Mary J. Clark

Discovery of positive allosteric modulators and silent allosteric modulators of the μ-opioid receptor

μ-Opioid receptors are among the most studied G protein-coupled receptors because of the therapeutic value of agonists, such as morphine, that are used to treat chronic pain. However, these drugs have significant side effects, such as respiratory suppression, constipation, allodynia, tolerance, and dependence, as well as abuse potential. Efforts to fine tune pain control while alleviating the side effects of drugs, both physiological and psychological, have led to the development of a wide variety of structurally diverse agonist ligands for the μ-opioid receptor, as well as compounds that target κ- and δ-opioid receptors. In recent years, the identification of allosteric ligands for some G protein-coupled receptors has provided breakthroughs in obtaining receptor subtype-selectivity that can reduce the overall side effect profiles of a potential drug. However, positive allosteric modulators (PAMs) can also have the specific advantage of only modulating the activity of the receptor when the orthosteric agonist occupies the receptor, thus maintaining spatial and temporal control of receptor signaling in vivo. This second advantage of allosteric modulators may yield breakthroughs in opioid receptor research and could lead to drugs with improved side-effect profiles or fewer tolerance and dependence issues compared with orthosteric opioid receptor agonists. Here, we describe the discovery and characterization of μ-opioid receptor PAMs and silent allosteric modulators, identified from high-throughput screening using a β-arrestin–recruitment assay.

Selectivity of ligand binding to opioid receptors in brain membranes from the rat, monkey and guinea pig

Conditions for the equilibrium binding to opioid receptor of [3H]sufentanil (mu selective), [3H][D-Pen2,D-Pen5]enkephalin (delta selective), and [3H]U69,593 (kappa selective) were established in membranes from rat brain cerebrum, monkey cortex, or guinea pig cerebellum. The selectivity index of various opioid alkaloids and peptides in binding to the mu, delta, or kappa opioid receptors was expressed as the ratio of their EC50 values in displacing two selective radiolabeled ligands: [3H]sufentanil/[3H](D-Pen2,D-Pen5)enkephalin (selectivity: mu/delta), [3H]sufentanil/[3H]U69,593 (selectivity: mu/kappa), or [3H][D-Pen2,D-Pen5]enkephalin/[3H]U69,593 (selectivity: delta/kappa). High resolution in binding selectivity was observed: in rat brain the mu/delta selectivity for Tyr-D-Ala-Gly-(Me)Phe-Gly-ol and sufentanil were 0.02 and 0.03, whereas for [D-Pen2,D-Pen5]enkephalin and ICI 174,864 they were 1,200 and 998. Compared to mu opiates, the specific binding of delta and kappa agonists was less sensitive to sodium. The results describe a routinely applicable methodological approach for the assessment of selective ligand binding to the mu, delta and kappa opioid receptors in rodent and monkey brain membranes.

Comparison of the Relative Efficacy and Potency of μ-Opioid Agonists to Activate Gαi/o Proteins Containing a Pertussis Toxin-Insensitive Mutation

Pertussis toxin (PTX)-insensitive mutants of Gαi/o proteins expressed in C6μ cells were used to examine the hypothesis that there are agonist-specific conformational states of the μ-opioid receptor with coupling preferences to different Gαi/o subtypes, as measured by the degree of stimulation of [35S]guanosine 5′-O-(3-thio)triphosphate (GTPγS) binding. Binding of [35S]GTPγS to endogenous Gαi/o proteins stimulated by the full μ-opioid agonist [d-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) was completely blocked by overnight treatment with 100 ng/ml PTX. Treatment for 4 h with lower concentrations led to a PTX-dependent reduction in the maximal effect of DAMGO but no alteration in the potency of DAMGO or morphine nor in the relative maximal effect (relative efficacy) of the partial agonists morphine and buprenorphine compared with the full agonist DAMGO. Using PTX-insensitive Gα mutants in which the PTX-sensitive cysteine was replaced with isoleucine, the potency for a series of μ-opioid agonists was highest in cells expressing Gαi3 and Gαo and lowest with Gαi1 and Gαi2, with no significant change in the order of potency, namely, etorphine >> endomorphin-1 = DAMGO = endomorphin-2 = fentanyl = morphine >> meperidine. The order of agonist relative efficacy, etorphine = DAMGO = endomorphin-1 = endomorphin-2 = fentanyl ≥ morphine ≥ meperidine > buprenorphine ≥ nalbuphine, was also the same across all of the PTX-insensitive Gαi/o subtypes. Highest relative efficacy to stimulate [35S]GTPγS binding was seen with Gαi3. Consequently, reported observations of agonist-directed trafficking at μ-opioid receptors most likely involve non-PTX-sensitive Gα protein mechanisms.

