Pain and RFamides

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

Pain, 2018

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.

Heteroarylguanidines as Allosteric Modulators of ASIC1a and ASIC3 Channels

ACS Chemical Neuroscience, 2018

Acid-sensing ion channels (ASICs) are neuronal Na+-selective ion channels that open in response to extracellular acidification. They are involved in pain, fear, learning, and neurodegeneration after ischemic stroke. 2-Guanidine-4-methylquinazoline (GMQ) was recently discovered as the first nonproton activator of ASIC3. GMQ is of interest as a gating modifier and pore blocker of ASICs. It has however a low potency, and exerts opposite effects on ASIC1a and ASIC3. To further explore the molecular mechanisms of GMQ action, we have used the guanidinium moiety of GMQ as a scaffold and tested the effects of different GMQ derivatives on the ASIC pH dependence and maximal current. We report that GMQ derivatives containing quinazoline and quinoline induced, as GMQ, an alkaline shift of the pH dependence of activation in ASIC3 and an acidic shift in ASIC1a. Another group of 2-guanidinopyridines shifted the pH dependence of both ASIC1a and ASIC3 to more acidic values. Several compounds induced an alkaline shift of the pH dependence of ASIC1a/2a and ASIC2a/3 heteromers. Compared to GMQ, guanidinopyridines showed a 20-fold decrease in the IC50 for ASIC1a and ASIC3 current inhibition at pH 5. Strikingly, 2-guanidino-quinolines and -pyridines showed a concentration-dependent biphasic effect that resulted at higher concentrations in ASIC1a and ASIC3 inhibition (IC50 > 100 μM), while causing at lower concentration a potentiation of ASIC1a, but not ASIC3 currents (EC50 ≈ 10 μM). In conclusion, we describe a new family of small molecules as ASIC ligands and identify an ASIC subtype-specific potentiation by a subgroup of these compounds.

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

ACS Chemical Neuroscience, 2018

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.

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Phenylpyridine-2-ylguanidines and rigid mimetics as novel inhibitors of TNFα overproduction: Beneficial action in models of neuropathic pain and of acute lung inflammation

European Journal of Medicinal Chemistry, 2018

4-phenylpyridin-2-yl-guanidine (5b): a new inhibitor of the overproduction of pro-inflammatory cytokines (TNFα and Il1β) was identified from a high-throughput screening of a chemical library on human peripheral blood mononuclear cells (PBMCs) after LPS stimulation. Derivatives, homologues and rigid mimetics of 5b were designed and synthesized, and their cytotoxicity and ability to inhibit TNFα overproduction were evaluated. Among them, compound 5b and its mimetic 12 (2-aminodihydroquinazoline) showed similar inhibitory activities, and were evaluated in vivo in models of lung inflammation and neuropathic pain in mice. In particular, compound 12 proved to be active (5 mg/kg, ip) in both models.

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Inhibition of neuronal FLT3 receptor tyrosine kinase alleviates peripheral neuropathic pain in mice

Nature Communications, 2018

Peripheral neuropathic pain (PNP) is a debilitating and intractable chronic disease, for which sensitization of somatosensory neurons present in dorsal root ganglia that project to the dorsal spinal cord is a key physiopathological process. Here, we show that hematopoietic cells present at the nerve injury site express the cytokine FL, the ligand of fms-like tyrosine kinase 3 receptor (FLT3). FLT3 activation by intra-sciatic nerve injection of FL is sufficient to produce pain hypersensitivity, activate PNP-associated gene expression and generate short-term and long-term sensitization of sensory neurons. Nerve injury-induced PNP symptoms and associated-molecular changes were strongly altered in Flt3-deficient mice or reversed after neuronal FLT3 downregulation in wild-type mice. A first-in-class FLT3 negative allosteric modulator, discovered by structure-based in silico screening, strongly reduced nerve injury-induced sensory hypersensitivity, but had no effect on nociception in non-injured animals. Collectively, our data suggest a new and specific therapeutic approach for PNP.

