In vitro pharmacological characterisation of a novel cyclic nociceptin/orphanin FQ analogue c[Cys(7,10)]N/OFQ(1-13)NH (2).
Nociceptin/orphanin FQ (N/OFQ) is the endogenous 17 amino acid peptide ligand for the G(i)-protein-coupled N/OFQ receptor (NOP). In an attempt to improve the metabolic stability of N/OFQ, we have produced a truncated cyclic analogue with cysteine residues at positions 7 and 10, c[Cys(7,10)]N/OFQ(1-13)NH(2) (c[Cys(7,10)]). c[Cys(7,10)], the template N/OFQ(1-13)NH(2) and N/OFQ displaced the binding of [(3)H]N/OFQ to Chinese hamster ovary cells expressing recombinant human NOP (CHO(hNOP)) with pK ( i ) values of 9.98, 9.83 and 9.18, respectively. In addition, c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ stimulated the binding of guanosine triphosphate gamma [(35)S] to CHO(hNOP) cells with pEC(50)/E (max) (stimulation factor) of 9.16/5.5, 9.11/4.9 and 8.35/5.5, respectively. c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation with pEC(50) values of 10.08, 10.11 and 9.78, respectively. All ligands produced complete inhibition of cAMP formation. In both functional assays, c[Cys(7,10)] was a full agonist. In a series of metabolism experiments, incubation of 1 nM c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ with a rat brain homogenate produced a time-dependent loss of peptide that was greatest for the native peptide N/OFQ. Amidation in N/OFQ(1-13)NH(2) produced some metabolic protection, but this was not significantly improved by further inclusion of c[Cys(7,10)]. In summary, c[Cys(7,10)] is a high-affinity, high-potency full agonist of the NOP receptor. However, we were unable to demonstrate clear metabolic protection.
Kitayama
M
M
Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, University of Leicester, Leicester, UK.
McDonald
J
J
Barnes
T A
TA
Calo'
G
G
Guerrini
R
R
Rowbotham
D J
DJ
Lambert
D G
DG
D000704:Analysis of Variance; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002540:Cerebral Cortex / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D066298:In Vitro Techniques; D018847:Opioid Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D000097629:Nociceptin
D000242:Cyclic AMP; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D018847:Opioid Peptides; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D000097629:Nociceptin; C524720:cyclo(Cys(7,14))nociceptin (1-13) amide