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


CONCLUSIONS
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.
Department of Cardiovascular Sciences, University of Leicester, Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE2 7LX, United Kingdom.
Mark F
MF
Bird
Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy.
Maria Camilla
MC
Cerlesi
Department of Cardiovascular Sciences, University of Leicester, Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE2 7LX, United Kingdom.
Mark
M
Brown
Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy.
Davide
D
Malfacini
Department of Pharmacology, Istituto Superiore di Sanità, Rome, 00161, Italy.
Vanessa
V
Vezzi
Department of Pharmacology, Istituto Superiore di Sanità, Rome, 00161, Italy.
Paola
P
Molinari
Department of Neuroscience, Psychology, Drug Research and Child Health-Neurofarba, Pharmacology and Toxicology Section, University of Florence, Florence, Italy.
Laura
L
Micheli
Department of Neuroscience, Psychology, Drug Research and Child Health-Neurofarba, Pharmacology and Toxicology Section, University of Florence, Florence, Italy.
Lorenzo
L
Di Cesare Mannelli
Department of Neuroscience, Psychology, Drug Research and Child Health-Neurofarba, Pharmacology and Toxicology Section, University of Florence, Florence, Italy.
Carla
C
Ghelardini
Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy.
Remo
R
Guerrini
Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy.
Girolamo
G
Calò
Department of Cardiovascular Sciences, University of Leicester, Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE2 7LX, United Kingdom.
David G
DG
Lambert
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D003412:Cricetulus; D006168:Guinea Pigs; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D018847:Opioid Peptides / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011957:Receptors, Opioid / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism; D000094942:Nociceptin Receptor
D018847:Opioid Peptides; D010455:Peptides; D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu; C030058:dermorphin; D002118:Calcium; D000094942:Nociceptin Receptor