Nalmefene is a biased partial agonist of the kappa opioid receptor that preferentially activates G-protein signaling over β-arrestin recruitment. In vitro experiments showed that nalmefene partially activated GTPγS binding as a measure of G-protein signaling but did not recruit β-arrestin. Nalmefene also blocked β-arrestin recruitment by the full kappa agonist U69,593. These properties suggest nalmefene is a G-protein biased partial agonist that could represent an approach for mitigating the negative side effects of kappa receptor stimulation in addiction therapies.

1. Nalmefene: G-protein biased partial agonist of the kappa opioid receptor
Joshua Hillman*, Amelia Dunn*, Eduardo Butelman, Brian Reed, and Mary Jeanne Kreek
Laboratory of the Biology of Addictive Diseases, Rockefeller University, New York, NY, USA
INTRODUCTION RESULTS
METHODS
FUTURE DIRECTIONS
CONCLUSIONS
G-protein β-arrestin
Full Agonist
G-protein β-arrestin
Biased Partial AgonistPartial Agonist
G-protein β-arrestin
Nalmefene has recently been approved for use in the European Union to treat
alcoholism.
Both antagonism of MOP-r and partial agonism of KOP-r may contribute to its
efficacy.
Nalmefene partially activates GTPγS binding Nalmefene does not recruit β-arrestin
Nalmefene
Compounds: U69,593, nor-BNI, and guanosine 5’-diphosphate (GDP) were obtained
from Sigma (St. Louis, MO, USA). Nalmefene was obtained from Baker Norton
Pharmaceuticals (Miami, FL, USA). Dynorphin A(1-17) was obtained from BACHEM
(Torrance, CA, USA). [35 S]GTPγS was obtained from Perkin Elmer, (Waltham, MA, USA).
GTPγS Assay: Membranes from HEK cells stably expressing human kappa opioid
receptors were used. Cells were scraped from tissue culture plates, homogenized with a
dounce homogenizer in membrane buffer (10mM Tris, 100mM NaCl, and 1mM EDTA;
pH 7.4), and centrifuged at 20,000 g for 30 minutes at 4°C and frozen at -80°C until use.
Prior to use, the pellets were resuspended in assay buffer (50mm Tris, 100mm NaCl,
5mM MgCl2, and 1mM EDTA; pH 7.4) and 50 μg incubated with 0.1 nM [35 S]GTPγS, 10
nM GDP, and the appropriate concentration of agonist for 60 minutes at 30°C.
Membranes with bound [35 S]GTPγS were collected on Whatman GF/B filter paper
(Brandel, Gaithersburg, MD, USA) utilizing a Brandel harvester. Bound [35 S]GTPγS was
quantified using a TriCarb-2900TR scintillation counter (Packard, Downers Grove, IL,
USA) following addition of 4 ml ReadySafe scintillation fluid (Beckman Coulter,
Indianapolis, IN, USA).
Functionally selective G-protein coupled receptor (GPCR) ligands may possess
enhanced therapeutic benefits.
In vitro pharmacological studies of G-protein coupled receptor ligands (GPCR)
have largely focused on G-protein mediated endpoints (e.g. cyclic AMP levels).
GPCR signaling can also occur via non-G-protein mediated pathways, specifically
β-arrestin mediated pathways.
Ligands can differentially activate these pathways, leading to functional
selectivity or biased agonism.
Nalmefene inhibition of U69,593 (300 nM) induced arrestin coupling. Inset shows U69,593 induced arrestin coupling
alone. Arrestin recruitment was measured using human KOPr-U2OS cells. Cells were treated with nalmefene and
U69,593 for 90 minutes. DiscoverX assay protocol was followed for arrestin recruitment detection. Nalmefene fully
blocks U69,593 recruitment of arrestin at concentrations over 100nm.
Arrestin coupling in human KOP-r-U2OS cells, in response to U69,593, dynorphin A (1-17), or nalmefene.
