Positive allosteric modulators of the µ-opioid receptor: a novel approach for future pain medications.

UNLABELLED: Morphine and other agonists of the µ-opioid receptor are used clinically for acute and chronic pain relief and are considered to be the gold standard for pain medication. However, these opioids also have significant side effects, which are also mediated via activation of the µ-opioid receptor. Since the latter half of the twentieth century, researchers have sought to tease apart the mechanisms underlying analgesia, tolerance and dependence, with the hope of designing drugs with fewer side effects. These efforts have revolved around the design of orthosteric agonists with differing pharmacokinetic properties and/or selectivity profiles for the different opioid receptor types. Recently, µ-opioid receptor-positive allosteric modulators (µ-PAMs) were identified, which bind to a (allosteric) site on the µ-opioid receptor separate from the orthosteric site that binds an endogenous agonist. These allosteric modulators have little or no detectable functional activity when bound to the receptor in the absence of orthosteric agonist, but can potentiate the activity of bound orthosteric agonist, seen as an increase in apparent potency and/or efficacy of the orthosteric agonist. In this review, we describe the potential advantages that a µ-PAM approach might bring to the design of novel therapeutics for pain that may lack the side effects currently associated with opioid therapy. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

G-protein coupling of mu-opioid receptors (OP3): elevated basal signalling activity.

To determine mu-opioid receptor (OP(3)) signalling activity, guanosine 5'-[gamma-[(35)S]thio]triphosphate (GTP[(35)S]) binding to G-proteins was measured in the membranes of human embryonic kidney cells (HEK-293) transfected with mu-opioid receptor (HEK-mu). GTP[(35)S] binding to HEK-mu membranes was significantly elevated compared with HEK-293 control membranes (without OP(3)), and this was abolished by pertussis-toxin pretreatment. The irreversible antagonist beta-chlornaltrexamine (beta-CNA) dose-dependently decreased elevated basal G-protein coupling of HEK-mu to control levels in cells devoid of OP(3). This characterizes beta-CNA as an inverse OP(3) agonist. Immunoprecipitation of solubilized G-proteins with G(i3)alpha antisera demonstrated that basal GTP[(35)S] binding to G(i3)alpha was also substantially elevated in HEK-mu membranes over the control, whereas G(i3)alpha protein levels were unchanged. Basal GTP[(35)S] binding to G(i1)alpha/G(i2)alpha and G(o)alpha was also increased twofold in HEK-mu membranes over the control. Morphine further increased coupling to each of these Galpha proteins with similar potency, but not to G(q)/(11)alpha or G(s)alpha. These results indicate that the wild-type OP(3) can couple constitutively to endogenously expressed G(i3)alpha, G(i1)alpha/G(i2)alpha and G(o)alpha subunits of G-proteins in HEK-293 cells.

Specific G protein activation and mu-opioid receptor internalization caused by morphine, DAMGO and endomorphin I.

Previous studies have shown that the agonist [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO) but not morphine induces mu-opioid receptor internalization [Arden, J.R., Segredo, V., Wang, Z., Lameh, J., Sadee, W., 1995. J. Neurochem. 65, 1636-1645]. In the present study we investigated the relationship between internalization of the mu-opioid receptor and the specific G proteins activated following treatment with morphine, DAMGO and endomorphin I (Tyr-Pro-Trp-Phe-NH2) (a putative endogenous mu-opioid receptor agonist) in human embryonic kidney (HEK) cells. Endomorphin I and DAMGO, but not morphine, caused mu-opioid receptor internalization. Morphine, DAMGO and endomorphin I each activated Gi1 alpha/Gi2 alpha, Go alpha and Gi3 alpha to a similar extent, but not Gq alpha/G11 alpha or Gs alpha in HEK membranes. Therefore, the three ligands tested differed in their ability to internalize mu-opioid receptors even though they were similar in activating individual G proteins.

A constitutively internalizing and recycling mutant of the mu-opioid receptor.

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in mu-opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr-rich domain (354ThrSerSerThrIleGluGlnGlnAsn362) unique to the C-terminus of the mu-opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these mu-receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N-terminus of the wild-type and mutant receptors. Both the wild-type mu-opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin-coated vesicles and recycled constitutively. Despite agonist-independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C-terminus, particularly the Ser/Thr domain, appears to suppress mu-receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist-dependent process, can occur spontaneously without concomitant G protein activation.

Binding and activity of the nine possible regioisomers of myo-inositol tetrakisphosphate at the inositol 1,4,5-trisphosphate receptor.

