. No base was added to avoid additional coupling at the unprotected Dmt side chain. After washing with DMF (3×), iPrOH (3×), and CH 2 Cl 2 (3×), the peptide was cleaved from the resin and purified (See General peptide synthesis).

Peptide Characterization

Ac

Synthesis of the Dipeptide Building Blocks

Fmoc-Aba-β-Ala-OH ( 14)-Phth-Aba-β-Ala-OH 19 (3.91 g, 10.3 mmol, 1 equiv) was dissolved in 110 mL of ethanol. Hydrazine hydrate (3.0 mL, 62.0 mmol, 6 equiv) was added, and the solution was refluxed for 1.5 h, after which the solvent was evaporated. The residue was dissolved in 60 mL of water, and the pH was carefully adjusted to pH 5 by dropwise addition of acetic acid and monitoring with a pH meter. The suspension was stirred for 1 h at room temperature and the phthalhydrazide side-product precipitated. This was filtered off, and the filtrate was evaporated. The residue was dissolved in 100 mL of a water/acetone mixture. Sodium carbonate (261 mg, 11.4 mmol, 1.1 equiv) was added together with Fmoc-OSu (3.49 g, 10.3 mmol, 1 equiv) for the Fmoc protection. The reaction mixture was stirred for 16 h. The solvent was evaporated, and the residue was brought to pH 2 with 6 N HCl.

Ethyl acetate was added, and the phases were separated. The organic phase was washed with a saturated NaHCO 3 solution (3×) and brine.

In Vitro Biological Evaluation

In Vitro Affinity

Radioligand Binding Assays: Membranes from HEK293 cells transiently expressing human MOP, DOP, and KOP, as well as NOP, were obtained as previously reported. [START_REF] Becker | Ligands for κ-Opioid and ORL1 Receptors Identified from a Conformationally Constrained Peptide Combinatorial Library[END_REF][START_REF] Simonin | RF9, a Potent and Selective Neuropeptide FF Receptor Antagonist, Prevents Opioid-induced Tolerance Associated with Hyperalgesia[END_REF] All membrane preparations were stored at -80 °C as aliquots (1 mg protein/mL) until use. Following their dilution in 50 mM Tris-HCl buffer (pH 7.4) supplemented with 0.1% BSA, opioid receptor-containing membranes were incubated for 1 h at 25 °C with [ 3 H]Diprenorphine (0.6 nM; MOP, DOP, and KOP) or [ 3 H]OFQ/nociceptin (0.15 nM; NOP) and increasing concentrations of compounds to be tested in a final volume of 0.2 mL.

Nonspecific binding was defined in the presence of 10 µM naloxone (MOP, DOP, and KOP) or OFQ/nociceptin (NOP). Membrane-bound radioactivity was separated from free radioligand by rapid filtration through a 96-well GF/B unifilter apparatus (PerkinElmer Life and Analytical Sciences, Courtaboeuf, France) and quantified using a TopCount scintillation counter (PerkinElmer).

cAMP Accumulation: The agonist/antagonist nature of MOP/NOP hybrids at NOP was examined by using the GloSensor cAMP assay according to manufacturer recommendations (Promega, Madison WI, USA) with a few modifications. HEK293 cells selected for stable expression of human NOP and GloSensor were harvested from culture dishes and suspended (10 6 cells per mL) in physiological Hepes buffer (10 mM HEPES, 0.4 mM NaH 2 PO 4 , 137.5 mM NaCl, 1.25 mM MgCl 2 , 1.25 mM CaCl 2 , 6 mM KCl, 10 mM glucose, and 1 mg/mL bovine serum albumin, pH 7.4) supplemented with 1 mM D-luciferin. Following preequilibration for 2 h at 25 °C, D-luciferin-loaded HEK cells were distributed (100000 cells per well) in white 96-well Greiner plates (Courtaboeuf, France) and kinetic recordings of their luminescence level were acquired using a FlexStation II. In the agonist mode, compounds to be tested were injected at various concentrations 15 min before forskolin addition (0.5 µM final concentration) and readings were pursued for 90 min. In the antagonist mode, different concentrations of compounds to be tested were preincubated with cells for 15 min before the addition of various concentrations of OFQ/nociceptin. Experiments were conducted at 25 °C in the presence of 0.5 mM IBMX to prevent the degradation of cAMP by phospodiesterases. pA 2 values were obtained by analyzing the antagonist-induced rightward shifts of dose-response curves according to the Arunlakshana and Schild equation: 79 log (DR-1) = s log [B] -log K, where dose ratios DR (EC 50,obs / EC 50,cont ) serve to quantify midpoint shifts at each antagonist concentration [B], K is an estimate of antagonist potency, and s is the Schild slope. A pA 2 value for the antagonist was estimated via the pA 2 = pK/s relationship.

In Vitro Activity: Functional Guinea Pig Ileum (GPI) and Mouse Vas Deferens (MVD) Assays:

The GPI and MVD bioassays were carried out as described in detail elsewhere. [START_REF] Dimaio | Synthesis amd Pharmacological Characterization in vitro of Cyclic Enkephalin Analogues: Effect of the Conformational Constraints on Opioid Receptor Selectivity[END_REF][START_REF] Waterfield | Opioid Activities of Fragments of β-Endorphin and of its Lucine65-analogue. Comparison of the Binding Properties of Methionineand Leucine-enkephalin[END_REF] A dose-response curve was determined with [Leu 5 ]enkephalin as standard for each ileum and vas preparation, and IC 50 values of the compounds being tested were normalized according to a published procedure. 82

Parallel Artificial Membrane Permeability Assay (PAMPA):

The PAMPA assay was used to determine passive diffusion capacity across the BBB. The assay was performed as described previously. [START_REF] Arranz-Gibert | Lipid Bilayer Crossing-The Gate of Symmetry. Water-Soluble Phenylproline-Based Blood-Brain Barrier Shuttles[END_REF] Compounds were dissolved with the System Solution (pION) containing 20% 1-propanol (1 mL) at a concentration of 500 µM. Propranolol was used as a positive control. Stirring magnets were added in the donor compartment, and 195 µL of each compound was added then to donor compartments. Subsequently, 4 µL of a phospholipid mixture (Porcine Brain Polar Lipid Extract from Avantis Polar Lipids; 20 mg/mL in dodecane) was added to the membrane, located at the bottom of the acceptor compartments. This phospholipid mixture comprised phosphatidylcholine (PC; 12.6%), phosphatidylethanolamine (PE; 33.1%), phosphatidylserine (PS; 18.5%), phosphatidylinositol (PI; 4.1%), phosphatidic acid (0.8%), and other compounds (30.9%).