Arrestin recruitment was measured using human KOPr-U2OS cells. Cells were treated with nalmefene,
dynorphin A or U69,593 and incubated for 30 minutes. DiscoverX assay protocol was followed for arrestin
recruitment detection. Nalmefene does not recruit arrestin.
(i) Prolactin
(ii)
Anhedonia
(iii) Aversion
(i) Motor incoordination
(ii) Sedation
Nalmefene blocks β-arrestin recruitment by full agonist
Stimulation of GTPγS binding of human KOPr by U69,593 and nalmefene. HEK cell membranes expressing KOPr were
incubated with U69,593 or nalmefene in the presence of [35S] GTPγS for one hour and bound [35S] GTPγS was measured.
Nalmefene partially stimulates GTPγS binding compared to U69,593 stimulation.
Biased partial agonists may represent a way to
mitigate the negative side effects of kappa
receptor stimulation.
Nalmefene could be used as a model partial
kappa agonist with a G-protein bias.
The effects of kappa stimulation in alcohol
addiction therapy is still not well understood.
This study and others suggests that perhaps a
G-protein bias could be beneficial in kappa
agonist therapies.
Bart et al., 2005, Neuropscyhopharm., 30:2254-62
Nalmefene exhibits partial agonism in in vivo
prolactin assays and in vitro GTPγS binding.
Nalmefene does not recruit β-arrestin, and blocks
β-arrestin recruitment by a full kappa agonist
Nalmefene is a biased, partial kappa agonist
Kappa agonists may be useful for addiction therapies, but with negative side
effects such as sedation and motor incoordination.
These negative side effects may be mediated through the β-arrestin mediated
pathway.2
ACKNOWLEDGEMENTS
We gratefully appreciate the support of the Dr. Miriam and Sheldon G. Adelson Medical
Research Foundation (M.J.K.), a grant from the Robertson Foundation (E.B.), and the
support of the National Institutes of Health-National Institute on Drug Addiction
(R21DA031990) (B.R.).
1. Bart et al., 2005, Neuropsychopharm., 30:2254-62
2. Roth et al., 2014, J Pharmacol Exp Ther, 352: 98-109
U69593:
EC50 = 4.25 nM
Maximal Stimulation: 37.1%
Nalmefene:
EC50 = 1.99 nM
Maximal Stimulation: 20.7% [56%
U69593]
U69,593:
EC50 = 155 ± 33 nM
Dynorphin A(1-17):
EC50 = 9.5 ± 1.6 nM
Nalmefene:
EC50 = N/A
Nalmefene stimulation of [35S]GTPγS
binding mediated by the KOP-r
Nalmefene inhibition of [35S]GTPγS
binding stimulated by U50,488
β-arrestin Assay: Experiments were conducted using an AssayComplete U2OS Cell
Culture Kit obtained from DiscoverX.(Fremont, CA, USA). The PathHunter U2OS
OPRK1 β-arrestin cell line was used. Cells were plated in 96-well plates and
stimulated with nalmefene, U69593, or both for 90 minute at 37°C followed by
incubation for 60 minutes in the presence of galoctosidase substrate, yielding
chemiluminescent product. Chemiluminescence was measured using a Synergy Neo
microplate reader (BioTek, Winooski, VT, USA).
Nalmefene has been shown to act as a partial agonist at the KOP-r in GTPγS
binding assays, but its effects on β-arrestin recruitment have not been
shown1.
U69,593 stimulation of β-arrestin recruitment
Max: 91.4
Min: -3.6
IC50: 30.4
Ki=8.70 nM
(nM)
(nM)
10.1 1010 100 1000 10000
β-arrestinG-protein
U69,593
Nalmefene
(nM)
GDP
GTPγS GDP
GTPγS
Add agonist that
stimulates G-protein
activity
Radioactive
filterBind receptor to filter
and wash
KOPr fused to
part of enzyme
B-arrestin
B-arrestin fused to
other half of enzyme
Add agonist that
stimulates B-arrestin
recruitment B-arrestin
Substrate
Light
G-protein β-arrestin
Nalmefene