Pharmacology of Kappa Opioid Receptors: Novel Assays and Ligands

Abstract

The present study investigated the in vitro pharmacology of the human kappa opioid receptor using multiple assays, including calcium mobilization in cells expressing chimeric G proteins, the dynamic mass redistribution (DMR) label-free assay, and a bioluminescence resonance energy transfer (BRET) assay that allows measurement of receptor interaction with G protein and β-arrestin 2. In all assays, dynorphin A, U-69,593, and [D-Pro¹⁰]dyn(1-11)-NH₂ behaved as full agonists with the following rank order of potency [D-Pro¹⁰]dyn(1-11)-NH₂ > dynorphin A ≥ U-69,593. [Dmt¹,Tic²]dyn(1-11)-NH₂ behaved as a moderate potency pure antagonist in the kappa-β-arrestin 2 interaction assay and as low efficacy partial agonist in the other assays. Norbinaltorphimine acted as a highly potent and pure antagonist in all assays except kappa-G protein interaction, where it displayed efficacy as an inverse agonist. The pharmacological actions of novel kappa ligands, namely the dynorphin A tetrameric derivative PWT2-Dyn A and the palmitoylated derivative Dyn A-palmitic, were also investigated. PWT2-Dyn A and Dyn A-palmitic mimicked dynorphin A effects in all assays showing similar maximal effects but 3-10 fold lower potency. In conclusion, in the present study, multiple in vitro assays for the kappa receptor have been set up and pharmacologically validated. In addition, PWT2-Dyn A and Dyn A-palmitic were characterized as potent full agonists; these compounds are worthy of further investigation in vivo for those conditions in which the activation of the kappa opioid receptor elicits beneficial effects e.g. pain and pruritus.

Keywords: BRET; G protein-coupled receptor; PWT2-dyn A; biased agonism; calcium mobilization; dyn A-palmitic; kappa opioid receptor; label-free.

FIGURE 1

Calcium mobilization experiments carried out in CHO cells stably expressing the human kappa opioid receptor and the Gα_qi5 chimeric G protein (A_i) Dyn A, U-69,593, and [D-Pro¹⁰], and (A_ii) Nor-BNI and [Dmt¹,Tic²] concentration-response curves (B _i ) Concentration-response curves to Dyn A in the absence and in the presence of increasing (10 p.m.—100 nM) concentrations of Nor-BNI, corresponding (B _ii ) Schild analysis and (B _iii ) computation of antagonist potency for non-competitive antagonists (C _i ) Concentration-response curves to Dyn A in the presence of increasing concentrations (0.1—1 µM) of [Dmt¹,Tic²] with corresponding (C _i ) Schild analysis. Data are mean +sem of three to six independent experiments performed in duplicate and Emax were analyzed statistically using one-way analysis of variance followed by Dunnett’s test for multiple comparisons. p values <0.05 were considered statistically significant and labelled with *.

FIGURE 2

DMR experiments in CHO cells stably expressing the human kappa opioid receptor (A _i-iii ) Averaged kinetics of increasing concentrations of Dyn A, U-69,593, and [D-Pro¹⁰], and (A _iv ) corresponding concentration-response curves (B _i-ii ) Averaged effects of Nor-BNI and [Dmt¹,Tic²], and (B _iii ) corresponding concentration-response curves. Data represented are mean +sem of four to seven independent experiments performed in duplicate and Emax were analyzed statistically using one-way analysis of variance followed by Dunnett’s test for multiple comparisons.

FIGURE 3

DMR experiments in CHO cells stably expressing the human kappa opioid receptor (A _i-iv ) Averaged kinetic traces of increasing (1 p.m.—1 µM) concentrations of Dyn A in the absence and in the presence of different (1–100 nM) concentrations of Nor-BNI, corresponding (B _i ) concentration-response curves and (B _ii ) Schild analysis (C _i-iv ) Averaged kinetic traces of increasing Dyn A concentrations in the absence and in the presence of different (0.1–1 µM) concentrations of [Dmt¹,Tic²] with corresponding (D _i ) concentration-response curves and (D _ii ) Schild analysis. Data represented are mean +sem of five to seven independent experiments performed in duplicate and Emax were analyzed statistically using one-way analysis of variance followed by Dunnett’s test for multiple comparisons. p values <0.05 were considered statistically significant and labelled with *.

FIGURE 4

BRET experiments in SH-SY5Y cells stably expressing the human kappa opioid-RLuc tagged receptor together with either the Gβ₁-RGFP or the β-Arrestin 2-RGFP fusoprotein. Concentration-response curves to Dyn A, U-69,593, and [D-Pro¹⁰] on the stimulation of kappa-G protein (A _i ) and kappa-β-Arrestin 2 (A _ii ) interaction. Concentration-response curves to Dyn A in the absence and in the presence of increasing concentrations of Nor-BNI on the stimulation of kappa-G protein (B _i ) and kappa-β-Arrestin 2 (B _iii ) interactions with corresponding Schild plots (B _ii and B _iv , respectively). Concentration-response curves to Dyn A in the absence and in the presence of increasing concentrations of [Dmt¹,Tic²] on the stimulation of kappa-G protein (C _i ) and kappa-β-Arrestin 2 (C _iii ) interaction, with corresponding Schild analysis (C _ii and C _iv , respectively). Data represented are mean +sem of three to five independent experiments performed in duplicate.

FIGURE 5

Pharmacological characterization of PWT2-Dyn A. Molecular structure (A _i ). Calcium mobilization experiments in CHO stably expressing the human kappa (B), mu (Bii)i and delta (Bii) opioid receptor and chimeric G proteins, concentration-response curves to Dyn A and PWT2-Dyn A. Averaged DMR traces obtained in CHO cells stably expressing the human kappa opioid for increasing concentration of Dyn A (C _i ) and PWT2-Dyn A (C _ii ), with corresponding concentration-response curves (C _iii ). Concentration response curves to Dyn A and PWT2-Dyn A performed with a BRET assay on SH-SY5Y cells stably expressing the human kappa opioid-RLuc tagged receptor together with either the Gβ₁-RGFP (D _i ) or the β-Arrestin 2-RGFP fusoprotein (D_ii). Data are mean +sem of three to six independent experiments performed in duplicate.

FIGURE 6

Pharmacological characterization of Dyn A-palmitic. Molecular structure (A _i ). Calcium mobilization experiments in CHO cells stably expressing the human kappa (B _i ), mu (B _ii ), and delta (B _iii ) opioid receptors and chimeric G proteins. Concentration-response curves to Dyn A and Dyn A-palmitic. Averaged DMR traces obtained in CHO cells stably expressing the human kappa opioid for increasing concentration of Dyn A (C _i ) and Dyn A-palmitic (C _ii ), with corresponding concentration-response curves (C _iii ). Concentration response curves to Dyn A and Dyn A-palmitic performed with a BRET assay on SH-SY5Y cells stably expressing the human kappa opioid-RLuc tagged receptor together with either the Gβ₁-RGFP (D _i ) or the β-Arrestin 2-RGFP fusoprotein (D _ii ). Data are mean +sem of three to six independent experiments performed in duplicate.