Acceptor wells were placed above the donor plate and filled with 200 µL of System Solution (20% 1-propanol). The PAMPA plate (pION) was placed into a GUTBOX (containing wet sponges) for 4 h at room temperature. Agitation was maintained in 25 µm of unstirred water layer (UWL). Compounds were then quantified by RP-HPLC and identified by MALDI-TOF. Effective permeability was calculated as shown in eq 1:

(1)

In Vitro Human BBB Cell-Based Model Assay: This assay was performed using the model developed by Cecchelli and co-workers. [START_REF] Cecchelli | A Stable and Reproducible Human Blood-Brain Barrier Model Derived from Hematopoietic Stem Cells[END_REF] Endothelial cells and pericytes were defrosted in gelatin-coated Petri dishes (Corning). Pericytes and endothelial cells were cultured in DMEM pH 6.8 or in supplemented endothelial cell growth medium (Sciencells), respectively. After 48 h, pericytes (50000 cells/well) and endothelial cells (80000 cells/well) were seeded in gelatin-coated 12-well plates or in Matrigel-coated 12-well Transwell inserts (Corning), respectively. Medium was changed every 2-3 days, and assays were performed 7-8 days after seeding. Lucifer Yellow (50 µM) was used as internal control (P app <15 × 10 -6 cm/s), and LY fluorescence was measured in a 96-well plate with a Fluoroskan Ascent microplate fluorometer (Thermo Fisher Scientific).

Compounds were dissolved in Ringer Hepes at the final concentration of 200 µM. Then, 500 µL of the compound and 1500 µL of Ringer HEPES alone were introduced in the apical or in the basolateral compartments, respectively. The plates were set on at 37 °C for 2 h. The solutions from both compartments were then recovered and quantified by UPLC and identified by UPLC-MS and MALDI-TOF. Apparent permeability was calculated using eq 2:

(2)

Behavioral Study in Mice

Animals-The male mice strain C57BL6 (20-25g), obtained from the Animal House Mossakowski Medical Research Centre, Polish Academy of Sciences, were housed 4-5 mice per cage in cages lined with sawdust. Animals was maintained under standard room temperature 22 ± 2 °C, humidity 50 ± 5%, 12/12 h light-dark cycle, with unlimited access to food and water. All experimental procedures were approved by The Local Committee for Ethics in Animal Experiments (permission number: 14/2013).

Acute Pain in Mice (Tail-Flick Test)-Analgesic activity of the compounds was measured on male mice strain C57BL6 in the tail-flick test. These experiments were conducted using a plantar test and Tail Flick Analgesia Meter apparatus (IITC Life Science Inc., USA), where a light beam is used as a thermal nociceptive stimulus. First, the mouse was restrained and either tested compound or saline (used as a control group) was administered by intravenous injection to the tail vain. During the measurements, the radiant heat was applied to the ventral side of animals' tail and the time latency for withdrawal or shaking of the tail was recorded. Measurements were performed before drugs administration (baseline) and at following time-points of 5, 15, 30, 60, and 120 min after injection. A cutoff of 10 s was set in the tail flick test in order to avoid tissue damage. Responses are expressed as a percentage of the maximum possible effect (%MPE) which is calculated as [(T 1 -T 0 )/(T 2 -T 0 )] × 100, where T 0 and T 1 are the tail-flick latencies before and after drug injection respectively and T 2 is the cutoff time. Drug-treated and saline-treated (control) groups consisted of 8-10 mice each. The significance is given in the graphs (no significance P > 0.05, significant * P < 0.05, ** P < 0.01, *** P < 0.001).

Neuropathic Pain

Animals-Male Albino Swiss CD- Chronic Constriction Injury in Mice-Chronic constriction injury (CCI) model was performed according to Bennet and Xie [START_REF] Bennett | A Peripheral Mononeuropathy in Rat that Produces Disorders of Pain Sensation Like Those Seen in Man[END_REF] and was modified for mice by Mika (2007). [START_REF] Mika | Minocycline and Pentoxifylline Attenuate Allodynia and Hyperalgesia and Potentiate the Effects of Morphine in Rat and Mouse Models of Neuropathic Pain[END_REF] Mice were preanaesthetized with chloral hydrate (500 mg/kg ip) to prolong and deepen anesthesia state. The surgical procedure was performed under isoflurane anesthesia. An incision was made below the mouse's right hipbone, and the sciatic nerve was exposed. Three ligatures with 4/0 silk thread were made around the nerve distal to the sciatic notch with 1 mm spacing until a brief twitch in the respective hind limb was observed. After 7 days of recovery, mice were tested to assess neuropathy development. All CCI mice developed allodynia and hyperalgesia. Main experiments were conducted on days 7-16 after CCI surgical procedure. Animals-Male Sprague-Dawley rats (Janvier, France) weighing between 250 and 350 g at the time of experimentation were used. Animals were housed for 7 days before experimentation in an environment maintained at 21 ± 0.5 °C with controlled humidity and light-dark cycle (lights on between 08:00 and 20:00). Food and tap water were provided ad libitum.

Drugs-Morphine sulfate was purchased from Francopia, France, diluted in 0.9% NaCl to obtain a solution of 3.5 mg/mL.

Jugular Catheterization-One week before the study, the rat jugular vein was catheterized using 20 cm silastic tubing with external and internal diameters of 0.94 and 0.51 mm, respectively (Dow Corning Co., Midland, MI). Catheter was tunneled subcutaneously and fixed at the back of the neck. Heparinized saline was injected into the catheter to avoid thrombosis and catheter obstruction. Rats were then returned to their individual cages for a recovery period of 7 days, allowing complete anesthesia washout. On the day of experimentation, rats were placed in horizontal Plexiglas cylinders (6.5 cm internal diameter, up to 20 cm adjustable length) (Harvard Apparatus, Inc., Holliston, MA, USA), modified by the addition of several holes at the cephalic end to avoid CO 2 rebreathing. Before drug administration, the catheter was exteriorized, purged, and its permeability checked.

