Characterization of anti-GASP motif antibodies that inhibit the interaction between GPRASP1 and G protein-coupled receptors.

G protein-coupled receptor associated sorting protein 1 (GPRASP1) belongs to a family of 10 proteins that display sequence homologies in their C-terminal region. Several members including GPRASP1 also display a short repeated sequence called the GASP motif that is critically involved in protein-protein interactions with G protein-coupled receptors (GPCRs). Here, we characterized anti-GASP motif antibodies and investigated their potential inhibitory functions. We first showed that our in-house anti-GPRASP1 rabbit polyclonal serum contains anti-GASP motif antibodies and purified them by affinity chromatography. We further showed that these antibodies can detect GPRASP1 and GPRASP2 in Western blot, immunoprecipitation and immunofluorescence experiments while a mutant of GPRASP2, in which the most conserved hydrophobic core of the GASP motifs is mutated, was no more detected. Further characterization of anti-GASP motif antibodies by ELISA and Surface Plasmon Resonance assays suggests that GASP motifs function as multivalent epitopes. Finally, we set-up an Amplified Luminescent Proximity Homogeneous AlphaScreen® assay to detect the interaction between purified ADRB2 receptor and the central domain of GPRASP1 and showed that anti-GASP motif antibodies efficiently inhibit this interaction. Altogether, our results suggest that anti-GASP motif antibodies could represent a valuable tool to neutralize the interaction of GPRASP1 and GPRASP2 with different GPCRs.

Neuropeptide FF and prolactin-releasing peptide decrease cortical excitability through activation of NPFF receptors.

OBJECTIVE: Drugs with a novel mechanism of action are needed to reduce the number of people with epilepsy that are refractory to treatment. Increasing attention is paid to neuropeptide systems and several anticonvulsant neuropeptides have already been described, such as galanin, ghrelin, and neuropeptide Y (NPY). Many others, however, have not been investigated for their ability to affect epileptic seizures. In this study, the potential anticonvulsant activities of three members of the RF-amide neuropeptide family, neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), and kisspeptin (Kp) and other receptor ligands (NPFF1/2 R, GPR10, and GRP54, respectively) were tested in the motor cortex stimulation model. METHODS: A train of pulses with increasing intensity (0-10 mA over 150 s, 50 Hz, pulse width 2 msec) was delivered to the motor cortex of rats. The threshold intensity for eliciting a motor response (i.e., motor threshold) was determined through behavioral observation and used as a measure for cortical excitability. The threshold was determined before, during, and after the intracerebroventricular (i.c.v.) administration of various NPFF1/2 R, GPR10, and GPR54 receptor ligands. RESULTS: NPFF and PrRP significantly increased the motor threshold by a maximum of 143 ± 27 and 83 ± 13 µA, respectively, for the doses of 1 nmol/h (p < 0.05). The increase of motor threshold by NPFF and PrRP was prevented by pretreatment and co-treatment with the NPFF1/2 R antagonist RF9. Pretreatment with a selective NPFF1 R antagonist also prevented the threshold increase induced by NPFF. Kp did not increase motor threshold. SIGNIFICANCE: Intracerebroventricular infusion of NPFF or PrRP decreases cortical excitability in rats through activation of NPFFRs. Furthermore, the NPFF1 R is required for the NPFF-induced decrease in cortical excitability.

Rapid and scalable synthesis of innovative unnatural α,ß or γ-amino acids functionalized with tertiary amines on their side-chains.

We report a selective ruthenium catalyzed reduction of tertiary amides on the side chain of Fmoc-Gln-OtBu derivatives, leading to innovative unnatural α,ß or γ-amino acids functionalized with tertiary amines. Rapid and scalable, this process allowed us to build a library of basic unnatural amino acids at the gram-scale and directly usable for liquid- or solid-phase peptide synthesis. The diversity of available tertiary amines allows us to modulate the physicochemical properties of the resulting amino acids, such as basicity or hydrophobicity.

Multitarget µ-Opioid Receptor Agonists─Neuropeptide FF Receptor Antagonists Induce Potent Antinociception with Reduced Adverse Side Effects.