Evidence for the involvement of TNF-α, IL-1β and IL-10 in the antinociceptive and anti-inflammatory effects of indole-3-guanylhydrazone hydrochloride, an aromatic aminoguanidine, in rodents

Chemico-Biological Interactions, 2018, 286, 1-10

Indole-3-guanylhydrazone hydrochloride (LQM01) is a new derivative of aminoguanidine hydrochloride, an aromatic aminoguanidine.

Mice were treated with LQM01 (5, 10, 25 or 50 mg/kg, i.p.), vehicle (0.9% saline i.p.) or a standard drug. The mice were subjected to carrageenan-induced pleurisy, abdominal writhing induced by acetic acid, the formalin test and the hot-plate test. The model of non-inflammatory chronic muscle pain induced by saline acid was also used. Mice from the chronic protocol were assessed for withdrawal threshold, muscle strength and motor coordination. LQM01 or vehicle treated mice were evaluated for Fos protein.

LQM01 inhibits TNF-α and IL-1β production, as well as leukocyte recruitment during inflammation process. The level of IL-10 in LQM01-treated mice increased in pleural fluid. In addition, LQM01 decreased the nociceptive behavior in the acetic acid induced writhing test, the formalin test (both phases) and increased latency time on the hot-plate. LQM01 treatment also decreased mechanical hyperalgesia in mice with chronic muscle pain, with no changes in muscle strength and motor coordination. LQM01 reduced the number of Fos positive cells in the superficial dorsal horn. This compound exhibited antioxidant properties in in vitro assays.

LQM01 has an outstanding anti-inflammatory and analgesic profile, probably mediated through a reduction in proinflammatory cytokines release, increase in IL-10 production and reduction in neuron activity in the dorsal horn of the spinal cord in mice.


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

Neuropharmacology, 2017, 118, 188-198

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 agonistactivity (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.

χ-Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds

ACS Medicinal Chemistry Letters, 2017, 8, 11, 1177-1182

Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these constricted scaffolds into the N-terminal tetrapeptide of dermorphin (i.e., H-Tyr-d-Ala-Phe-Gly-NH2) induced significant shifts in binding affinity, selectivity, and in vitro activity at the μ- and δ-opioid receptors (MOP and DOP, respectively). A reported constrained μ-/δ-opioid lead tetrapeptide H-Dmt-d-Arg-Aba-Gly-NH2 was modified through application of various constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when the aromatic moieties were turned toward the C-terminus of the peptide sequences, (partial) (ant)agonism at MOP and weak (ant)agonism at DOP were noticed, whereas the incorporation of the 1-Ana residue led toward balanced low nanomolar MOP/DOP binding and in vitro agonism.

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Opioid adjuvant strategy: improving opioid effectiveness

Future Medicinal Chemistry, 2016, 8(3), 339-354

Opioid analgesics continue to be the mainstay of pharmacologic treatment of moderate to severe pain. Many patients, particularly those suffering from chronic pain, require chronic high-dose analgesic therapy. Achieving clinical efficacy and tolerability of such treatment regimens is hampered by the appearance of opioid-induced side effects such as tolerance, hyperalgesia and withdrawal syndrome. Among the therapeutic options to improve the opioid effectiveness, this current review focuses on strategies combining opioids to other drugs that can modulate opioid-mediated effects. We will discuss about experimental evidences reported for several potential opioid adjuvants, including N-methyl-d-aspartate receptor antagonists, 5-HT7 agonists, sigma-1 antagonists, I2-R ligands, cholecystokinin antagonists, neuropeptide FF-R antagonists and toll-like receptor 4 antagonists.

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

Pharmacology & Therapeutics, 2016, 160, 84-132

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.

Development of Dipeptidic hGPR54 Agonists

ChemMedChem, 2016, 11, 19, 2147-2154

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.