Respiratory Effects Measurement Using the Whole Body Plethysmography-Four days before the study, temperature transmitters (TA-F10, DSI, Chatillon, France) were implanted in the peritoneal cavity. Ventilatory parameters were recorded in a whole body plethysmograph by the barometric method described and validated in the rat. [START_REF] Bartlett | Control of Breathing in Experimental Anemia[END_REF] The first measurement was performed after a 30 min period of accommodation, while the animal was quiet and not in deep or rapid eye movement sleep, as roughly estimated from their behavior, response to noise, and pattern of breathing. Then, the animal was gently removed from the chamber for iv injection of the tested drug at T 0 and replaced in the chamber for the remaining measurements. Ventilation was recorded at -30, -15, -5, 5, 10, 15, 30, 45, 60, 90, and 120 min, each record lasting about 60 s. The following parameters were measured: the tidal volume (V T ), the inspiratory time (T I ), the expiratory time (T E ), and the respiratory cycle duration (T TOT = T I + T E ). Additional parameters were calculated: the respiratory frequency (f) and the minute volume (V E = V T × f). T 0 values were the mean of the three baseline measurements.

Study Design-Rats were randomized into three groups receiving iv 0.9% NaCl, morphine (5 mg/kg) or 13a (0.5 mg/kg) to investigate antinociception (N = 7) and respiratory effects (N = 6).

Data Analysis

Results are expressed as mean ± SEM. To permit simultaneous analysis of the effect of time and treatments on %MPE and plethysmography parameters, for each animal and for each studied parameter, the area under the curve (AUC) from T 0 to the completion of the measurement (240 and 120 min, respectively) was calculated using the trapezoid method.

For each parameter, we compared the AUCs using Kruskal-Wallis tests for comparisons between the three groups. All tests were performed using Prism version 6.0 (GraphPad Software, Inc., San Diego, CA), and P-values of less than 0.05 were considered as significant. Effect of intrathecal (it) administration of hybrids 13a-13d and nociceptin parents 8 and 20 and morphine (all doses in nmol, 6-8 animals per group) on allodynia in CCI (A) and STZ (B) mice. As naive mice do not react to non-noxious stimuli, their reaction threshold may be established close to (5.9 ± 0.1 g) cutoff level (6 g). In comparison, the reaction threshold in CCI mice is 1.03 ± 0.07 g. Solid Phase Synthesis of the Bifunctional Opioid-Nociceptin Peptides Synthesis of Dipeptidomimetic Fmoc-Aba-β-Ala-OH ( 14) Compound Numbers, Sequences, and Yields of the Bifunctional Peptides Binding Affinity and Activity at Nociceptin and Opioid Receptors

  -Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 (8): Preparative HPLC yielded the desired compound (white powder, 34%). HPLC: t R = 9.4 min. TLC R f 0.23 (EBAW). HRMS (ESP + ) found m/z 939.5515 [M + H] + , C 44 H 71 N 14 O 9 + requires 939.5523. H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 (13a): Preparative HPLC yielded the desired compound (white powder, 25%). HPLC: t R = 10.0 min. TLC R f 0.60 (EBAW). HRMS (ESP + ) found m/z 1474.8403 [M + H] + , C 72 H 107 N 21 O 13 + requires 1474.8430. H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-(pF-Phe)-Arg-Trp-Arg-NH 2 (13b): Preparative HPLC yielded the desired compound (white powder, 17%

Figure 1 .

 1 Figure 1.Examples of reported opioid-NOP bifunctional ligands: 1 38,47-49 2 28,42 3 43 4[START_REF] Schunk | Discovery of a Potent Analgesic NOP and Opioid Receptor Agonist: Cebranopadol[END_REF][START_REF] Linz | Cebranopadol: A Novel Potent Analgesic Nociceptin/Orphanin FQ Peptide and Opioid Receptor Agonist[END_REF] a chimeric peptide 5[START_REF] Kawano | Synthesis and Receptor Binding Properties of Chimeric Peptides Containing a μ-Opioid Receptor Ligand and Nociceptin/Orphanin FQ Receptor Ligand Ac-RYYRIK-amide[END_REF][START_REF] Kawano | Receptor Binding Properties and Antinociceptive Effects of Chimeric Peptides Consisting of a μ-Opioid Receptor Agonist and an ORL1 Receptor Antagonist[END_REF] and 6[START_REF] Becker | Ligands for κ-Opioid and ORL1 Receptors Identified from a Conformationally Constrained Peptide Combinatorial Library[END_REF][START_REF] Bigoni | In vitro Pharmacological Profile of Peptide III-BTD: A Novel Ligand for Nociceptin/Orphanin FQ and Opioid Receptors[END_REF]

Figure 2 .

 2 Figure 2.Inhibition of nociceptin effect on forskolin-stimulated cAMP accumulation by hybrids 13a-13d in HEK293 stably expressing human NOP receptor. Dose-response curves of nociceptin were performed in absence or presence of 2 and 20 µM of each compound. This figure shows a representative experiment. Evaluation were performed at least two times in duplicate.

Figure 3 .

 3 Figure 3. Peptide transport in an in vitro cell-based model of the BBB.

Figure 4 .

 4 Figure 4. Time and dose dependent analgesic effect of 13a after iv administration compared to morphine. Ordinal values represent tail-flick latency measurements which were normalized as %MPE (mean ± SEM).

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.Effect of intrathecal (it) administration of hybrids 13a-13d and nociceptin parents 8 and 20 and morphine (all doses in nmol, 6-8 animals per group) on hyperalgesia in CCI (A) and STZ (B) mice. The naive mice very poorly respond to noxious thermal stimuli in cold plate test (26.8 ± 0.45 s), and the value is close to cutoff level (30 s). In comparison, the reaction threshold in CCI mice is 5.4 ± 0.3 s.

Figure 7 .

 7 Figure 7.Effects in Sprague-Dawley rats of intravenous 13a (0.5 mg/kg, open circles), morphine (5 mg/kg, gray triangles), and 0.9% NaCl (black triangles) on plethysmography parameters (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using Kruskal-Wallis tests. *p < 0.05, **p < 0.01.

  Scheme 2.

