An Immunocytochemical-Derived Correlate for Evaluating the Bridging of Heteromeric Mu-Delta Opioid Protomers by Bivalent Ligands

Bivalent ligands that contain two pharmacophores linked by a spacer are promising tools to investigate the pharmacology of opioid receptor heteromers. Evidence for occupation of neighboring protomers by two phamacophores of a single bivalent ligand (bridging) has relied mainly on pharmacological data. In the present study, we have employed an immunocytochemical correlate to support <i>in vivo</i> biological studies that are consistent with bridging. We show that a bivalent mu agonist/delta antagonist (MDAN-21) that is devoid of tolerance due to possible bridging of mu and delta protomers prevents endocytosis of the heteromeric receptors in HEK-293 cells. Conversely, a bivalent ligand (MDAN-16) with a short spacer or monovalent mu agonist give rise to robust internalization. The data suggest that the immobilization of proximal mu and delta protomers is due to bridging by MDAN-21. The finding that MDAN-21 and its shorter spacer homologue MDAN-16 possess equivalent activity in HEK-293 cells, but produce dramatically divergent internalization of mu-delta heteromer, is relevant to the role of internalization and tolerance

Clinically Employed Opioid Analgesics Produce Antinociception via μ‑δ Opioid Receptor Heteromers in Rhesus Monkeys

Morphine and related drugs are widely employed as analgesics despite the side effects associated with their use. Although morphine is thought to mediate analgesia through mu opioid receptors, delta opioid receptors have been implicated in mediating some side effects such as tolerance and dependence. Here we present evidence in rhesus monkeys that morphine, fentanyl, and possibly methadone selectively activate mu-delta heteromers to produce antinociception that is potently antagonized by the delta opioid receptor antagonist, naltrindole (NTI). Studies with HEK293 cells expressing mu-delta heteromeric opioid receptors exhibit a similar antagonism profile of receptor activation in the presence of NTI. In mice, morphine was potently inhibited by naltrindole when administered intrathecally, but not intracerebroventricularly, suggesting the possible involvement of mu-delta heteromers in the spinal cord of rodents. Taken together, these results strongly suggest that, in primates, mu-delta heteromers are allosterically coupled and mediate the antinociceptive effects of three clinically employed opioid analgesics that have been traditionally viewed as mu-selective. Given the known involvement of delta receptors in morphine tolerance and dependence, our results implicate mu-delta heteromers in mediating both antinociception and these side effects in primates. These results open the door for further investigation in humans

The δ Opioid Receptor Agonist SNC80 Selectively Activates Heteromeric μ–δ Opioid Receptors

Coexpressed and colocalized μ- and δ-opioid receptors have been established to exist as heteromers in cultured cells and <i>in vivo</i>. However the biological significance of opioid receptor heteromer activation is less clear. To explore this significance, the efficacy of selective activation of opioid receptors by SNC80 was assessed <i>in vitro</i> in cells singly and coexpressing opioid receptors using a chimeric G-protein-mediated calcium fluorescence assay, SNC80 produced a substantially more robust response in cells expressing μ–δ heteromers than in all other cell lines. Intrathecal SNC80 administration in μ- and δ-opioid receptor knockout mice produced diminished antinociceptive activity compared with wild type. The combined <i>in vivo</i> and <i>in vitro</i> results suggest that SNC80 selectively activates μ–δ heteromers to produce maximal antinociception. These data contrast with the current view that SNC80 selectively activates δ-opioid receptor homomers to produce antinociception. Thus, the data suggest that heteromeric μ–δ receptors should be considered as a target when SNC80 is employed as a pharmacological tool <i>in vivo</i>

Inhibition of Inflammatory and Neuropathic Pain by Targeting a Mu Opioid Receptor/Chemokine Receptor5 Heteromer (MOR-CCR₅)

Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR₅ heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR₅ antagonist pharmacophores linked through homologous spacers (14–24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (<b>MCC22</b>; <b>3e</b>) with a 22-atom spacer exhibited profound antinociception (i.t. ED₅₀ = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, <b>MCC22</b> was ∼3500× more potent than a mixture of mu agonist and CCR₅ antagonist monovalent ligands. These data strongly suggest that <b>MCC22</b> acts by bridging the protomers of a MOR-CCR₅ heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR₅ heteromer as a novel target for the treatment of chronic pain

Inhibition of Inflammatory and Neuropathic Pain by Targeting a Mu Opioid Receptor/Chemokine Receptor5 Heteromer (MOR-CCR₅)

Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR₅ heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR₅ antagonist pharmacophores linked through homologous spacers (14–24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (<b>MCC22</b>; <b>3e</b>) with a 22-atom spacer exhibited profound antinociception (i.t. ED₅₀ = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, <b>MCC22</b> was ∼3500× more potent than a mixture of mu agonist and CCR₅ antagonist monovalent ligands. These data strongly suggest that <b>MCC22</b> acts by bridging the protomers of a MOR-CCR₅ heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR₅ heteromer as a novel target for the treatment of chronic pain

Putative Kappa Opioid Heteromers As Targets for Developing Analgesics Free of Adverse Effects

It is now generally recognized that upon activation by an agonist, β-arrestin associates with G protein-coupled receptors and acts as a scaffold in creating a diverse signaling network that could lead to adverse effects. As an approach to reducing side effects associated with κ opioid agonists, a series of β-naltrexamides <b>3</b>–<b>10</b> was synthesized in an effort to selectively target putative κ opioid heteromers without recruiting β-arrestin upon activation. The most potent derivative <b>3</b> (INTA) strongly activated KOR-DOR and KOR-MOR heteromers in HEK293 cells. In vivo studies revealed <b>3</b> to produce potent antinociception, which, when taken together with antagonism data, was consistent with the activation of both heteromers. <b>3</b> was devoid of tolerance, dependence, and showed no aversive effect in the conditioned place preference assay. As immunofluorescence studies indicated no recruitment of β-arrestin2 to membranes in coexpressed KOR-DOR cells, this study suggests that targeting of specific putative heteromers has the potential to identify leads for analgesics devoid of adverse effects