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Effects of systematic N-terminus deletions and benzoylations of endogenous RF-amide peptides on NPFF1R, NPFF2R, GPR10, GPR54 and GPR103

 Peptides, 2015, 71, 156-161

 Mammalian RF-amide peptides including RF-amide-related peptides-1 and -3, neuropeptides AF and FF, Prolactin releasing peptides, Kisspeptins and RFa peptides are currently considered endogenous peptides for the GPCRs NPFF1R, NPFF2R, GPR10, GPR54 and GPR103, respectively. While NPFF1R and NPFF2R displayed high affinity for all the RF-amide peptides, GPR10, GPR54 and GPR103 only bind their cognate ligands. Through a systematic and sequential N-terminus deletion and benzoylation of either RF-amide neuropeptide (RFRP-3, NPFF, Kp-10, PrRP20, and 26RFa), we report the corresponding impact on affinity and activity towards all the RF-amide receptors (NPFF1R, NPFF2R, GPR10, GPR54 and GPR103). Our results highlight the difficulty to develop selective peptide ligands for GPR10, GPR54 or GPR103 without a modification of the C-terminus RF-amide signature, but open the door to the design of new RF-amide peptides acting as agonist for one receptor and antagonist for another one.

Development of a Peptidomimetic Antagonist of Neuropeptide FF Receptors for the Prevention of Opioid-Induced Hyperalgesia, modulate nociception and morphine analgesia via NPFF receptors

ACS Chemical Neuroscience, 2015, 6(3), 438-445

Through the development of a new class of unnatural ornithine derivatives as bioisosteres of arginine, we have designed an orally active peptidomimetic antagonist of neuropeptide FF receptors (NPFFR). Systemic low-dose administration of this compound to rats blocked opioid-induced hyperalgesia, without any apparent side-effects. Interestingly, we also observed that this compound potentiated opioid-induced analgesia. This unnatural ornithine derivative provides a novel therapeutic approach for both improving analgesia and reducing hyperalgesia induced by opioids in patients being treated for chronic pain.

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ACS Editor's choice ACS Editors' Choice

Effects of systematic N-terminus deletions and benzoylations of endogenous RF-amide peptides on NPFF1R, NPFF2R, GPR10, GPR54 and GPR103

Peptides, 2015, 71, 156-161

Mammalian RF-amide peptides including RF-amide-related peptides-1 and -3,neuropeptides AF and FF, Prolactin releasing peptides, Kisspeptins and RFa peptides are currently considered endogenous peptides for the GPCRs NPFF1R, NPFF2R, GPR10, GPR54 and GPR103, respectively. While NPFF1R and NPFF2R displayed high affinity for all the RF-amide peptides, GPR10, GPR54 and GPR103 only bind their cognate ligands. Through a systematic and sequential N-terminus deletion and benzoylation of either RF-amide neuropeptide (RFRP-3, NPFF, Kp-10, PrRP20, and 26RFa), we report the corresponding impact on affinity and activity towards all the RF-amide receptors (NPFF1R, NPFF2R, GPR10, GPR54 and GPR103). Our results highlight the difficulty to develop selective peptide ligands for GPR10, GPR54 or GPR103 without a modification of the C-terminus RF-amide signature, but open the door to the design of new RF-amide peptides acting as agonist for one receptor andantagonist for another one.


Neuropeptide FF receptors as novel targets for limbic seizure attenuation

Neuropharmacology, 2015, 95, 415-423

Neuropeptide Y (NPY) is a well established anticonvulsant and first-in-class antiepilepticneuropeptide. 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 NPFF1receptor 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.

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

Epilepsia, 2015, 56(3), 489-498

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 (NPFF1/2R, GPR10, and GRP54, respectively) were tested in the motor cortex stimulation model.
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 NPFF1/2R, GPR10, and GPR54 receptor ligands.
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 NPFF1/2R antagonist RF9. Pretreatment with a selective NPFF1R antagonist also prevented the threshold increase induced by NPFF. Kp did not increase motor threshold.
Intracerebroventricular infusion of NPFF or PrRP decreases cortical excitability in rats through activation of NPFFRs. Furthermore, the NPFF1R is required for the NPFF-induced decrease in cortical excitability.

