John R. Traynor

Structural insights into µ-opioid receptor activation

Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

Nonpeptidic δ-opioid Receptor Agonists Reduce Immobility in the Forced Swim Assay in Rats

The present study examined the effect of opioid receptor agonists in the rat forced swim assay. The δ-opioid receptor agonists SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide) and (+)BW373U86 ((+)-[1(S*),2α,5β]-4-[[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl] (3-hydroxyphenyl)methyl]-N,N-diethyl-benzamide dihydrochloride) produced a decrease in immobility indicating an antidepressant-like effect. At antinociceptive doses, neither the κ-opioid selective agonist CI977 (5R-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl-1-oxaspiro[4,5]dec-8-yl]-4-benzofuranacetamide) showed a change in immobility that was identifiable by dose, nor were changes in immobility seen with morphine. A δ-opioid mechanism of action in the forced swim assay was likely since naltrindole prevented the effects of both δ-agonists. When compared to desipramine and fluoxetine, SNC80 was more active with a single dose whereas both desipramine and fluoxetine produced greater effects with subchronic dosing (3 doses). All three compounds were active when administered before the initial swim exposure. SNC80 was, however, more effective following a single dose than by subchronic administration demonstrating both a fast onset of activity and potential tolerance. Thus, δ-agonists differ from typical antidepressants in the forced swim assay.

Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay.

Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein α-subunits (Gα) and thus shorten the time course and reduce the magnitude of G-protein α- and βγ-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Gα-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Gαo binding with 3 to 5 μM potency. It binds to RGS4, inhibits RGS4 stimulation of Gαo GTPase activity in vitro, and prevents RGS4 regulation of μ-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Gα protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.

A Spatial Focusing Model for G Protein Signals REGULATOR OF G PROTEIN SIGNALING (RGS) PROTEIN-MEDIATED KINETIC SCAFFOLDING

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding to Gi by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH2- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPγS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic “spatial focusing” of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Gα-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Gα-GDP is not depleted there. Thus, a novel RGS-mediated “kinetic scaffolding” mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of Gi protein signals.

Comparison of the Antinociceptive Response to Morphine and Morphine-Like Compounds in Male and Female Sprague-Dawley Rats

Male rats are more sensitive to the antinociceptive effects of morphine than female rats. This difference is seen across several rat strains using a variety of nociceptive stimuli. However, the literature in regard to sex differences in antinociceptive responses to μ-opioids other than morphine is less consistent. The present study was designed to examine whether there is a structure-activity rationale that determines which μ-opioids will show a differential antinociceptive response between male and female rats. A series of morphinans closely related in structure to morphine, namely, codeine, heroin, hydrocodone, hydromorphone, oxymorphone, and oxycodone, were examined for their antinociceptive activity in male and female Sprague-Dawley rats and compared with the structurally unrelated μ-opioid agonists methadone and fentanyl. Antinociception was measured by the warm-water tail-withdrawal assay. The results show that morphine is more potent in males compared with females > hydromorphone = hydrocodone = oxymorphone, but there was no observable sex difference in the antinociceptive potency of codeine, heroin, oxycodone, methadone, or fentanyl. The potency to stimulate guanosine 5′-O-(3-[35 S]thio)triphosphate ([35S]GTPγS) binding and binding affinity of the various morphinans was compared in rat glioma C6 cells expressing the rat μ-opioid receptor; relative efficacy was also compared by stimulation of [35S]GTPγS binding in slices of rat brain thalamus. The presence of a sex difference in antinociceptive responsiveness was not related to drug potency, efficacy, or affinity. Consequently, it is likely that differential metabolism of the opioid, possibly by glucuronidation, determines the presence or absence of a sex difference.

Differential binding properties of oripavines at cloned μ- and δ-opioid receptors

This study examines the possibility that oripavine opioid receptor agonists bind equally to both high and low affinity states of the μ-opioid receptor. Studies were performed in C6 cells expressing μ- or δ-opioid receptors; high and low agonist affinity states of the receptors were defined by the absence and presence, respectively of Na+ ions and the GTP analog Gpp(NH)p. At the μ-opioid receptor dihydroetorphine and etorphine were full agonists, buprenorphine had moderate efficacy while diprenorphine was an antagonist. At the δ-opioid receptor, dihydroetorphine, etorphine, and diprenorphine had moderate efficacy while buprenorphine was an antagonist. The binding affinities of the oripavines at the μ-opioid receptor decreased only one to 2-fold in the presence of NaCl and Gpp(NH)p. In contrast, decreases in oripavine affinity at the δ-opioid receptor correlated with δ-opioid receptor efficacy. The ability of oripavine agonists to bind with high affinity to the low agonist affinity state of the ν-opioid receptor may explain the high potencies of these compounds in vivo.

The use of [3H]-[D-Pen2,D-Pen5]enkephalin as a highly selective ligand for the δ-binding site

1 The characteristics of the binding of [3H]‐[d‐Pen2,d‐Pen5]enkephalin were determined in homogenates of guinea‐pig and rat brain. 2 In the guinea‐pig, the maximum binding capacity for [3H]‐[d‐Pen2,d‐Pen5]enkephalin was 4.19 pmol g−1 and the KD 1.61 nm. In the rat, the corresponding values were 2.47 pmol g−1 and 5.42 nm. In both species, the maximum binding capacity and the affinity were not altered when μ‐binding was suppressed with [d‐Ala2, MePhe4, Gly‐ol5]enkephalin. 3 The μ‐agonists, [d‐Ala2, MePhe4, Gly‐ol5]enkephalin and morphine, displaced a small portion of the binding of [3H]‐[d‐Pen2,d‐Pen5]enkephalin with high affinities.