The design of bifunctional compounds is a promising approach toward the development of strong analgesics with reduced side effects. We here report the optimization of the previously published lead peptide KGFF09, which contains opioid receptor agonist and neuropeptide FF receptor antagonist pharmacophores and is shown to induce potent antinociception and reduced side effects. We evaluated the novel hybrid peptides for their in vitro activity at MOP, NPFFR1, and NPFFR2 and selected four of them (DP08/14/32/50) for assessment of their acute antinociceptive activity in mice. We further selected DP32 and DP50 and observed that their antinociceptive activity is mostly peripherally mediated; they produced no respiratory depression, no hyperalgesia, significantly less tolerance, and strongly attenuated withdrawal syndrome, as compared to morphine and the recently FDA-approved TRV130. Overall, these data suggest that MOP agonist/NPFF receptor antagonist hybrids might represent an interesting strategy to develop novel analgesics with reduced side effects.

Development of sub-nanomolar dipeptidic ligands of neuropeptide FF receptors.

Based on our earlier reported neuropeptide FF receptors antagonist (RF9), we carried out an extensive structural exploration of the N-terminus part of the amidated dipeptide Arg-Phe-NH(2) in order to establish a structure-activity relationships (SAR) study towards both NPFF receptor subtypes. This SAR led to the discovery of dipeptides (12, 35) with subnanomolar affinities towards NPFF1 receptor subtype, similar to endogenous ligand NPVF. More particularly, compound 12 exhibited a potent in vivo preventive effect on opioid-induced hyperalgesia at low dose. The significant selectivity of 12 toward NPFF1-R indicates that this receptor subtype may play a critical role in the anti-opioid activity of NPFF-like peptides.

Behavioral characterization, potential clinical relevance and mechanisms of latent pain sensitization.

Chronic pain is a debilitating disorder that can occur as painful episodes that alternates with bouts of remission and occurs despite healing of the primary insult. Those episodes are often triggered by stressful events. In the last decades, a similar situation has been evidenced in a wide variety of rodent models (including inflammatory pain, neuropathy and opioid-induced hyperalgesia) where animals develop a chronic latent hyperalgesia that silently persists after behavioral signs of pain resolution. This state, referred as latent pain sensitization, is due to the compensatory activation of antinociceptive systems, such as the opioid system or NPY and its receptors. A transitory phase of hyperalgesia can then be reinstated by pharmacological or genetic blockade of these antinociceptive systems or by submitting animals to acute stress. Those observations reveal that there is a constant endogenous analgesia responsible for chronic pain inhibition that might paradoxically contribute to maintain this maladaptive state and could then participate to the transition from acute to chronic pain. Thus, demonstration of the existence of this phenomenon in humans and a better understanding of the mechanisms by which latent pain sensitization develops and maintains over long periods of time will be of particular interest to help identifying new therapeutic strategies and targets for chronic pain treatment. The present review aims to recapitulate behavioral expression, potential clinical relevance, cellular mechanisms and intracellular signaling pathways involved so far in latent pain sensitization.

GPRASP/ARMCX Protein Family: Potential Involvement in Health and Diseases Revealed by their Novel Interacting Partners.

GPRASP (GPCR-associated sorting protein)/ARMCX (ARMadillo repeat-Containing proteins on the X chromosome) family is composed of 10 proteins, whose genes are located on a small locus of the X chromosome except one. They possess at least two armadillo-like repeats on their carboxylterminal homologous sequence, but they can be subdivided on specific sequence features. Subfamily 1 (GPRASP1, GPRASP2, GPRASP3, ARMCX4 and ARMCX5) displays additional repeated motifs while a mitochondrial targeting transmembrane domain is present in subfamily 2 (ARMC10, ARMCX1, ARMCX2, ARMCX3 and ARMCX6). Although their roles are not yet fully understood, the recent identification of several interacting partners has shed new light on the processes in which GPRASP/ARMCX proteins are implicated. Among the interacting partners of proteins from subfamily 1, many are GPCRs. GPRASP1 binds trafficking proteins, such as Beclin2 and the Dysbindin-HRS-Gαs complex, to participate in GPCR post-endocytic sorting. Moreover, in vitro as well as in vivo experiments indicate that GPRASP1 is a critical player in the adaptive responses related to chronic treatments with GPCR agonists. GPRASP2 seems to play a key role in the signaling of the hedgehog pathway in the primary cilium through a Smoothened-GPRASP2-Pifo complex. Identified small compound inhibitors of this complex could treat drug-resistant smoothened derived cancer forms. Deletion of GPRASP2 in mice causes neurodevelopmental alteration and affects mGluR5 regulation, reflected by autism-like behavior. Several members of subfamily 2, in complex with TRAK2 and MIRO, are involved in the trafficking of mitochondria in axons and in the regulation of their size and division, influencing the cell cycle. The essential role of GPRASP/ARMCX proteins in cellular physiology is supported by human cases of deletions, causing male neonatal lethality by pulmonary delayed development, dysmorphic face, and psychiatric and intellectual impacts in females.

