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.

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.

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.

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.

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.

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.

A strategy to discover decoy chemokine ligands with an anti-inflammatory activity.

Excessive signaling by chemokines has been associated with chronic inflammation or cancer, thus attracting substantial attention as promising therapeutic targets. Inspired by chemokine-clearing molecules shaped by pathogens to escape the immune system, we designed a generic screening assay to discover chemokine neutralizing molecules (neutraligands) and unambiguously distinguish them from molecules that block the receptor (receptor antagonists). This assay, called TRIC-r, combines time-resolved intracellular calcium recordings with pre-incubation of bioactive compounds either with the chemokine or the receptor-expressing cells. We describe here the identification of high affinity neutraligands of CCL17 and CCL22, two chemokines involved in the Th2-type of lung inflammation. The decoy molecules inhibit in vitro CCL17- or CCL22-induced intracellular calcium responses, CCR4 endocytosis and human T cell migration. In vivo, they inhibit inflammation in a murine model of asthma, in particular the recruitment of eosinophils, dendritic cells and CD4(+)T cells. Altogether, we developed a successful strategy to discover as new class of pharmacological tools to potently control cell chemotaxis in vitro and in vivo.

Neuropeptide FF receptors as novel targets for limbic seizure attenuation.

Neuropeptide Y (NPY) is a well established anticonvulsant and first-in-class antiepileptic neuropeptide. In this study, the controversial role of NPY1 receptors in epilepsy was reassessed by testing two highly selective NPY1 receptor ligands and a mixed NPY1/NPFF receptor antagonist BIBP3226 in a rat model for limbic seizures. While BIBP3226 significantly attenuated the pilocarpine-induced seizures, neither of the highly selective NPY1 receptor ligands altered the seizure severity. Administration of the NPFF1/NPFF2 receptor antagonist RF9 also significantly attenuated limbic seizure activity. To further prove the involvement of NPFF receptors in these seizure-modulating effects, low and high affinity antagonists for the NPFF receptors were tested. We observed that the low affinity ligand failed to exhibit anticonvulsant properties while the two high affinity ligands significantly attenuated the seizures. Continuous NPFF1 receptor agonist administration also inhibited limbic seizures whereas bolus administration of the NPFF1 receptor agonist was without effect. This suggests that continuous agonist perfusion could result in NPFF1 receptor desensitization and mimic NPFF1 receptor antagonist administration. Our data unveil for the first time the involvement of the NPFF system in the management of limbic seizures.

Exploration of the orthosteric/allosteric interface in human M1 muscarinic receptors by bitopic fluorescent ligands.

Bitopic binding properties apply to a variety of muscarinic compounds that span and simultaneously bind to both the orthosteric and allosteric receptor sites. We provide evidence that fluorescent pirenzepine derivatives, with the M1 antagonist fused to the boron-dipyrromethene [Bodipy (558/568)] fluorophore via spacers of varying lengths, exhibit orthosteric/allosteric binding properties at muscarinic M1 receptors. This behavior was inferred from a combination of functional, radioligand, and fluorescence resonance energy transfer binding experiments performed under equilibrium and kinetic conditions on enhanced green fluorescent protein-fused M1 receptors. Although displaying a common orthosteric component, the fluorescent compounds inherit bitopic properties from a linker-guided positioning of their Bodipy moiety within the M1 allosteric vestibule. Depending on linker length, the fluorophore is allowed to reach neighboring allosteric domains, overlapping or not with the classic gallamine site, but distinct from the allosteric indolocarbazole "WIN" site. Site-directed mutagenesis, as well as molecular modeling and ligand docking studies based on recently solved muscarinic receptor structures, further support the definition of two groups of Bodipy-pirenzepine derivatives exhibiting distinct allosteric binding poses. Thus, the linker may dictate pharmacological outcomes for bitopic molecules that are hardly predictable from the properties of individual orthosteric and allosteric building blocks. Our findings also demonstrate that the fusion of a fluorophore to an orthosteric ligand is not neutral, as it may confer, unless carefully controlled, unexpected properties to the resultant fluorescent tracer. Altogether, this study illustrates the importance of a "multifacet" experimental approach to unravel and validate bitopic ligand binding mechanisms.

An antedrug of the CXCL12 neutraligand blocks experimental allergic asthma without systemic effect in mice.

The chemokine receptor CXCR4 and its chemokine CXCL12 are involved in normal tissue patterning but also in tumor cell growth and survival as well as in the recruitment of immune and inflammatory cells, as successfully demonstrated using agents that block either CXCL12 or CXCR4. In order to achieve selectivity in drug action on the CXCR4/CXCL12 pair, in particular in the airways, drugs should be delivered as selectively as possible in the treated tissue and should not diffuse in the systemic circulation, where it may reach undesired organs. To this end, we used a previously unexploited Knoevenagel reaction to create a short lived drug, or soft drug, based on the CXCL12-neutralizing small molecule, chalcone 4, which blocks binding of CXCL12 to CXCR4. We show that the compound, carbonitrile-chalcone 4, blocks the recruitment of eosinophils to the airways in ovalbumin-sensitized and challenged mice in vivo when administered directly to the airways by the intranasal route, but not when administered systemically by the intraperitoneal route. We show that the lack of effect at a distant site is due to the rapid degradation of the molecule to inactive fragments. This approach allows selective action of the CXCL12 neutraligands although the target protein is widely distributed in the organism.

Allosteric functional switch of neurokinin A-mediated signaling at the neurokinin NK2 receptor: structural exploration.

The neurokinin NK2 receptor is known to pre-exist in equilibrium between at least three states: resting-inactive, calcium-triggering, and cAMP-producing. Its endogeneous ligand, NKA, mainly induces the calcium response. Using a FRET-based assay, we have previously discovered an allosteric modulator of the NK2 receptor that has the unique ability to discriminate among the two signaling pathways: calcium-signaling is not affected while cAMP signaling is significantly decreased. A series of compounds have been prepared and studied in order to better understand the structural determinants of this allosteric functional switch of a GPCR. Most of them display the same allosteric profile, with smooth pharmacomodulation. One compound however exhibits significantly improved modulatory properties of NKA induced signaling when compared to the original modulator.