Endogenous mammalian RF-amide peptides, including PrRP, kisspeptin and 26RFa, modulate nociception and morphine analgesia via NPFF receptors

Neuropharmacology, 2013, 75, 164-171

Mammalian RF-amide peptides are encoded by five different genes and act through five different G protein-coupled receptors. RF-amide-related peptides-1 and -3, neuropeptides AF and FF, Prolactin releasing peptides, Kisspeptins and RFa peptides are currently considered endogenous peptides for NPFF1, NPFF2, GPR10, GPR54 and GPR103 receptors, respectively. However, several studies suggest that the selectivity of these peptides for their receptors is low and indicate that expression patterns for receptors and their corresponding ligands only partially overlap. In this study, we took advantage of the cloning of the five human RF-amide receptors to systematically examine their affinity for and their activation by all human RF-amide peptides. Binding experiments, performed on membranes from CHO cells expressing GPR10, GPR54 and GPR103 receptors, confirmed their high affinity and remarkable selectivity for their cognate ligands. Conversely, NPFF1 and NPFF2 receptors displayed high affinity for all RF-amide peptides. Moreover, GTPγS and cAMP experiments showed that almost all RF-amide peptides efficiently activate NPFF1 and NPFF2 receptors. As NPFF is known to modulate morphine analgesia, we undertook a systematic analysis in mice of the hyperalgesic and anti morphine-induced analgesic effects of a representative set of endogenous RF-amide peptides. All of them induced hyperalgesia and/or prevented morphine analgesia following intracerebroventricular administration. Importantly, these effects were prevented by administration of RF9, a highly selective NPFF1/NPFF2 antagonist. Altogether, our results show that all endogenous RF-amide peptides display pain-modulating properties and point to NPFF receptors as essential players for these effects.

Development of sub-nanomolar dipeptidic ligands of neuropeptide FF receptors

Bioorganic & Medicinal Chemistry Letters 2012, 22, 7471-7474

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-NH2 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.

Graphical abstract : Development of sub-nanomolar dipeptidic ligands of neuropeptide FF receptors

Involvement of neuropeptide FF receptors in neuroadaptive responses to acute and chronic opiate treatments

British Journal of Pharmacology 2012, 165, 424-435

Opiates remain the most effective compounds for alleviating severe pain across a wide range of conditions. However, their use is associated with significant side effects. Neuropeptide FF (NPFF) receptors have been implicated in several opiate-induced neuroadaptive changes including the development of tolerance. In this study, we investigated the consequences of NPFF receptor blockade on acute and chronic stimulation of opioid receptors in mice by using RF9, a potent and selective antagonist of NPFF receptors that can be administered systemically.
The effects of RF9 were investigated on opioid pharmacological responses including locomotor activity, antinociception, opioid-induced hyperalgesia, rewarding properties and physical dependence.
RF9 had no effect on morphine-induced horizontal hyperlocomotion and slightly attenuated the decrease induced in vertical activity. Furthermore, RF9 dose-dependently blocked the long-lasting hyperalgesia produced by either acute fentanyl or chronic morphine administration. RF9 also potentiated opiate early analgesic effects and prevented the development of morphine tolerance. Finally, RF9 increased morphine-induced conditioned place preference without producing any rewarding effect by itself and decreased naltrexone-precipitated withdrawal syndrome following chronic morphine treatment.
The NPFF system is involved in the development of two major undesirable effects: tolerance and dependence, which are clinically associated with prolonged exposure to opiates. Our findings suggest that NPFF receptors are interesting therapeutic targets to improve the analgesic efficacy of opiates by limiting the development of tolerance, and for the treatment of opioid dependence.