Differential Effect of Membrane Cholesterol Removal on μ- and δ-Opioid Receptors A PARALLEL COMPARISON OF ACUTE AND CHRONIC SIGNALING TO ADENYLYL CYCLASE

According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-β-cyclodextrin (MβCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged μ-opioid receptors (HEK FLAG-μ) or δ-opioid receptors (HEK FLAG-δ) with MβCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the μ-opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MβCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing μ-opioid receptors. The effects of MβCD may be due to uncoupling of the μ receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-μ cells, MβCD treatment of HEK FLAG-δ cells had no effect on acute inhibition or sensitization of adenylyl cyclase by δ-opioid agonists. The differential responses of μ- and δ-opioid agonists to cholesterol depletion suggest that μ-opioid receptors are more dependent on cholesterol for efficient signaling than δ receptors and can be partly explained by localization of μ- but not δ-opioid receptors in cholesterol- and caveolin-enriched membrane domains.According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-beta-cyclodextrin (MbetaCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged mu-opioid receptors (HEK FLAG-mu) or delta-opioid receptors (HEK FLAG-delta) with MbetaCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MbetaCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing mu-opioid receptors. The effects of MbetaCD may be due to uncoupling of the mu receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-mu cells, MbetaCD treatment of HEK FLAG-delta cells had no effect on acute inhibition or sensitization of adenylyl cyclase by delta-opioid agonists. The differential responses of mu- and delta-opioid agonists to cholesterol depletion suggest that mu-opioid receptors are more dependent on cholesterol for efficient signaling than delta receptors and can be partly explained by localization of mu- but not delta-opioid receptors in cholesterol- and caveolin-enriched membrane domains.

Mu and Delta opioid receptors activate the same G proteins in human neuroblastoma SH-SY5Y cells

There is evidence for interactions between mu and delta opioid systems both in vitro and in vivo. This work examines the hypothesis that interaction between these two receptors can occur intracellularly at the level of G protein in human neuroblastoma SH‐SY5Y cells. The [35S]GTPγS binding assay was used to measure G protein activation following agonist occupation of opioid receptors. The agonists DAMGO (EC50, 45 nM) and SNC80 (EC50, 32 nM) were found to be completely selective for stimulation of [35S]‐GTPγS binding through mu and delta opioid receptors respectively. Maximal stimulation of [35S]‐GTPγS binding produced by SNC80 was 57% of that seen with DAMGO. When combined with a maximally effective concentration of DAMGO, SNC80 caused no additional [35S]‐GTPγS binding. This effect was also seen when measured at the level of adenylyl cyclase. Receptor activation increased the dissociation of pre‐bound [35S]‐GTPγS. In addition, the delta agonist SNC80 promoted the dissociation of [35S]‐GTPγS from G proteins initially labelled using the mu agonist DAMGO. Conversely, DAMGO promoted the dissociation of [35S]‐GTPγS from G proteins initially labelled using SNC80. Tolerance to DAMGO and SNC80 in membranes from cells exposed to agonist for 18 h was homologous and there was no evidence for alteration in G protein activity. The findings support the hypothesis that mu‐ and delta‐opioid receptors share a common G protein pool, possibly through a close organization of the two receptors and G protein at the plasma membrane.