  -D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 18(A)/25(B) 13b H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-(pF-Phe)-Arg-Trp-Arg-NH 2 17 13c H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Dmt-Arg-Trp-Arg-NH 2 23 13d H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Trp-Arg-NH 2 Table 2

  of 3-4 experiments. The GPI functional assay is representative of MOP activation, whereas the MVD is a DOP representative assay. b Reported as NOP agonists 57 and antagonists. 33,55 Here, no agonist activity up to 10 µM nor antagonist activity up to 20 µM. K i , pA 2 , and EC 50 values represent means ± SEM for at least two experiments performed in duplicates. nd: not determined IC

  Therefore, simultaneous modulation can cause a much greater change of the input signal than a single compound can evoke. The hybrid strategy offers the advantage over the administration of the two substances which can give way to a different distribution profile.Nevertheless, also the NOP parent compounds remained effective at low doses, especially with regard to allodynia [e.g., ED 50 (8) = 0.008 nmol, ED 50 (20) = 0.004 nmol]. The Nterminally acetyl-capped parent analogue 8 was clearly less potent than the noncapped 20, which was most pronounced in the cold plate assay [e.g., ED 50 (8) = 0.69 nmol, ED 50 (20) = 0.06 nmol]) despite its higher NOP affinity (IC 50 = 0.53 vs 39 nM, Table2). Both NOP parents (8 and 20) were less potent in the cold plate test (thermal stimulus). Generally, thermal hyperalgesia and tactile allodynia do not correlate neither in mice nor in humans,74 and these effects are mediated not only by distinct cellular mechanisms, which is corroborated by a huge amount of data of assorted nature from electrophysiological to clinical observations

Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Dmt-Arg-Trp-Arg-NH 2 (13c): Preparative

	). HPLC: t R = 11.2 min. TLC R f 0.63
	(EBAW). HRMS (ESP + ) found m/z 1577.8538 [M + H] + , C 77 H 106 FN 24 O 12 + requires
	1577.8401.
	HPLC
	yielded the desired compound (white powder, 23%). HPLC: t R = 10.7 min. TLC R f 0.61
	(EBAW). HRMS (ESP + ) found m/z 802.4427 [M/2 + H] + , C 79 H 111 N 24 O 13 + /2 requires
	802.4418.
	H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Trp-Arg-NH 2 (13d): Preparative HPLC
	yielded the desired compound (white powder, 22%). HPLC: t R = 10.5 min. TLC R f 0.60
	(EBAW). HRMS (ESP + ) found m/z 788.4251 [M/2 + H] + , C 77 H 107 N 24 O 13 + /2 requires
	788.4261.
	H-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 (20): Preparative HPLC yielded the desired compound
	(white powder, 33%). HPLC: t R = 9.0 min. TLC R f 0.07 (EBAW). HRMS (ESP + ) found m/z
	897.5428 [M + H] + , C 42 H 69 N 14 O 8 + requires 897.5417.

H-

  The organic phase was dried with MgSO 4 , filtered, and evaporated. The resulting residue was crystallized from a minimum amount of hot ethanol. After cooling down and filtration, white crystals were obtained with a yield of 70% after two subsequent crystallizations. In a two-neck flask, equipped with a Dean-Stark apparatus, P 2 O 5 (10 g, 70.5 mmol, 11.1 equiv), 85% H 3 PO 4 (6.5 mL, 95.0 mmol, 15 equiv), 100 mL of acetic acid, and 150 mL of benzene were mixed. The solution was refluxed for 30 min, then the starting material 17 (2.5 g, 6.34 mmol, 1 equiv) and trioxane (3.75 g, 41.6 mmol, 6.6 equiv) were added. The mixture was refluxed until completion of the reaction (4 h) and every 30 min, trioxane (3.75 g, 41.6 mmol, 6.6 equiv) was added. After 2 h, P 2 O 5 (10.0 g, 70.5 mmol, 11.1 equiv) was added. After completion, benzene was evaporated and the residue was diluted with ether. This mixture was washed three times with 1 N HCl, saturated NaHCO 3 , and brine. The organic phase was dried, filtered, and evaporated to obtain a yellow oil. This residue was then purified with flash chromatography with 30% EtOAc in petroleum ether to yield a yellow oil in 65%.

	Yield: 65% (1.68 g). Formula: C 23 H 22 N 2 O 5 . MW: 406.44 g/mol. R f = 0.61 (EtOAc/ The residue was purified by column petroleum ether 1:1). HPLC: t R = 16.6 min. MS (ES +): 407 [M + H] + , 429 [M + Na] + , 361 chromatography with 1% methanol in CH 2 Cl 2 (+1% AcOH) and a yellow solid was [M -OEt] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 1.22 (3H, t, J = 7.2 Hz), 2.62 (2H, m), obtained in 54% yield. 3.12 (1H, dd, J = 15.6 Hz, 3 J = 4.7 Hz), 3.74 (1H, m), 3.89 (1H, m), 4.11 (3H, m), 4.71 (1H,
	d, J = 15.8 Hz), 4.78 (1H, d, 2 J = 15.9 Hz), 5.36 (1H, dd, J = 13.1 Hz, J = 4.9 Hz), 7.25-7.30 Yield: 54% (2.66 g). Formula: C 28 H 26 N 2 O 5 . MW: 470.53 g/mol. R f = 0.75 (EtOAc + 1% (4H, m), 7.75 (2H, m), 7.89 (2H, m). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 14.1, 33.2, AcOH). HPLC: t R = 17.3 min. HRMS (ESP + ): found m/z 471.1909 [M + H] + , + requires 471.1914; melting interval, 165.0-170.0 °C (decomposition). 1 H 34.1, 46.6, 52.0, 53.0, 60.7, 123.5, 127.1, 128.5, 128.7, 130.0, 132.0, 134.1, 135.6, 135.9, C 44 H 71 N 14 O 9 168.0, 168.6, 172.0. Yield: 89% (3.91 g). Formula: C 21 H 18 N 2 O 5 . MW: 378.38 g/mol. R f = 0.62 (EtOAc + 1% AcOH). HPLC: t R = 15.7 min. MS (ES+): 379 [M + H] + , 401 [M + H] + , 417 [M + K] + ; melting interval, 201.5-203.0 °C. 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 2.68 (2H, m), 3.10 (1H, dd, J = 15.5 Hz, J = 4.8 Hz), 3.72 (1H, m), 3.88 (1H, m), 4.11 (1H, dd, J = 15.5 Hz, J = 13.7 Hz), 4.73 (2H, pseudo-s), 5.32 (1H, dd, J = 13.0 Hz, J = 4.9 Hz), 7.22-7.32 (4H, m), 7.73 (2H, m), 7.87 (2H, m). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 32.9, 34.2, 46.6, 52.1, 53.2, 123.6, 127.3, 128.4, 128.8, 130.0, 132.0, 134.2, 135.6, 135.8, 167.9, 168.8, NMR (CDCl Yield: 70% (4.649 g, white crystals). Formula: C 22 H 22 N 2 O 5 . MW: 394.43 g/mol. R f = 0.56 175.7.
	(EtOAc/petroleum ether 1:1). HPLC: t R = 15.9 min. MS (ES+): 395 [M + H] + , 349 [M -
	OEt] + ; melting interval, 121.1-121.9 °C. 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 1.23 (3H,
	t, J = 7.1 Hz), 2.53 (2H, m), 3.54 (4H, m), 4.09 (2H, q, J = 7.1 Hz), 5.08 (1H, dd, J = 10.4
	Hz, J = 6.3 Hz), 6.70 (1H, m), 7.04-7.23 (5H, m), 7.63-7.82 (4H, m). 13 C NMR (CDCl 3 ,
	125 MHz): δ (ppm) = 14.1, 33.7, 34.7, 35.2, 55.7, 60.8, 123.5, 126.9, 128.6, 128.9, 131.4,
	134.2, 136.7, 167.8, 168.4, 172.5.

