2. Academic Validation
  3. Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo)

Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo)

  • PLoS One. 2016 Jun 7;11(6):e0156897. doi: 10.1371/journal.pone.0156897.
Mark F Bird 1,  Maria Camilla Cerlesi 2,  Mark Brown 1,  Davide Malfacini 2,  Vanessa Vezzi 3,  Paola Molinari 3,  Laura Micheli 4,  Lorenzo Di Cesare Mannelli 4,  Carla Ghelardini 4,  Remo Guerrini 5,  Girolamo Calò 2,  David G Lambert 1
Affiliations

Affiliations

  • 1 Department of Cardiovascular Sciences, University of Leicester, Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE2 7LX, United Kingdom.
  • 2 Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy.
  • 3 Department of Pharmacology, Istituto Superiore di Sanità, Rome, 00161, Italy.
  • 4 Department of Neuroscience, Psychology, Drug Research and Child Health-Neurofarba, Pharmacology and Toxicology Section, University of Florence, Florence, Italy.
  • 5 Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy.
PMID: 27272042 DOI: 10.1371/journal.pone.0156897
Abstract

Introduction: Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ.

Methods: We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and Arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats.

Results: DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and Arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats.

Conclusion: Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties.

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