Journal: Frontiers in Pain Research

Article Title: In vivo and in vitro Characterization of a Partial Mu Opioid Receptor Agonist, NKTR-181, Supports Future Therapeutic Development

doi: 10.3389/fpain.2021.695962

Figure Lengend Snippet: The profile of NKTR-181 inhibition of voltage-activated calcium channel currents (VACCs) in dorsal root ganglia (DRG) neurons. (A) Inhibition of VACCs was measured in dissociated and cultured adult DRG neurons. The current was obtained by a step voltage protocol from the holding voltage of −70 mV to +10 mV to open the Ca 2+ channels, as shown in the top panel. This step protocol is repeated every 15 s to obtain a baseline Ca 2+ current amplitude, after which mu opioid receptor (MOPr) agonist such as oxycodone or NKTR-181 is applied (shown in red) to inhibit the VACCs, and the agonist is then washed off. (B) In comparison with known MOPr agonists, NKTR-181 is both less efficacious and potent in this assay . (C) Rate of inhibition. [C(i)] These exemplar recordings of the change in amplitude across time (each dot represents a 15 s sweep interval) induced by oxycodone (10 μM) or NKTR-181 (30 μM) were analyzed to obtain the rate of inhibition and the percentage inhibition obtained during the first sweep interval. NKTR-181 inhibited VACCs more slowly than oxycodone [C(ii)]; ** p < 0.01 vs. oxycodone and obtained less percentage inhibition over time [C(iii)]; * p < 0.05 vs. oxycodone. Data are shown as mean ± SEM. Refer to and for statistics.

Article Snippet: HEK293 cells, untransfected or transfected with fluorogen-activated peptide- (FAP-) tagged MOPr (Spectragenetics, Pittsburgh, PA, USA) were cultured in Dulbecco's Modified Eagle Medium (Gibco-Life Technologies, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Gemini Bioproducts, Sacramento, CA, USA), 1% 100X Gibco® GlutaMAXTM supplement (Thermo Fischer, Life Technologies, Waltham, MA, USA), 0.5% 100X antibiotic-antimycotic (Thermo Fischer, Life Technologies, Waltham, MA, USA).

Techniques: Inhibition, Cell Culture, Comparison

Journal: Frontiers in Pain Research

Article Title: In vivo and in vitro Characterization of a Partial Mu Opioid Receptor Agonist, NKTR-181, Supports Future Therapeutic Development

doi: 10.3389/fpain.2021.695962

Figure Lengend Snippet: NKTR-181-induced G-protein and β-arrestin recruitment to MOPr. HEK293 cells, transfected with MOPr-venus and either GNAi1-luciferase, luciferase-βarr2, or luciferase-βarr1, were treated with [D-Ala 2 , N-MePhe 4 , Gly-ol]-enkephalin (DAMGO) (0–10 βM), NKTR-181 (0–100 βM), or oxycodone (0–100 βM). (A–C) Dose-response curves showing agonist-activated NetBRET values [E 535 /E 490 minus the background signal observed with luciferase-tagged constructs alone and normalized to baseline bioluminescence resonance energy transfer (BRET) in untreated cells] as a percentage of the maximum DAMGO-induced response on the y -axis, and log agonist concentration on the x -axis for βarr2 (A) , βarr1 (B) , and Gαi (C) . Data were fitted with a four-parameter, variable slope model with Hill Slopes constrained to one. (D–F) Time course of βarr2 (D) and βarr1 (E) recruitment at DAMGO (10 μM), NKTR-181 (100 μM), and oxy (100 μM), fitted with a one-phase decay model, and recruitment rate comparison (F) . Bias factors were calculated for Gαi vs. β-arrestin (G) or βarr1 vs. βarr2 (H) . Refer to , , for statistics. Refer to for the same analysis with morphine and fentanyl.

Article Snippet: HEK293 cells, untransfected or transfected with fluorogen-activated peptide- (FAP-) tagged MOPr (Spectragenetics, Pittsburgh, PA, USA) were cultured in Dulbecco's Modified Eagle Medium (Gibco-Life Technologies, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Gemini Bioproducts, Sacramento, CA, USA), 1% 100X Gibco® GlutaMAXTM supplement (Thermo Fischer, Life Technologies, Waltham, MA, USA), 0.5% 100X antibiotic-antimycotic (Thermo Fischer, Life Technologies, Waltham, MA, USA).

Techniques: Transfection, Luciferase, Construct, Bioluminescence Resonance Energy Transfer, Concentration Assay, Comparison

Journal: Frontiers in Pain Research

Article Title: In vivo and in vitro Characterization of a Partial Mu Opioid Receptor Agonist, NKTR-181, Supports Future Therapeutic Development

doi: 10.3389/fpain.2021.695962

Figure Lengend Snippet: NKTR-181 induces limited internalization. Internalization was assessed in HEK cells expressing FAP-labeled MOPrs and quantified by flow cytometry. (A) After 10 min of agonist incubation at 37°C, DAMGO and fentanyl, but no other agonist, induced dose-dependent internalization. (B) After 30 min of incubation, DAMGO, fentanyl, and morphine induced dose-dependent internalization. (C) After 60 min of incubation, DAMGO, fentanyl, and NKTR-181 had induced dose-dependent internalization. Oxycodone did not induce dose-dependent MOPr internalization and could not be curve-fitted. The control samples indicated as a single broken black line, received vehicle at each of these timepoints. Data are shown as mean ± SEM. Refer to and for statistics.

Article Snippet: HEK293 cells, untransfected or transfected with fluorogen-activated peptide- (FAP-) tagged MOPr (Spectragenetics, Pittsburgh, PA, USA) were cultured in Dulbecco's Modified Eagle Medium (Gibco-Life Technologies, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Gemini Bioproducts, Sacramento, CA, USA), 1% 100X Gibco® GlutaMAXTM supplement (Thermo Fischer, Life Technologies, Waltham, MA, USA), 0.5% 100X antibiotic-antimycotic (Thermo Fischer, Life Technologies, Waltham, MA, USA).

Techniques: Expressing, Labeling, Flow Cytometry, Incubation, Control