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. 2017 Aug;5(4):e00333.
doi: 10.1002/prp2.333.

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

Federica Ferrari  1 , Davide Malfacini  1 , Blair V Journigan  2 , Mark F Bird  3 , Claudio Trapella  4 , Remo Guerrini  4 , David G Lambert  3 , Girolamo Calo'  1 , Nurulain T Zaveri  2
Affiliations

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

Federica Ferrari et al. Pharmacol Res Perspect. 2017 Aug.

Abstract

Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP), a member of the opioid receptor family. We recently identified a new high affinity and highly selective NOP agonist AT-403. In this study, we characterized the functional profile of AT-403 and compared it to other known nonpeptide NOP agonists Ro 65-6570, Ro 2q, SCH-221510, MCOPPB, AT-202 and SCH-486757, using the following assays: GTPγ[35 S] stimulated binding, calcium mobilization assay in cells-expressing human NOP or classical opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, and the electrically stimulated mouse vas deferens bioassay. All compounds behaved as NOP full agonists consistently showing the following rank order of potency MCOPPB > AT-403 > Ro 65-6570 = Ro 2q > SCH-221510 > AT-202 > SCH-486757. AT-403 and MCOPPB displayed the highest NOP selectivity both at human and murine receptors. Interestingly, while all the other nonpeptide NOP agonists displayed bias toward G protein-mediated signaling in the BRET assay, AT-403, similar to the natural ligand N/OFQ, behaved as an unbiased agonist, activating G-protein-mediated function as well as arrestin recruitment. AT-403 may be a useful nonpeptide tool compound to study the pharmacology of NOP activation in disease states.

Keywords: BRET arrestin assay; GPCR signaling bias; NOP receptor; functional selectivity; ligand bias; nonpeptide NOP agonists.

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Figures

Figure 1
Figure 1
Chemical structures of the AT‐403 and other non‐peptide NOP agonists characterized in the present study.
Figure 2
Figure 2
GTPγ[35S]‐binding experiments. Concentration response curves to N/OFQ, Ro 65‐6570, Ro 2q, SCH‐221510 and MCOPPB (panel A), and to N/OFQ, AT‐403, AT‐202 and SCH‐486757 (panel B) in membranes of CHONOP cells. Data are the mean ± SEM of 6 separate experiments.
Figure 3
Figure 3
Calcium mobilization assay performed in Chinese Hamster Ovary cells expressing the NOP receptor and the chimeric Gα qi5 protein. Concentration response curves to N/OFQ, Ro 65‐6570, Ro 2q, SCH‐221510 and MCOPPB (panel A) and to N/OFQ, AT‐403, AT‐202 and SCH‐486757 (panel B). Data are expressed as mean ± SEM. of at least 4 separate experiments made in duplicate.
Figure 4
Figure 4
Calcium mobilization assay performed in Chinese Hamster Ovary cells expressing the NOP receptor and the chimeric Gα qi5 protein. Concentration response curves to N/OFQ (panel A), Ro 65‐6570 (panel B), Ro 2q (panel C), SCH‐221510 (panel D) and MCOPPB (panel E) in absence (control) and in presence of SB‐612111 100 nmol/L. Data are expressed as mean ± SEM. of at least 4 separate experiments made in duplicate.
Figure 5
Figure 5
Calcium mobilization assay performed in Chinese Hamster Ovary cells expressing the NOP receptor and the chimeric Gα qi5 protein. Concentration response curves to N/OFQ (panel A), AT‐403 (panel B), AT‐202 (panel C) and SCH‐486757 (panel D) in absence (control) and in presence of SB‐612111 100 nmol/L. Data are expressed as mean ± SEM. of at least 4 separate experiments made in duplicate.
Figure 6
Figure 6
Calcium mobilization assay performed in Chinese Hamster Ovary cells expressing opioid receptors and chimeric G proteins. Concentration response curves to opioid receptor standard agonists and to NOP receptor agonists in cells expressing the mu (panels A and B), the kappa (panels C and D), and delta (panels E and F) opioid receptors. Data are expressed as mean ± SEM of at least 4 separate experiments made in duplicate.
Figure 7
Figure 7
Bioluminescence resonance energy transfer assay.Concentration response curve to N/OFQ and NOP agonists in promoting NOP/G protein interaction (panels A and B) and NOP/β‐arrestin 2 interaction (panels C and D). Data are the mean ± SEM of 5 separate experiments made in duplicate.
Figure 8
Figure 8
Mouse vas deferens bioassay. Concentration response curves to N/OFQ and to NOP agonists. Data are the mean ± SEM. of at least 5 experiments. *P < 0.05 versus N/OFQ (Student t test for paired samples).
Figure 9
Figure 9
Representative tracings of the concentration response curve to N/OFQ and Ro 65‐6570 in the electrically mouse vas deferens.
Figure 10
Figure 10
Mouse vas deferens bioassay. Concentration response curve to DPDPE (panel A), N/OFQ (panel B), Ro 65‐6570 (panel C), Ro 2q (panel D), SCH‐221510 (panel E), MCOPPB (panel F), AT‐202 (panel G) and AT‐403 (panel H) in tissues taken from NOP(+/+) and NOP(−/−) mice. Data are the mean ± SEM. of at least 5 experiments.
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