3 , 500 MHz): δ (ppm) = 2.32 (2H, m), 2.94 (1H, dd, J = 17.1 Hz, J = 13.5 Hz), 3.18 (1H, dd, J = 17.3 Hz, J = 4.5 Hz), 3.56 (2H, m), 4.14 (1H, d, J = 16.7 Hz), 4.24 (1H, m), 4.31 (2H, m), 5.08 (1H, m), 5.13 (1H, d).

13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 33.3, 35.6, 43.9, 47.1, 50.2, 51.4, 66.3, 120.6, 125.8, 126.4, 127.6, 128.1, 128.1, 129.2, 131.0, 135.2, 135.9, 141.2, 144.4, 156.1, 171.5, 173.0.

Phth-Phe-β-Ala-OEt (17)-Phthaloyl protected phenylalanine 16 (5 g, 16.9 mmol, 1 equiv) was dissolved in 80 mL of CH 2 Cl 2 . β-Alanine ethylester hydrochloride (β-Ala-OEt.HCl, 2.86 g, 18.6 mmol, 1.1 equiv) and coupling reagent TBTU (5.98 g, 18.6 mmol, 1.1 equiv) were added. Et 3 N (7.08 mL, 50.8 mmol, 3 equiv) was added to the solution, and the pH was kept at pH 8 by use of Et 3 N. The solution was then stirred for 1 h. The solvent was evaporated, and the residue was dissolved in ethyl acetate. The solution was then washed with 1 N HCl solution (3×), saturated NaHCO 3 solution (3×), and brine (3×).

Phth-Aba-β-Ala-OEt (18)-Phth-Aba-β-Ala-OH (19)-Phth-Aba-β-Ala-OEt 18 (4.74 g, 11.7 mmol, 1 equiv) was dissolved in 60 mL of acetone. Then 60 mL of a 1 N HCl solution was slowly added. The mixture was refluxed in an oil bath at 90 °C for 16 h and then cooled to room temperature, and the solvent was evaporated. A white solid was obtained in 89% yield and was used in the next step without purification.

  1 IGS mice (30-35 g) obtained from Charles River Breeding Laboratories, Germany, were housed six per cage in cages lined with sawdust under a standard 12/12 h light/dark cycle (lights on at 6:00 am) with food and water available ad libitum. All experiments were performed according to the recommendations of IASP, the NIH Guide for Care and Use of Laboratory Animals, and were approved by the local Bioethics Committee (Krakow, Poland).

Type 1 Diabetes Model in Mice-The type

  1 diabetes model was obtained by a single intraperitoneal (ip) injection of streptozocin (STZ; 200 mg/kg; Sigma-Aldrich, USA) prepared in water for injection (Polpharma, Poland). The glucose concentration was measured in blood collected from the tail vein. Mice with serum glucose levels above 300 mg/dL were considered diabetic. Mice that did not develop diabetes, or allodynia and hyperalgesia, were excluded from the study. The behavioral tests were conducted on day 10 after STZ injection. Mechanical sensitivity to non-noxious stimuli was measured by applying a set of calibrated nylon monofilaments (0.6-6 g; Stoelting) in serial increments on a tested hind paw plantar surface until a behavioral response was observed. Response considered as pain behavior included paw withdrawal, shaking, and licking. In the von Frey test, results are expressed as pressure [g] applied with a calibrated plastic filament to the midplantar surface of the mouse's injured hind paw (cutoff: 6 g), which elicited a foot withdrawal response. Sensitivity to noxious thermal stimuli was assessed with usage of Cold/Hot Plate Analgesia Meter, Columbus Instruments. The temperature of the plate was kept at 2 °C, and the cutoff latency was 30 s. The mice were placed on the cold plate, and the time until the hind paw was lifted is recorded. The lifting of the injured hind paw in the CCI model, or reaction of one of the hind paws in the STZ model were considered as a reaction to noxious cold stimulus. To assess motor coordination, Rota Rod 47600 for mice was used (Ugo Basile, Italy). Mice were placed on a horizontal rod which is rotating at accelerating speed, starting with 2 rpm and reaching 40 rpm within 300 s (cutoff). The experiment was conducted after three training sessions with minimum 15 min pause between each session. The time was recorded until a mouse fell off the rod.

	Behavioral Tests in Mice
	Von Frey Test: Cold Plate Test: Rota Rod Test:
	Drugs Administration-Drugs were dissolved in water for injection (Polpharma, Poland)
	and administered intrathecally (it) in 5 µL dose volume through lumbar puncture between L5
	and L6 to non-anesthetized mice according to upgraded Hylden and Wilcox 86 model
	described with modifications by Fairbanks. 87 The it injections were performed with
	disposable 30 gauge ½ in. needles (Becton Dickinson and Company, Rutherford, NJ,
	U.S.A.) matched to a 25 µL syringe (Hamilton, Reno, NV, USA). The von Frey and cold
	plate tests were performed at 30, 90, and 180 min after drug administration, while Rota Rod
	test was performed at 15 and 120 min.

Respiratory Depression-All the experimental protocols were carried out within the ethical guidelines established by the European Union Legislation. Protocols were approved by the local ethics committee for animal experimentation of Paris-Descartes University.

Table 1

 1

Table 3

 3 Transport in BBB-PAMPA Assay

	P e (cm/s) ×	SD P e (cm/s) ×	transport	SD transport
	no.	10 -6	10 -6	(%)	(%)
	7	0.9	0.6	1.8	1.3
	20	nd a	nd a	nd a	nd a
	8	0.07	0.05	0.15	0.10
	13a	0.11	0.02	0.23	0.04
	propranolol	8.59	0.8	15.2	1.4
	a nd: not detected.