Comprehensive overview of biased pharmacology at the opioid receptors: biased ligands and bias factors.

One of the main challenges in contemporary medicinal chemistry is the development of safer analgesics, used in the treatment of pain. Currently, moderate to severe pain is still treated with the "gold standard" opioids whose long-term often leads to severe side effects. With the discovery of biased agonism, the importance of this area of pharmacology has grown exponentially over the past decade. Of these side effects, tolerance, opioid misuse, physical dependence and substance use disorder (SUD) stand out, since these have led to many deaths over the past decades in both USA and Europe. New therapeutic molecules that induce a biased response at the opioid receptors (MOR, DOR, KOR and NOP receptor) are able to circumvent these side effects and, consequently, serve as more advantageous therapies with great promise. The concept of biased signaling extends far beyond the already sizeable field of GPCR pharmacology and covering everything would be vastly outside the scope of this review which consequently covers the biased ligands acting at the opioid family of receptors. The limitation of quantifying bias, however, makes this a controversial subject, where it is dependent on the reference ligand, the equation or the assay used for the quantification. Hence, the major issue in the field of biased ligands remains the translation of the in vitro profiles of biased signaling, with corresponding bias factors to in vivo profiles showing the presence or the lack of specific side effects. This review comprises a comprehensive overview of biased ligands in addition to their bias factors at individual members of the opioid family of receptors, as well as bifunctional ligands.

Identification of an N-acylated-DArg-Leu-NH2 Dipeptide as a Highly Selective Neuropeptide FF1 Receptor Antagonist That Potently Prevents Opioid-Induced Hyperalgesia.

RFamide-related peptide-3 (RFRP-3) and neuropeptide FF (NPFF) target two different receptor subtypes called neuropeptide FF1 (NPFF1R) and neuropeptide FF2 (NPFF2R) that modulate several functions. However, the study of their respective role is severely limited by the absence of selective blockers. We describe here the design of a highly selective NPFF1R antagonist called RF3286, which potently blocks RFRP-3-induced hyperalgesia in mice and luteinizing hormone release in hamsters. We then showed that the pharmacological blockade of NPFF1R in mice prevents the development of fentanyl-induced hyperalgesia while preserving its analgesic effect. Altogether, our data indicate that RF3286 represents a useful pharmacological tool to study the involvement of the NPFF1R/RFRP-3 system in different functions and different species. Thanks to this compound, we showed that this system is critically involved in the development of opioid-induced hyperalgesia, suggesting that NPFF1R antagonists might represent promising therapeutic tools to improve the use of opioids in the treatment of chronic pain.

Prolactin-releasing peptide effects in the rat brain are mediated through the Neuropeptide FF receptor.

