In vitro functional characterization of novel nociceptin/orphanin FQ receptor agonists in recombinant and native preparations

Abstract

Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [35S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65-6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127≥AT-090≥AT-035 > AT-004= AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [35S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists.

NOP receptor pharmacological profile – A dynamic mass redistribution study

“…In the DMR assay all these compounds produced maximal effects that were not statistically different to those of N/OFQ. Similar results were obtained in calcium mobilization studies performed in cells co-expressing the NOP receptor and chimeric G proteins [ 54 , 55 ]. On the contrary these same compounds consistently displayed significantly lower efficacy than N/OFQ in GTPγS binding and NOP/G protein interaction experiments [ 3 , 5 , 54 ].…”

“…Similar results were obtained in calcium mobilization studies performed in cells co-expressing the NOP receptor and chimeric G proteins [ 54 , 55 ]. On the contrary these same compounds consistently displayed significantly lower efficacy than N/OFQ in GTPγS binding and NOP/G protein interaction experiments [ 3 , 5 , 54 ]. As discussed in [ 56 ], this apparent discrepancy is probably due to the fact that the estimated efficacy of partial agonists strongly depends on the efficiency of the stimulus–response coupling which is different in the different assays.…”

“…In the second set of experiments a series of compounds with known partial agonist activity at the NOP receptor, the peptides [F/G]N/OFQ(1–13)-NH 2 [ 52 ] and Ac-RYYRIK-NH 2 [ 53 ] and the non peptides AT-090 and AT-127 [ 54 ], were evaluated. In the DMR assay all these compounds produced maximal effects that were not statistically different to those of N/OFQ.…”

Anti‐Parkinsonian and anti‐dyskinetic profiles of two novel potent and selective nociceptin/orphanin FQ receptor agonists

“…The width of the therapeutic window may thus be compound‐dependent and relate to several pharmacodynamic or pharmacokinetic considerations, among which selectivity or activity against different populations of NOP receptors (or NOP receptor‐regulated motor pathways), different brain penetrance in brain areas mediating the two effects, or different off‐targets. Moreover, recent studies pointed out that Ro 65‐6570 is a G protein biased ligand, being less efficient in activating the β‐arrestin2 pathway than N/OFQ (Ferrari et al, ) or AT‐403 (Ferrari et al, ). A different efficacy towards the Gi and β‐arrestin2 pathways may thus result in a dissociation between the anti‐dyskinetic and sedative effects.…”

In vitro pharmacological characterization of a novel unbiased NOP receptor‐selective nonpeptide agonist AT‐403

“…) and a different series of AT compounds (Ferrari et al. ). The rank order of agonist potency obtained in the mouse vas deferens assay was superimposable to that obtained at the human recombinant receptor further corroborating our proposal that the human and murine NOP receptor display very similar if not identical pharmacological profiles (Calo and Guerrini ; Toll et al.…”