Table 4

 4 Peptide Transport in an in Vitro Cell-Based Model of the BBB 68

no.

J Med Chem. Author manuscript; available in PMC 2016 April 28.

Acknowledgments

We thank Yannick van Wanseele and Dr. Ann Van Eeckhaut for the determination of plasma stability for compound 13a. The work of J.S., E.R., J.M., and B.P. was supported by a MAESTRO NCN2012/06/A/NZ4/00028; statutory funds; J.S. is a holder of KNOW scholarship sponsored by Ministry of Science and Higher Education, Poland. The work of M.T. and P.A. was supported by MINECO-FEDER (BIO2013-40716-R). V.U. and F.S. were supported by CNRS, Strasbourg University and LABEX ANR-10-LABX-0034-Medalis and received financial support from the French government managed by "Agence Nationale de la Recherche" under "Programme d'investissement d'avenir". The work of P.W.S. was supported by NIH grants DA004443 and DA015353, and by CIHR grant MOP-89716.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

ABBREVIATIONS USED

%MPE percent of maximal possible effect

Author Manuscript


Lucie Hartmann

Estelle Metzger

Noémie Ottelard

Renaud Wagner
email: renaud.wagner@unistra.fr

Direct extraction and purification of recombinant membrane proteins from Pichia pastoris protoplasts

Keywords: Pichia pastoris, yeast, membrane protein, protoplast, detergent solubilization, purification

In the past decade, the methylotrophic yeast Pichia pastoris has proved to be one of the most efficient systems for mass production of recombinant eukaryotic membrane proteins (MPs), leading to the crystallization and structure determination for a variety of them. The actual overexpression of functional MPs achieved with this system is however often accompanied by the formation of a variable but significant proportion of misfolded and/or aggregated proteins that are co-extracted and copurified during the purification process. In order to minimize this unwanted phenomenon, we devised a novel procedure in which MPs produced in Pichia pastoris are directly solubilized from whole cells instead of crude membrane preparation. This approach aims at favouring the extraction of correctly folded membrane proteins that have been targeted to the plasma membrane, limiting the solubilization of the misfolded proteins and protein aggregates that are stored in internal membrane compartments. The method described herewith is based on the formation of protoplasts through enzymatic treatment prior to protein solubilization. This chapter details a set of protocols going from yeast cell preparation and protein solubilization to purification using affinity and size exclusion chromatography.

Introduction

When coping with membrane protein (MP) solubilization from any cellular expression system, the most common approach consists in the preliminary preparation of the membrane fraction. This membrane enrichment step can be quite valuable during the downstream extraction and purification phases by reducing the amount of soluble proteases as well as potential soluble contaminants from the sample.

However, membrane fractionation necessarily involves cell lysis procedures that could be more or less drastic. When harsh mechanical conditions are used, undesired and poorly controlled events such as heat and shear forces may occur that are often detrimental to the integrity of membrane proteins.

In addition, standard MP protocols usually recover the whole membrane fractions issuing from the different cellular compartments and containing a mixture of properly folded and misfolded proteins. Even if this phenomenon is still poorly characterized in the context of membrane protein production, it is actually widely recognised that overexpression overwhelms the cell biosynthesis and translocation machineries that often elicits improper MP folding. Such events cause a number of stresses and responses with various outcomes including the retention of misfolded proteins within the secretory pathway and the formation of protein aggregates before their eventual degradation [START_REF] Schlebach | The safety dance: biophysics of membrane protein folding and misfolding in a cellular context[END_REF]2). As a result, when preparing membranes for protein purification purposes, the following solubilization step may lead to the co-extraction of various heterogeneous forms of the protein of interest that are further difficult to separate.

The protocol presented here proposes to limit these phenomena by performing a short solubilization step directly on whole cells in order to favour the extraction of the undamaged, correctly folded MPs that have been targeted to the plasma membrane.

This method has been developed in our lab with the yeast Pichia pastoris, a robust and versatile system that we routinely use for the recombinant expression of various MPs [START_REF] André | Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen[END_REF](4)(5)(6)(7). Because this cellular host possesses a very thick and robust cell wall that is detergent-resistant, a preliminary enzymatic treatment is required to obtain yeast protoplasts (i.e. yeast cells devoid of their cell wall) before the detergent solubilization and purification steps.

The whole procedure has been successfully applied to a variety of eukaryotic (mainly human) MPs recombinantly expressed in P. pastoris, encompassing GPCRs, ion channels and other integral MPs. By comparison with the standard procedure where the same proteins were extracted from membrane preparations after cell lysis, the method not only proved to be much more rapid, but, most importantly, allowed to significantly reduce the presence of protein aggregates after purification in several cases (manuscript in preparation).

Here, we illustrate this method with the solubilization and purification of the human adenosine A2A receptor extracted from yeast protoplasts. The starting biological material consists of yeast pellets obtained after the expression of a N-Flag-10His-HUMAN_AA2A-Biotag construct, following the conditions reported in [START_REF] André | Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen[END_REF]. A full description of the generation and selection of Pichia pastoris clones, as well as the subsequent procedures for recombinant expression can be found in other recent and comprehensive chapters of the Methods in Molecular Biology series (8,9).

The present protocol reports the enzymatic treatment of yeast cells expressing recombinant protein to obtain protoplasts, which are then directly subjected to detergent solubilization (Figure 1). A subsequent two-step purification procedure (Ion Metal Affinity Chromatography and Size Exclusion Chromatography) is also presented to illustrate the yield and purity of the MPs that can be isolated with this method. 6. Zymolyase® 20T (Amsbio). It can be resuspended in water at 200 U/ml and stored at -20°C for a few months.

Materials

Protein solubilization

1. Solubilization buffer: 50 mM Tris HCl pH 7.4, 500 mM NaCl, 10% glycerol, 20 mM imidazole (see Note 1), detergent (see Note 2), inhibitor protease cocktail (added extemporaneously, e.g. cOmplete TM EDTA-free, Roche).

2. Ultracentrifuge equipped with an appropriate fixed-angle rotor and adapted polycarbonate tubes.

Purification of solubilized material

1. Automated protein purification system (ÄKTA Purifier, GE Healthcare, or equivalent). A sample pump or a superloop is required for the affinity chromatography step.

Methods

Protoplasting: Zymolyase® treatment of recombinant cell paste

Zymolyase® is a mixture of several carbohydrolases (i.e., ß-1,3 glucan laminaripentaohydrolase, ß-1,3 glucanase, protease and mannanase) extracted from Arthrobacter luteus. These enzymes work in conjunction to degrade efficiently the cell wall of a large spectrum of yeasts, thus leading to the formation of yeast protoplasts.