All 9 racemic regioisomers (15 enantiomerically) of myo-inositol tetrakisphosphates (IP4s): DL-Ins(1,2,4,5)P4 [A], DL-Ins(1,2,4,6)P4 [B], Ins(1,2,3,5)P4 [C], Ins(1,3,4,6)P4 [D], Ins(2,4,5,6)P4 [E], DL-Ins(1,3,4,5)P4 [F], DL-Ins(1,2,5,6)P4 [G], DL-Ins(1,2,3,4)P4 [H] and DL-Ins(1,4,5,6)P4 [I] [Chung S-K., Chang Y-T. Synthesis of all possible regioisomers of myo-inositol tetrakisphosphate. J Chem Soc Chem Commun 1995; 11-13] were investigated for their ability to bind to the D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor in bovine adrenal cortical membranes, and for their ability to mobilize 45Ca2+ from Ins(1,4,5)P3-sensitive Ca2+ stores in permeabilized Chinese hamster ovary (CHO) cells. DL-Ins(1,2,4,5)P4 (Ki = 11 nM) bound to Ins(1,4,5)P3 receptors with an affinity only 2-fold lower than Ins(1,4,5)P3 (Ki = 6 nM). Ins(1,2,3,5)P4, Ins(1,3,4,6)P4, Ins(2,4,5,6)P4, DL-Ins(1,3,4,5)P4, DL-Ins(1,2,3,4)P4 and DL-Ins(1,4,5,6)P4 bound with affinities of between 0.4-0.7 microM. DL-Ins(1,2,4,6)P4 and DL-Ins(1,2,5,6)P4 bound to the Ins(1,4,5)P3 receptor with low affinity (approximately 2-3 microM). All but one of the IP4s mediated release of 45Ca2+ from stores of permeabilized CHO cells with a similar rank order of potency as that for Ins(1,4,5)P3 receptor binding, being between 16-fold and 50-fold less potent at releasing 45Ca2+ compared with their apparent binding affinities to the Ins(1,4,5)P3 receptor. The notable exception was Ins(1,2,3,5)P4, which showed an approximately 200-fold lower potency compared with its affinity for the Ins(1,4,5)P3 receptor. Ins(1,2,3,5)P4 may be a useful lead compound for the rational design of novel synthetic Ins(1,4,5)P3 analogues possessing structure-activity profiles with relatively high binding affinity, but low intrinsic efficacy, and hence partial agonists and antagonists at the Ins(1,4,5)P3 receptor.

Residues specifically involved in down-regulation but not internalization of the m1 muscarinic acetylcholine receptor.

Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [35S]GTP gamma S binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.

Muscarinic m1 receptor-stimulated adenylate cyclase activity in Chinese hamster ovary cells is mediated by Gs alpha and is not a consequence of phosphoinositidase C activation.

The mechanism underlying muscarinic m1 receptor-mediated increases in adenosine 3',5'-cyclic monophosphate (cAMP) was investigated in Chinese hamster ovary (CHO) cells expressing human recombinant m1 muscarinic receptors (CHO-ml cells). Stimulation of CHO-ml cells with carbachol resulted in marked accumulation of Ins(1,4,5)P3 and cAMP, in an atropine-sensitive manner, with EC50 values (log M) of -5.16 +/- 0.06 and -3.93 +/- 0.07 respectively. Basal and agonist-stimulated cAMP accumulation were unaffected by a 5 min pretreatment with l microM phorbol 12,13-dibutyrate and were not attenuated by pertussis toxin (100 ng/ml, 20h). Agonist-stimulated cAMP accumulation was also observed in CHO-ml cell membranes incubated in a buffer containing 100 nM free Ca2+. Guanosine 5'- [gamma-thio]triphosphate (10 microM) potentiated agonist-stimulated cAMP accumulation in CHO-ml cell membranes, implicating a G-protein involvement in this response. Co-incubation of carbachol with forskolin (10 microM) produced a greater than additive accumulation of cAMP in CHO-ml cells. Furthermore, a C-terminal-directed anti-Gs alpha serum attenuated both carbachol-stimulated (in CHO-ml cell membranes) and isoprenaline-stimulated (in CHO-beta 2 cell membranes) cAMP accumulation with a similar dose-dependency. These results suggest that muscarinic agonist-stimulated cAMP accumulation in CHO-ml cells occurs via activation of Gs alpha and not as a consequence of phosphoinositidase C activation.

Relationship between agonist binding, phosphorylation and immunoprecipitation of the m3-muscarinic receptor, and second messenger responses.

