NOP receptor pharmacological profile – A dynamic mass redistribution study

Abstract

The Nociceptin/Orphanin FQ (N/OFQ) peptide NOP receptor is coupled to pertussis toxin (PTX)-sensitive G proteins (Gi/o) whose activation leads to the inhibition of both cAMP production and calcium channel activity, and to the stimulation of potassium currents. The label free dynamic mass redistribution (DMR) approach has been demonstrated useful for investigating the pharmacological profile of G protein-coupled receptors. Herein, we employ DMR technology to systematically characterize the pharmacology of a large panel of NOP receptor ligands. These are of peptide and non-peptide nature and display varying degrees of receptor efficacy, ranging from full agonism to pure antagonism. Using Chinese hamster ovary (CHO) cells expressing the human NOP receptor we provide rank orders of potency for full and partial agonists as well as apparent affinities for selective antagonists. We find the pharmacological profile of NOP receptor ligands to be similar but not identical to values reported in the literature using canonical assays for Gi/o-coupled receptors. Our data demonstrate that holistic label-free DMR detection can be successfully used to investigate the pharmacology of the NOP receptor and to characterize the cellular effects of novel NOP receptor ligands.

Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies

“…It should, however, be mentioned that in absolute terms the potency of NPSR antagonists estimated in the DMR assay was on average 3-fold lower than that estimated in calcium mobilization studies. A similar trend has been obtained in our laboratory with a series of antagonists selective for the nociceptin/orphanin receptor and with naloxone on classical opioid receptors evaluated in the DMR 20 and in calcium mobilization studies performed in cells expressing chimeric G proteins. [42][43][44] Further studies are clearly needed to corroborate these initial findings and eventually to investigate the reasons underlying this tendency of the DMR assay to underestimate antagonist potency.…”

“…NPS fragments (NPS (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), NPS (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), NPS(1-6), NPS(1-10) 23…”

“…These results are summarized in Table 1. Specifically, NPS (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and [Bip 2 ]NPS at 10 μmol L −1 , but not at 1 μmol L −1 , elicited a significant DMR response in wild-type cells, thus for these compounds, 1 μmol L −1 was selected as highest concentration for further studies. PWT1-NPS caused a significant DMR effect at 1 and 0.1 μmol L −1 in wild-type cells, thus further experiments in HEK293 mNPSR were performed using 0.01 μmol L −1 as highest concentration for this ligand.…”

“…DMR has been already applied to study the pharmacological properties of new ligands acting at various GPCRs, such as histamine H 1 , β 2 adrenergic, muscarinic M 3 , purinergic P2Y, formyl peptide, and protease‐activated receptors. Classical opioid and the nociceptin/orphanin FQ peptide (NOP) receptors were also investigated in DMR studies. Additionally, the DMR assay, together with different biochemical tools, has been successfully used for GPCRs signaling deconvolution studies …”

“…These findings suggest that the DMR test can provide more exhaustive and T A B L E 2 Agonists potencies (pEC 50 ), intrinsic activity (α), and antagonist potencies (pA 2 ) of compounds tested in DMR assay in HEK293 mNPSR cells Data are the mean ± SEM of three experiments performed in duplicate complete information regarding ligand selectivity compared to classical assays. It is worth mentioning that Rizzi et al reported a reduced selectivity of PWT derivatives of nociceptin/orphanin FQ compared to the natural peptide in bioassay studies performed with tissues from receptor knockout mice,41 moreover PWT derivatives of nociceptin/orphanin FQ produced off-target effects in DMR studies20 ; thus a certain loss of selectivity may be a common feature of tetrabranched peptide derivatives synthesized using the PWT technology and DMR can be useful approach to reveal this aspect.…”