Nalfurafine is a G-protein biased agonist having significantly greater bias at the human than rodent form of the kappa opioid receptor
Graphical abstract
Introduction
Mu opioid agonists including morphine-like opiates remain the principal analgesics for the treatment severe pain, however the respiratory depression, constipating, and addiction risks of these drugs are well known liabilities and diversion of prescription opioids is currently a major public health crisis [1]. Kappa opioid analgesics have been developed as safer alternatives to mu opioids for control of pain, however the selective kappa opioid receptor (KOR) analgesics that were initially developed produce profound dysphoric and psychotomimetic actions [2]. Attempts to reduce the dysphoric properties of KOR agonists by restricting their brain penetrance have been made; however these peripherally restricted compounds have demonstrated only modest analgesic activity [3], [4]. Recent studies have suggested that the analgesic effects of KOR are mediated by G protein regulation of ion channels, including increased G protein gated potassium channel and decreased calcium channel conductance resulting in reduced neuronal excitability; whereas the aversive effects of KOR agonists require G protein-coupled receptor kinase 3 (GRK3)-dependent arrestin activation [5], [6]. The concept that functionally selective agonists that preferentially activate one signaling cascade can be developed has re-energized opioid drug development [7], [8]. Furthermore the hypothesis that a centrally active G protein biased KOR agonist that does not induce arrestin-dependent signaling might be a safer analgesic has stimulated the field [8], [9].
Functionally selective KOR agonists have been recently identified [10], [11], [12], however these compounds lack drug-like qualities. In contrast, the moderately receptor selective KOR agonist nalfurafine (TRK820) is currently in clinical trials for the treatment of uremic pruritis [13], and has pharmacological properties suggestive of functional selectivity (e.g. analgesic, low incidence of dysphoria) [14], [15], [16]. In the present study, we assessed the functional selectivity of nalfurafine. Identification of a drug-like lead compound in the development of functionally selective KOR agonists would be a significant advance in the generation of novel analgesics potentially lacking the addictive effects of mu opioids and the dysphoric effects of conventional KOR agonists.
Section snippets
Chemicals/reagents
For cell culture experiments KOR agonists (−)U50,488 (Tocris) and nalfurafine hydrochloride ((2E)-N-[(5α,6β)-17-(cyclopropylmethyl)-3,14-dihydroxy-4,5-epoxymorphinan-6-yl]-3-(3-furyl)-N-methylacrylamide) (NIDA Drug Supply) and the MOR agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin) (Sigma-Aldrich) were dissolved in water. For in vivo studies, the KOR agonists nalfurafine, (+/−)U50,488, and the KOR antagonists 5′-GNTI, and norBNI were obtained from the NIDA Drug Supply and were freshly
Does nalfurafine differentially activate p38 and ERK1/2 MAPK pathways by rKOR?
KOR agonists have previously been shown to promote ERK1/2 phosphorylation at 5 min and p38 phosphorylation at 30 min via different signaling cascades [21], [26]. We first sought to establish whether nalfurafine activates these cascades with similar efficacy and potency. HEK293 cells stably expressing rKOR were treated for 5 or 30 min with nalfurafine (10 pM-100 nM) or U50,488 (1 μM) prior to lysis and phospho-ERK1/2 (5 min) or phospho-p38 (30 min) was quantified by western blot (Fig. 1A–B). Nalfurafine
Discussion
The major finding of this study is that nalfurafine is a G protein biased ligand with greater bias at the human than rodent KOR. Nalfurafine is biased towards ERK1/2 activation (G protein-dependent), with 20 × and 250 × lower potency for p38 activation by rKOR and hKOR, respectively. These findings have implications for the development of KOR agonists with therapeutic use, as p38 contributes the aversive properties of KOR agonists [5], [6], [33]. We also found that nalfurafine-induced thermal
Conclusions
Despite lacking optimal receptor selectivity, nalfurafine is a centrally-active prototype G protein biased agonist and therefore provides a base structure for future analgesic drug development. An ideal KOR agonist would be highly efficacious for G protein-mediated pathways with minimal efficacy or greatly reduced potency for GRK3 mediated-p38 activation. Additional qualities would be low affinity and minimal efficacy for other opioid receptors, notably MOR, and being centrally active. This
Funding
This work was supported by the National Institute on Drug Abuse [Grants: PO1-DA035764 and T32-DA07278].
Authorship contributions
Participated in research design: Schattauer, Kuhar, Song, Chavkin.
Conducted experiments: Schattauer, Kuhar, Song.
Performed data analysis: Schattauer, Kuhar, Song, Chavkin.
Wrote or contributed to the writing of the manuscript: Schattauer, Chavkin.
Acknowledgements
We thank Dr. John Pintar for providing the original MOR−/− and KOR−/− mice. We thank Dan Messinger for managing the mouse breeding and genotyping. We thank Dr. Peter Groblewski for technical assistance.
References (36)
Kappa-opioid receptors and analgesia
Trends Pharmacol. Sci.
(1990)- et al.
Biased agonism at kappa opioid receptors: Implication in pain and mood disorders
Eur. J. Pharmacol.
(2015) - et al.
6′-Guanidinonaltrindole (6′-GNTI) is a G protein-biased kappa-opioid receptor agonist that inhibits arrestin recruitment
J. Biol. Chem.
(2012) - et al.
Functional selectivity of 6′-guanidinonaltrindole (6′-GNTI) at kappa-opioid receptors in striatal neurons
J. Biol. Chem.
(2013) - et al.
Nalfurafine prevents 5′-guanidinonaltrindole- and compound 48/80-induced spinal c-fos expression and attenuates 5′-guanidinonaltrindole-elicited scratching behavior in mice
Neuroscience
(2009) - et al.
Ligand directed signaling differences between rodent and human kappa-opioid receptors
J. Biol. Chem.
(2012) - et al.
Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes
J. Biol. Chem.
(2006) - et al.
Pharmacological properties of TRK-820 on cloned mu-, delta- and kappa-opioid receptors and nociceptin receptor
Eur. J. Pharmacol.
(1999) - et al.
Potent antinociceptive effects of TRK-820, a novel kappa-opioid receptor agonist
Life Sci.
(1999) - et al.
Characterization of the antinociceptive effects of TRK-820 in the rat
Eur. J. Pharmacol.
(2000)