1. Phosphoinositidase C-linked m3-muscarinic receptors expressed in Chinese hamster ovary cells (CHO-m3 cells) are phosphorylated on serine following agonist stimulation. 2. m3-Muscarinic receptor phosphorylation is concentration-dependent requiring a carbachol concentration of 13.2 microM for half maximal stimulation. 3. The phosphorylation concentration-response curve lies to the left of the curve for carbachol binding to muscarinic receptors (KD = 100 microM) in membranes from CHO-m3 cells. In contrast, receptor phosphorylation closely correlates with receptor-mediated phosphoinositidase C activation (EC50 for inositol 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 microM and 5.92 microM respectively) but not with rapid agonist-mediated calcium elevation (EC50 = 0.32 microM) measured in fura-2-AM loaded cells. 4. These data suggest a dissociation of receptor phosphorylation from agonist occupation. Such an apparent 'receptor reserve' for m3-muscarinic receptor phosphorylation may be indicative of a mechanism that is dependent on a small amplification of the receptor signal, though probably dissociated from the calcium signal.

Differential coupling of m1, m2 and m3 muscarinic receptor subtypes to inositol 1,4,5-trisphosphate and adenosine 3',5'-cyclic monophosphate accumulation in Chinese hamster ovary cells.

Agonist-stimulated accumulation of inositol 1,4,5-trisphosphate and adenosine 3',5'-cyclic monophosphate (cAMP) were measured in Chinese hamster ovary (CHO) cells expressing m1 (CHO-m1), m2 (CHO-m2) or m3 (CHO-m3) muscarinic receptors. At similar levels of expression (approximately 1000 fmol of receptor per mg of protein), m1 and m3 muscarinic receptors mediated similar carbachol-stimulated, biphasic accumulation of inositol-1,4,5-trisphosphate in intact cells and similar release of preloaded 45Ca++ from permeabilized cells. However, CHO-m1 cells produced a 4-fold greater agonist-stimulated accumulation of cAMP compared with CHO-m3 cells, in a pertussis toxin-insensitive manner. CHO-m2 cells (expressing approximately 100 fmol of receptor per mg of protein) coupled to the inhibition of adenylyl cyclase in a pertussis toxin-sensitive manner. However, after pertussis toxin pretreatment, agonist stimulation mediated a 50% potentiation of forskolin-stimulated cAMP accumulation. Muscarinic m1, m2 and m3 receptor-mediated stimulation of cAMP accumulation, correlated with the apparent binding affinity of carbachol for these receptors, suggesting a lack of an apparent receptor reserve for this response. Reducing the level of m3 muscarinic receptors by approximately 50% resulted in no detectable stimulation of cAMP accumulation. The results suggest that m1 and m3 muscarinic receptors, expressed at similar levels in CHO cells, couple to the activation of phospholipase C with similar efficiency. However, m1 muscarinic receptors couple with greater efficiency to the stimulation of adenylyl cyclase compared with m3 muscarinic receptors. Muscarinic m1, m2 and m3 receptor-mediated cAMP accumulation in CHO cells does not appear to be a consequence of phospholipase C activation.

Coupling of muscarinic m1, m2 and m3 acetylcholine receptors, expressed in Chinese hamster ovary cells, to pertussis toxin-sensitive/insensitive guanine nucleotide-binding proteins.

Chinese hamster ovary (CHO) cells expressing recombinant human m1 (CHO-m1 cells), m2 (CHO-m2 cells), or m3 (CHO-m3 cells) muscarinic receptors were characterised pharmacologically with [3H]N-methylscopolamine. Agonist-stimulated coupling of these receptors with guanine nucleotide-binding proteins (G proteins) was measured by guanine nucleotide- and pertussis toxin-modification of carbachol competition-binding curves, and pertussis toxin-sensitivity of agonist-stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) binding, in membrane preparations of the CHO cell clones. High affinity agonist binding and agonist-stimulated [35S]GTP gamma S binding was abolished in CHO-m2 cell membranes (expressing 99 +/- 25 fmol of [3H]N-methylscopolamine binding sites/mg protein) after pertussis toxin pretreatment of cells, suggesting that muscarinic m2 receptors expressed in these cell membranes couple predominantly with pertussis toxin-sensitive G proteins. CHO-m1 (713 +/- 102 fmol/mg protein) and CHO-m3 (1212 +/- 279 fmol/mg protein) cell membranes produced smaller elevations in agonist-stimulated [35S]GTP gamma S binding considering the higher receptor levels, compared with CHO-m2 cell membranes. Pertussis toxin pretreatment of these clones also resulted in a significant attenuation of agonist-stimulated [35S]GTP gamma S binding suggesting that, under these experimental conditions, muscarinic m1 and m3 receptors can couple with both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. Guanine nucleotide-modification of agonist binding in CHO-m1 and CHO-m3 cell membranes was comparatively smaller than in CHO-m2 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)