A number of other enzymes for yeast cell wall degradation are commercially available but Zymolyase® has proved to be the most efficient in our hands for this protocol with Pichia pastoris.

Cell paste preparation

The following proportions are given for 3 g of wet cell paste, obtained from about 250 ml of Pichia pastoris culture.

Resuspend the cell pellet in 50 ml

Milli-Q H 2 O.
2. Harvest the cells by centrifuging for 5 min at 5,000 g, 4°C.

3. Discard the supernatant and wash the cell pellet with 50 ml SED buffer.

4. Harvest the cells by centrifuging for 5 min at 5,000 g, 4°C.

5. Discard the supernatant and wash the cell pellet with 50 ml 1 M sorbitol.

6. Harvest the cells by centrifuging for 5 min at 5,000 g, 4°C.

7. Discard the supernatant and resuspend the cell pellet in 37.5 ml of CG buffer, in order to reach a cell paste concentration of 80 g/L.

Enzymatic cell wall degradation

1. Add 20 U of Zymolyase® per gram of cell paste initially prepared.

2. Incubate for 15 min (see Note 3) at room temperature under gentle agitation (see

Note 4).

3. Harvest the protoplasts formed by a soft centrifugation for 5 min at 750 g, room temperature. In order to avoid protoplast lysis, reduce the acceleration and deceleration speed of the centrifuge. 4. Carefully discard the supernatant using a pipette.

Protein solubilization

As for every MP solubilization, the successful selection of the best detergent to use is a fine-tuned combination between the extraction efficiency and the preservation of the protein fold and function. It both depends on the protein of interest and the expression system. The choice of the detergent, here DDM for AA2A, has thus to be adjusted accordingly (see Note 2).

1. Gently resuspend the pellet in 37.5 ml of solubilization buffer (no vortexing). The cell paste concentration should be close to 80 g/L.

2. Incubate for 30 min at room temperature under gentle agitation.

3. Separate the solubilized fraction by ultracentrifuging for 30 min at 100,000 g and 4°C. Store 20 µl of the suspension before ultracentrifugation at 4°C for SDS-PAGE analysis.

4. After ultracentrifugation, recover the supernatant containing the solubilized fraction. Store 20 µl at 4°C for SDS-PAGE analysis.

5. The ultracentrifuge pellet can also be analyzed by SDS-PAGE after resuspension in 37.5 ml of solubilization buffer without detergents, imidazole and protease inhibitor cocktail. Use a glass Teflon homogenizer to obtain a homogeneous suspension.

Purification of solubilized material

Nickel affinity chromatography

The following protocol is designed for automated purification (e.g. using ÄKTA protein purification systems). It can however be easily adapted for batch/gravity flow purification. The whole purification process can be performed at a flow rate of 1 ml/min. 1. Filter the ultracentrifuge supernatant using a 0.22 µm syringe filter.

2. Add imidazole at a final concentration of 20 mM.

3. Equilibrate a 1 ml pre-packed nickel affinity column with buffer A.

4. Inject the filtrated supernatant onto the column. Store 20 µl of the injected sample at 4°C for SDS-PAGE analysis.

5. Wash with 20 ml of buffer A, or until the 280 nm-absorbance signal returns to the baseline. 6. Wash for 10 ml with 20% buffer B (corresponding to about 100 mM imidazole) (see Note 5).

7. Elute the protein of interest either with a gradient of buffer B or with a step of 100% buffer B (see Note 6). Collect 0.5 ml elution fractions. 8. Pool the fractions representative of one elution peak and concentrate them, using a centrifugal concentrator, to a volume suited for injection onto the size exclusion chromatography column (according to the manufacturer's recommendations, typically a maximum of 500 µl for a 24 ml column). For concentration, choose a cut-off adapted to the size of the protein of interest (typically 30 or 50 kDa MWCO). 9. Store 20 µl of the concentrated pool of interest and all other representative elution fractions (flow through and wash fractions) at 4°C for SDS-PAGE analysis.

Size Exclusion Chromatography (SEC)

The following protocol is designed for automated purification (e.g. using ÄKTA protein purification systems). Superdex 200 10/300 SEC column or equivalent is generally well suited for small-scale preparative purification of MPs between 10 kDa and 600 kDa.

1. Equilibrate the SEC column with at least 1.5 column-volume (CV) of running buffer.

2. Inject the concentrated sample. Set the flow rate according to the manufacturer's recommendations. Collect 0.5 ml elution fractions.

Store 20 µl of each peak-representative fraction at 4°C for SDS-PAGE analysis.

Figure 2 shows a typical SEC elution profile obtained during the purification of the adenosine A2A receptor using the procedure described above. SEC enables the separation of oligomeric objects and contaminants from monomeric receptor of interest. Oligomeric and/or aggregated objects are eluted close to the void volume of the column (corresponding to peak 1) and correspond to the inactive protein. The monomeric and ligand-binding active receptor is mainly eluted in peak 2. In typical similar experiments performed on proteins extracted from whole membrane fractions after cell lysis (standard procedure), peak 1 has systematically about 3 to 5 times the intensity of peak 2 (data not shown). These data exemplify how this method may be highly beneficial to reduce the amount of aggregated MPs during extraction. In terms of yield, the protocol described here enables to recover about 1.5 mg of monomeric adenosine A2A receptor from 1 liter of yeast culture. However, protein extraction from yeast protoplasts does not significantly improve the final amount of active purified receptors compared to more classical methods.

Fractions representative of each elution peak (respectively 1, 2 and 3) have to be analysed using biochemical methods in order to assess their protein content. In this chapter we describe SDS-PAGE analyses, but functional tests such as radioligand binding assay may be also performed if available for the protein of interest.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

10% SDS tricine polyacrylamide gel preparation

The following proportions are given for casting two gels of 1 mm thickness in a standard Mini-Protean system from Bio-Rad.

1. Prepare the separating gel by mixing 2.5 ml of acrylamide solution, 3.3 ml of Tris-HCl SDS buffer, 1.25 ml of 80% (v/v) glycerol, 2.9 ml H 2 O. Add 90 µl of APS and 6 µl of TEMED, mix, and immediately cast the gel. Allow space for the stacking gel.