Prolactin-releasing peptide (PrRP), an RF amide peptide present in the brain, generates a wide variety of centrally generated autonomic responses, including increases in arterial blood pressure and heart rate. The identity of the receptor mediating the effects of PrRP is unknown. In addition to GPR10, which is its putative endogenous receptor, PrRP demonstrates a high binding affinity for Neuropeptide FF (NPFF) receptors, specifically the NPFF2 receptor. In the present study, we examined whether the central cardiovascular effects of PrRP in the intact animal and its cellular effects on parvocellular paraventricular nucleus (PVN) neurons are mediated via NPFF receptors. In conscious rats, intracerebroventricular (i.c.v.) PrRP caused an increase in arterial blood pressure and heart rate, which was blocked with RF9, a specific NPFF receptor antagonist. These PrRP-evoked cardiovascular effects were preserved in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat strain, in which the GRP10 receptor gene was mutated. In rat brain slices, whole-cell patch clamp recordings of parvocellular paraventricular nucleus neurons show PrRP caused a decrease in evoked and miniature GABAergic inhibitory postsynaptic currents (IPSCs), effects that were antagonized by RF9, but not neuropeptide Y, a putative GPR10 receptor antagonist. The effects of PrRP on IPSCs in OLETF rats were similar to those in wild-type rats. Both in vivo and in vitro data strongly suggest that certain PrRP effects in the brain are expressed via NPFF receptors, probably NPFF2, rather than the GPR10 receptor. These observations may assume clinical relevance as RF amide peptides such NPFF and PrRP become therapeutic targets for a variety of autonomically related disorders.

Attenuated behavioural responses to acute and chronic cocaine in GASP-1-deficient mice.

G protein-coupled receptor (GPCR) associated sorting protein 1 (GASP-1) interacts with GPCRs and is implicated in their postendocytic sorting. Recently, GASP-1 has been shown to regulate dopamine (D(2)) and cannabinoid (CB1) receptor signalling, suggesting that preventing GASP-1 interaction with GPCRs might provide a means to limit the decrease in receptor signalling upon sustained agonist treatment. In order to test this hypothesis, we have generated and behaviourally characterized GASP-1 knockout (KO) mice and have examined the consequences of the absence of GASP-1 on chronic cocaine treatments. GASP-1 KO and wild-type (WT) mice were tested for sensitization to the locomotor effects of cocaine. Additional mice were trained to acquire intravenous self-administration of cocaine on a fixed ratio 1 schedule of reinforcement, and the motivational value of cocaine was then assessed using a progressive ratio schedule of reinforcement. The dopamine and muscarinic receptor densities were quantitatively evaluated in the striatum of WT and KO mice tested for sensitization and self-administration. Acute and sensitized cocaine-locomotor effects were attenuated in KO mice. A decrease in the percentage of animals that acquired cocaine self-administration was also observed in GASP-1-deficient mice, which was associated with pronounced down-regulation of dopamine and muscarinic receptors in the striatum. These data indicate that GASP-1 participates in acute and chronic behavioural responses induced by cocaine and are in agreement with a role of GASP-1 in postendocytic sorting of GPCRs. However, in contrast to previous studies, our data suggest that upon sustained receptor stimulation GASP-1 stimulates recycling rather than receptor degradation.

Aminoguanidine Hydrazone Derivatives as Nonpeptide NPFF1 Receptor Antagonists Reverse Opioid Induced Hyperalgesia.

Neuropeptide FF receptors (NPFF1R and NPFF2R) and their endogenous ligand neuropeptide FF have been shown previously to display antiopioid properties and to play a critical role in the adverse effects associated with chronic administrations of opiates including the development of opioid-induced hyperalgesia and analgesic tolerance. In this work, we sought to identify novel NPFF receptors ligands by focusing our interest in a series of heterocycles as rigidified nonpeptide NPFF receptor ligands, starting from already described aminoguanidine hydrazones (AGHs). Binding experiments and functional assays highlighted AGH 1n and its rigidified analogue 2-amino-dihydropyrimidine 22e for in vivo experiments. As shown earlier with the prototypical dipeptide antagonist RF9, both 1n and 22e reduced significantly the long lasting fentanyl-induced hyperalgesia in rodents. Altogether these data indicate that AGH rigidification maintains nanomolar affinities for both NPFF receptors, while improving antagonist character toward NPFF1R.

A bifunctional-biased mu-opioid agonist-neuropeptide FF receptor antagonist as analgesic with improved acute and chronic side effects.