RGS inhibition at Gαi2 selectively potentiates 5-HT1A–mediated antidepressant effects

Elevating serotonin (5-HT) levels with selective serotonin reuptake inhibitors (SSRIs) is the most widely used treatment for depression. However, current therapies are ineffective, have delayed benefit, or cause side effects in many patients. Here, we define a mechanism downstream of 5-HT1A receptors that mediates antidepressant-like behavior and is profoundly and selectively enhanced by genetic disruption of regulators of G protein signaling (RGS) activity at Gαi2. Animals rendered insensitive to RGS protein regulation through a mutation in Gαi2 (G184S) exhibited spontaneous antidepressant- and anxiolytic-like behaviors. Mice expressing RGS-insensitive Gαi2 also exhibited increased cortical and hippocampal phosphorylation of glycogen synthase kinase-3β, a constitutively active proapoptotic kinase that is inhibited through phosphorylation in response to serotonin, SSRIs, and 5-HT1 receptor agonists. Both behavioral and biochemical phenotypes were blocked by treatment with WAY 100635, a 5-HT1A–selective antagonist. RGS-insensitive mice were also 5–10 times more responsive to the antidepressant-like effects of the SSRI fluvoxamine and 5-HT1A–selective agonist 8-hydroxy-2-dipropylaminotetralin. In contrast, the antidepressant potency of agents acting through nonserotonergic mechanisms was unchanged as was 5-HT1A action on body temperature. The findings point to a critical role for endogenous RGS proteins to suppress the antidepressant-like effects of 5-HT1A receptor activation. By selectively enhancing the beneficial effects of serotonin, inhibition of RGS proteins represents a therapeutic approach for the treatment of mood disorders.

Antinociceptive and toxic effects of (+)-epibatidine oxalate attributable to nicotinic agonist activity

1 Epibatidine is an analgesic substance, isolated from the skin of the poisonous frog Epipedobates tricolor, for which the mechanism of action was previously unknown. 2 The IC50 of synthetic (+)‐epibatidine oxalate (the naturally occurring isomer) for [3H]‐nicotine binding to rat whole‐brain membranes was 0.1 nm. The (−)−isomer also exhibited high affinity (IC50 = 0.2 nm). 3 (+)‐ and (−)−Epibatidine exhibited much lower affinity for displacement of the muscarinic ligand [3H]‐N‐methylscopolamine binding to rat cortical membranes (Kapp − 6.9μm and 16.0 μm respectively). The (+)‐enantiomer of epibatidine had an antagonist/agonist (NMS/oxo‐M) binding ratio of 4.2 This is consistent with a muscarinic antagonist profile. 4 (+)‐Epibatidine oxalate (10 μm) did not cause significant (>30%) displacement of radioligand binding to opioid, excitatory amino acid, benzodiazepine, 5‐HT, dopamine, adrenaline or peptide receptors. 5 (+)‐ and (−)−Epibatidine (5–20 μg kg−1 s.c.) doubled response latency in the mouse hot‐plate test. Antinociception and behavioural depression induced by (+)‐epibatidine (5 μg kg−1) was fully blocked by the nicotinic antagonists mecamylamine (2 mg kg−1 s.c.) or dihydro‐β‐erythroidine (2 mg kg−1 s.c). The muscarinic antagonist scopolamine (0.4 and 10 mg kg−1 s.c.) caused partial reversal of antinociception induced by (+)‐epibatidine in mice, but not in rats. 6 These findings demonstrate that (+)‐epibatidine oxalate salt is a highly selective and potent nicotinic analgesic agent.

Orphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors

Orphanin FQ (OFQ), an endogenous peptide for ORL1 receptors, has been identified. Although the actions of OFQ have much in common with those of opioid peptides at the cellular level, behavioral studies in rodents seem conflicting. The aim of this study was to investigate the potential pronociceptive or antinociceptive function of peripheral ORL1 receptors in primates. Experiments were conducted to verify whether local administration of OFQ can attenuate capsaicin‐induced nociception and whether peripheral ORL1 receptors selectively mediate the local action of OFQ in monkeys. Capsaicin (100 μg) was administered subcutaneously in the tail to locally evoke a nociceptive response (thermal allodynia/hyperalgesia), which was manifested as a reduced tail‐withdrawal latency in normally innocuous 46°C warm water. Co‐administration of OFQ (1 – 30 μg) with capsaicin in the tail dose‐dependently inhibited thermal nociception. However, a locally effective dose of OFQ (30 μg), when applied in the back, did not inhibit capsaicin‐induced nociception. OFQ‐induced local antinociception was antagonized by a small dose (10 μg) of J‐113397, a selective ORL1 receptor antagonist, in the tail. Similarly, s.c. administration of 10 μg of J‐113397 in the back did not antagonize local antinociception of OFQ. In addition, s.c. administration of either OFQ or J‐113397 in the tail alone did not change its thermal nociceptive threshold. Local administration of opioid receptor antagonists selective for mu, kappa, and delta opioid receptors did not antagonize OFQ‐induced local antinociception. Local administration of J‐113397 also did not interfere with the local actions of mu, kappa, and delta opioid agonists in the tail. These results provide the first functional evidence that activation of peripheral ORL1 receptors produces thermal antinociception in primates and this action is independent of antinociception produced at classical opioid receptors.