2. Prepare the stacking gel by mixing 0.6 ml of acrylamide solution, 1.6 ml of Tris-HCl SDS buffer, 4.1 ml H 2 O. Add 90 µl of APS and 6 µl of TEMED, mix, and cast carefully over the separating gel. The presence of glycerol in the separating part avoids the need to wait for its complete polymerization before casting the stacking gel. Insert a 10-well gel comb immediately without introducing air bubbles.

3. Let polymerize for about 30 min.

Gel electrophoresis and Coomassie staining

1. Incubate 20 µl of the elution fractions of interest in 20 µl of SB 2X for about 10 min at room temperature (see Note 7).

2. Load in parallel two 10% SDS-polyacrylamide gels with respectively 20 µl (first gel) and 10 µl (second gel) of the sample in its sample buffer. The first gel will be used for total protein staining with Coomassie blue, the second one will be transferred on a nitrocellulose membrane for immunodetection of the protein of interest.

Proceed to electrophoresis using Tris-Tricine-SDS cathode running buffer and

Tris anode running buffer in a tank unit. Run for about 1 h 30 min at 100 V.

Stain the first gel with Coomassie blue following the manufacturer's instructions.

Figure 3A presents a typical Commassie blue SDS-PAGE analysis obtained for adenosine A2A receptor purification. A number of contaminant proteins are still present in the IMAC elution pool but SEC enables to obtain highly purified proteins.

Protein transfer and Western Blot Immunodetection

1. Transfer the proteins from the second gel to a nitrocellulose membrane by electroblotting in Tris-glycine transfer buffer for about 1 h 30 min at 100 V. An ice pack can be added into the electroblotting device to mitigate the heat produced.

2. Incubate the membrane in 50 ml blocking buffer for 1 h at room temperature on an orbital shaker. Alternatively, incubate the membrane overnight at 4 °C.

3. Remove the blocking solution and incubate the membrane with the selected antibody diluted in blocking buffer (for instance a monoclonal anti-flag antibody at a final concentration of 0.1 µg/ml) for 1 h at room temperature on an orbital shaker.

4. Wash the membrane 3 times, each time with 50 ml of PBST on an orbital shaker for 5 min at room temperature. 5. Remove the PBST and incubate the membrane with the adapted ant-IgG antibody diluted in blocking buffer (typical final concentration of 0.1 µg/ml) for 1 h at room temperature on an orbital shaker.

6. Wash the membrane 3 times, each time with 50 ml of PBST on an orbital shaker for 5 min at room temperature.

7. Remove the PBST and wash the membrane with 50 ml of PBS on an orbital shaker for 5 min at room temperature.

8. Store the membrane in PBS until revelation. 9. Proceed to membrane revelation according to the reporter system selected and following the manufacturer's recommendations.

Figure 3B shows the western blot analysis of adenosine A2A receptor purification, using an anti-flag secondary antibody. Several bands corresponding to the receptor of interest can be identified. The major signal, at the apparent molecular weight of 55 kDa, corresponds to the complete form of the receptor. SEC peak 2 thus contains the complete and monomeric form of the adenosine A2A receptor. A radioactive ligand binding assay further confirms the functional state of this fraction of the purified receptor (data not shown).

Notes

1. Imidazole can be added to the solubilized sample after the ultracentrifugation step. The use of high quality imidazole (e.g. I0250, Sigma), which shows a very limited absorption signal at 280 nm, is strongly recommended for ion immobilized metal affinity chromatography.

2. The detergent used in the buffers has to be selected according to its solubilization and stabilization properties toward your protein of interest. We recommend a preliminary solubilization screening using different detergents before large-scale purification. In terms of concentration, we recommend using about 10 to 20 times the critical micellar concentration (CMC) during the solubilization step, and at least twice the CMC for subsequent purification steps.

3. The duration of Zymolyase® treatment and/or amount of Zymolyase® used may be critical for protein integrity and can thus be adjusted to the protein of interest. 4. Whereas yeast cells are very resistant to mechanical stress, yeast protoplasts are as fragile as mammalian cells and shall thus be handled very carefully in order to avoid mechanical cell lysis.

5. This first step enables to get rid of unspecific contaminants, and can be adapted according to the protein of interest and the cellular context. In our experience, G protein coupled receptors labeled with a decahistidine tag elute from Ni Sepharose High Performance nickel affinity columns from about 150 mM imidazole.

6. Protein elution using an imidazole gradient (typically 0 to 100% of B buffer spread in 25 ml) can be of great use to separate monomeric from oligomeric or aggregated MPs. Based on our experience, aggregated proteins show a higher affinity for the resin and are thus eluted later than monomeric proteins.

2. 1 . Zymolyase® treatment 1 .

 11 Recombinant cell pellet. 2. Milli-Q water. 3. SED buffer: 1 M sorbitol, 25 mM ethylenediaminetetraacetic acid (EDTA), 1 M dithiothreitol (DTT) (added extemporaneously). 4. 1 M sorbitol. 5. CG buffer: 20 mM trisodium citrate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF) (added extemporaneously). Adjust the pH to 5.8 with hydrochloric acid.

7 .

 7 It is usually not recommended to boil MP samples prior to electrophoresis. When compact and highly hydrophobic proteins such as GPCRs are boiled, they usually aggregate and stay stuck in the concentrating gel.

Figure 1 :

 1 Figure 1: Protoplasting and protein solubilization procedure. Recombinant yeast cell pellet is first submitted to Zymolyase® treatment for cell wall degradation. The protoplasts formed are then directly used for protein solubilization using detergents.An ultracentrifugation step enables to recover the solubilized fraction that will undergo the purification process.

Figure 2 :

 2 Figure 2: Size exclusion chromatography elution profile of the adenosine A2A receptor. Profile obtained on a Superdex 200 Increase 10/300 GL SEC column with absorbance measured at 280 nm. In this case, SEC enables the separation of 3 main peaks named respectively 1, 2 and 3.

Figure 3 :

 3 Figure 3: SDS-PAGE analysis of representative fractions from adenosine A2A receptor purification. P: ultracentrifugation pellet; S: solubilized fraction loaded onto nickel affinity chromatography column; FT: IMAC flow through; E: IMAC elution pool; W1 and W2: IMAC washes before elution; 1, 2 and 3: SEC elution peaks. A: Analysis of the total protein content of 10 µl of each sample on a 10% polyacrylamide Tris-Tricine gel stained with Coomassie blue. B: Western Blot analysis of the Adenosine A2A receptor content of 5 µl of each sample. Immunoblotting realized with a M2 anti-flag antibody (0.1 µg/ml).