Membrane Microviscosity Modulates μ‐Opioid Receptor Conformational Transitions and Agonist Efficacy

Abstract: The influence of membrane microviscosity on μ‐opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH‐SY5Y cells and from a C6 glioma cell line stably expressing the rat μ‐opioid receptor (C6μ). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D‐Ala2,N‐MePhe4,Gly‐ol5]enkephalin (DAMGO) for the μ‐opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis‐vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol—receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine‐5′‐O‐(3‐[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS‐treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist‐stimulated GTPase activity in control and CHS‐modified membranes. These findings support a regulatory role for membrane microviscosity in receptor‐mediated G protein activation.

Opioid Peptidomimetics: Leads for the Design of Bioavailable Mixed Efficacy μ Opioid Receptor (MOR) Agonist/δ Opioid Receptor (DOR) Antagonist Ligands

We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a μ opioid receptor (MOR) agonist, δ opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood-brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance.

COUPLING OF MULTIPLE OPIOID RECEPTORS TO GTPase FOLLOWING SELECTIVE RECEPTOR ALKYLATION IN BRAIN MEMBRANES

Opioid agonists of the mu, kappa and delta types stimulated low-Km guanosine triphosphatase (GTPase) in membranes, from the brain of the rat by up to 34%, with potencies the rank order of which corresponded to the respective binding affinities to opioid receptor. In general, kappa ligands stimulated GTPase to a lesser degree than mu or delta opiates. The coupling of a given type of opioid receptor to GTPase was resolved by direct or protective alkylation of the other receptors. Treatment of the membranes with beta-funaltrexamine abolished the stimulation of GTPase by sufentanil and levorphanol (mu), but not by bremazocine (kappa) or DSLET (delta). On the other hand, prior incubation with Superfit, an alkylating agent with selectivity for the delta opioid receptor, specifically eliminated the effect of DSLET. Partial alkylation by increasing concentrations of Superfit gradually reduced the extent of stimulation of GTPase by DSLET. The successive treatment of membranes with Superfit and beta-funaltrexamine blocked the actions of DSLET, sufentanil and levorphanol, but had no effect on the stimulation of the GTPase by bremazocine. Selective coupling of an opioid receptor to GTPase was also obtained after incubation of membranes with beta-chlornaltrexamine in the presence of protective concentrations of mu, kappa or delta opioid ligands. Alkylation resolved the coupling of the non-selective opiate etorphine: the sum of stimulation of GTPase in the receptor-selective membranes equalled maximal stimulation of enzyme in untreated membranes. Naloxone blocked the stimulation of GTPase by mu, kappa or delta agonists, but ICI-174,864 specifically inhibited the effect of DSLET.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for coupling of the κ opioid receptor to brain GTPase

Abstract In membranes from guinea pig cerebellum, a tissue which predominantly contains κ opioid receptors, exogenous and endogenous κ-selective opioid agonists stimulated low-Km GTPase activity by 11–20% with concentrations for half-maximal stimulation of 3–23 μM. Opioid ligands of the μ and δ type had no effect on GTPase in these membranes. Similar stimulation of GTPase by κ opiates was obtained in rat and monkey brain membranes pretreated with β-funaltrexamine (β-FNA) and ci s -(±)-3-methylfentanyl isothiocyanate (superfit) to alkylate the μ and δ receptors, respectively. The stimulation of brain GTPase by κ opiates in both types of membranes was inhibited by naloxone with IC50s of 0.35 μM and 0.40 μM. The results demonstrate the coupling of the κ opioid receptor to high affinity GTPase, the Ni regulatory protein of the adenylate cyclase complex.

Orvinols with Mixed Kappa/Mu Opioid Receptor Agonist Activity

Dual-acting kappa opioid receptor (KOR) agonist and mu opioid receptor (MOR) partial agonist ligands have been put forward as potential treatment agents for cocaine and other psychostimulant abuse. Members of the orvinol series of ligands are known for their high binding affinity to both KOR and MOR, but efficacy at the individual receptors has not been thoroughly evaluated. In this study, it is shown that a predictive model for efficacy at KOR can be derived, with efficacy being controlled by the length of the group attached to C20 and by the introduction of branching into the side chain. In vivo evaluation of two ligands with the desired in vitro profile confirms both display KOR, and to a lesser extent MOR, activity in an analgesic assay suggesting that, in this series, in vitro measures of efficacy using the [35S]GTPγS assay are predictive of the in vivo profile.