Opioid analgesics, such as morphine, oxycodone, and fentanyl, are the cornerstones for treating moderate to severe pain. However, on chronic administration, their efficiency is limited by prominent side effects such as analgesic tolerance and dependence liability. Neuropeptide FF (NPFF) and its receptors (NPFF1R and NPFF2R) are recognized as an important pronociceptive system involved in opioid-induced hyperalgesia and analgesic tolerance. In this article, we report the design of multitarget peptidomimetic compounds that show high-affinity binding to the mu-opioid receptor (MOPr) and NPFFRs. In vitro characterization of these compounds led to identification of KGFF03 and KGFF09 as G-protein-biased MOPr agonists with full agonist or antagonist activity at NPFFRs, respectively. In agreement with their biased MOPr agonism, KGFF03/09 showed reduced respiratory depression in mice, as compared to the unbiased parent opioid agonist KGOP01. Chronic subcutaneous administration of KGOP01 and KGFF03 in mice rapidly induced hyperalgesia and analgesic tolerance, effects that were not observed on chronic treatment with KGFF09. This favorable profile was further confirmed in a model of persistent inflammatory pain. In addition, we showed that KGFF09 induced less physical dependence compared with KGOP01 and KGFF03. Altogether, our data establish that combining, within a single molecule, the G-protein-biased MOPr agonism and NPFFR antagonism have beneficial effects on both acute and chronic side effects of conventional opioid analgesics. This strategy can lead to the development of novel and potent antinociceptive drugs with limited side effects on acute and chronic administration.

Morphine-induced hyperalgesia involves mu opioid receptors and the metabolite morphine-3-glucuronide.

Opiates are potent analgesics but their clinical use is limited by side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). The Opiates produce analgesia and other adverse effects through activation of the mu opioid receptor (MOR) encoded by the Oprm1 gene. However, MOR and morphine metabolism involvement in OIH have been little explored. Hence, we examined MOR contribution to OIH by comparing morphine-induced hyperalgesia in wild type (WT) and MOR knockout (KO) mice. We found that repeated morphine administration led to analgesic tolerance and hyperalgesia in WT mice but not in MOR KO mice. The absence of OIH in MOR KO mice was found in both sexes, in two KO global mutant lines, and for mechanical, heat and cold pain modalities. In addition, the morphine metabolite morphine-3beta-D-glucuronide (M3G) elicited hyperalgesia in WT but not in MOR KO animals, as well as in both MOR flox and MOR-Nav1.8 sensory neuron conditional KO mice. M3G displayed significant binding to MOR and G-protein activation when using membranes from MOR-transfected cells or WT mice but not from MOR KO mice. Collectively our results show that MOR is involved in hyperalgesia induced by chronic morphine and its metabolite M3G.

RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes effects of RF-amide-related peptide-3 and opioid-induced hyperalgesia in rodents.

Although opiates represent the most effective analgesics, their use in chronic treatments is associated with numerous side effects including the development of pain hypersensitivity and analgesic tolerance. We recently identified a novel orally active neuropeptide FF (NPFF) receptor antagonist, RF313, which efficiently prevents the development of fentanyl-induced hyperalgesia in rats. In this study, we investigated the properties of this compound into more details. We show that RF313 exhibited a pronounced selectivity for NPFF receptors, antagonist activity at NPFF1 receptor (NPFF1R) subtype both in vitro and in vivo and no major side effects when administered in mice up to 30 mg/kg. When co-administered with opiates in rats and mice, it improved their analgesic efficacy and prevented the development of long lasting opioid-induced hyperalgesia. Moreover, and in marked contrast with the dipeptidic NPFF receptor antagonist RF9, RF313 displayed negligible affinity and no agonist activity (up to 100 µM) toward the kisspeptin receptor. Finally, in male hamster, RF313 had no effect when administered alone but fully blocked the increase in LH induced by RFRP-3, while RF9 per se induced a significant increase in LH levels which is consistent with its ability to activate kisspeptin receptors. Altogether, our data indicate that RF313 represents an interesting compound for the development of therapeutic tools aiming at improving analgesic action of opiates and reducing adverse side effects associated with their chronic administration. Moreover, its lack of agonist activity at the kisspeptin receptor indicates that RF313 might be considered a better pharmacological tool, when compared to RF9, to examine the regulatory roles of RF-amide-related peptides and NPFF1R in reproduction.

Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages.

The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet-fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage-associated genes IL-4 receptor α (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs.

RF-amide neuropeptides and their receptors in Mammals: Pharmacological properties, drug development and main physiological functions.

RF-amide neuropeptides, with their typical Arg-Phe-NH2 signature at their carboxyl C-termini, belong to a lineage of peptides that spans almost the entire life tree. Throughout evolution, RF-amide peptides and their receptors preserved fundamental roles in reproduction and feeding, both in Vertebrates and Invertebrates. The scope of this review is to summarize the current knowledge on the RF-amide systems in Mammals from historical aspects to therapeutic opportunities. Taking advantage of the most recent findings in the field, special focus will be given on molecular and pharmacological properties of RF-amide peptides and their receptors as well as on their implication in the control of different physiological functions including feeding, reproduction and pain. Recent progress on the development of drugs that target RF-amide receptors will also be addressed.

Opioid-induced hyperalgesia: Cellular and molecular mechanisms.

Opioids produce strong analgesia but their use is limited by a paradoxical hypersensitivity named opioid-induced hyperalgesia (OIH) that may be associated to analgesic tolerance. In the last decades, a significant number of preclinical studies have investigated the factors that modulate OIH development as well as the cellular and molecular mechanisms underlying OIH. Several factors have been shown to influence OIH including the genetic background and sex differences of experimental animals as well as the opioid regimen. Mu opioid receptor (MOR) variants and interactions of MOR with different proteins were shown important. Furthermore, at the cellular level, both neurons and glia play a major role in OIH development. Several neuronal processes contribute to OIH, like activation of neuroexcitatory mechanisms, long-term potentiation (LTP) and descending pain facilitation. Increased nociception is also mediated by neuroinflammation induced by the activation of microglia and astrocytes. Neurons and glial cells exert synergistic effects, which contribute to OIH. The molecular actors identified include the Toll-like receptor 4 and the anti-opioid systems as well as some other excitatory molecules, receptors, channels, chemokines, pro-inflammatory cytokines or lipids. This review summarizes the intracellular and intercellular pathways involved in OIH and highlights some mechanisms that may be challenged to limit OIH in the future.

Development of Dipeptidic hGPR54 Agonists.

A series of dipeptides were designed as potential agonists of the human KiSS1-derived peptide receptor (hGPR54). While the sequence Arg-Trp-NH2 was the most efficient in terms of affinity, we established a convergent synthetic strategy to optimize the N terminus. Using two successive Sonogashira cross-coupling reactions on a solid-supported peptide, we were able to introduce various alkynes at the N terminus to afford compounds with sub-micromolar affinities for hGPR54. However, functional assays indicated the benzoylated dipeptide Bz-Arg-Trp-NH2 as the most promising compound in terms of agonistic properties. Interestingly, this compound appeared much more stable than the endogenous neuropeptide kisspeptin, both in serum and in liver microsomes of rats. This compound was also found to be able to induce a significant in vivo increase in testosterone levels in male rats.

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

Herein, the opioid pharmacophore H-Dmt-d-Arg-Aba-ß-Ala-NH2 (7) was linked to peptide ligands for the nociceptin receptor. Combination of 7 and NOP ligands (e.g., H-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) led to binding affinities in the low nanomolar domain. In vitro, the hybrids behaved as agonists at the opioid receptors and antagonists at the nociceptin receptor. Intravenous administration of hybrid 13a (H-Dmt-d-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) to mice resulted in potent and long lasting antinociception in the tail-flick test, indicating that 13a was able to permeate the BBB. This was further supported by a cell-based BBB model. All hybrids alleviated allodynia and hyperalgesia in neuropathic pain models. Especially with respect to hyperalgesia, they showed to be more effective than the parent compounds. Hybrid 13a did not result in significant respiratory depression, in contrast to an equipotent analgesic dose of morphine. These hybrids hence represent a promising avenue toward analgesics for the dual treatment of acute and neuropathic pain.