ERNEST COST action overview on the (patho)physiology of GPCRs and orphan GPCRs in the nervous system.

G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. This overview emphasises the GPCRs of the nervous system, which are the research focus of the members of ERNEST COST action (CA18133) working group 'Biological roles of signal transduction'.

The κ-opioid receptor-induced autophagy is implicated in stress-driven synaptic alterations.

Recent evidence has shown that G protein-coupled receptors (GPCRs) are direct sensors of the autophagic machinery and opioid receptors regulate neuronal plasticity and neurotransmission with an as yet unclarified mechanism. Using and experimental approaches, this study aims to clarify the potential role of autophagy and κ-opioid receptor (κ-OR) signaling in synaptic alterations. We hereby demonstrate that the selective κ-OR agonist U50,488H, induces autophagy in a time-and dose-dependent manner in Neuro-2A cells stably expressing the human κ-OR by upregulating microtubule-associated protein Light Chain 3-II (LC3-II), Beclin 1 and Autophagy Related Gene 5 (ATG5).

LP1 and LP2: Dual-Target MOPr/DOPr Ligands as Drug Candidates for Persistent Pain Relief.

Although persistent pain is estimated to affect about 20% of the adult population, current treatments have poor results. Polypharmacology, which is the administration of more than one drug targeting on two or more different sites of action, represents a prominent therapeutic approach for the clinical management of persistent pain. Thus, in the drug discovery process the "one-molecule-multiple targets" strategy nowadays is highly recognized.

Novel -Substituted Benzomorphan-Based Compounds: From MOR-Agonist/DOR-Antagonist to Biased/Unbiased MOR Agonists.

Modifications at the basic nitrogen of the benzomorphan scaffold allowed the development of compounds able to segregate physiological responses downstream of the receptor signaling, opening new possibilities in opioid drug development. Alkylation of the phenyl ring in the -substituent of the MOR-agonist/DOR-antagonist LP1 resulted in retention of MOR affinity. Moreover, derivatives , , and were biased MOR agonists toward ERK1,2 activity stimulation, whereas derivative was a low potency MOR agonist on adenylate cyclase inhibition.

A highly conserved δ-opioid receptor region determines RGS4 interaction.

The δ-opioid receptor (δ-OR) couples to Gi/Go proteins to modulate a variety of responses in the nervous system. Τhe regulator of G protein signalling 4 (RGS4) was previously shown to directly interact within the C-terminal region of δ-OR using its N-terminal domain to negatively modulate opioid receptor signalling. Herein, using molecular dynamics simulations and in vitro pull-down experiments we delimit this interaction to 12 helix 8 residues of δ-ΟR and to the first 17 N-terminal residues (NT) of RGS4.

Synthesis and Structure-Activity Relationships of (-)---Normetazocine-Based LP1 Derivatives.

(−)---Normetazocine represents a rigid scaffold able to mimic the tyramine moiety of endogenous opioid peptides, and the introduction of different -substituents influences affinity and efficacy of respective ligands at MOR (mu opioid receptor), DOR (delta opioid receptor), and KOR (kappa opioid receptor). We have previously identified LP1, a MOR/DOR multitarget opioid ligand, with an -phenylpropanamido substituent linked to (−)---Normetazocine scaffold. Herein, we report the synthesis, competition binding and calcium mobilization assays of new compounds ⁻ that differ from LP1 by the nature of the -substituent.

Synthesis and Structure-Activity Relationships of LP1 Derivatives: N-Methyl-N-phenylethylamino Analogues as Novel MOR Agonists.

The opioid pharmacological profile of -(-)--normetazocine derivatives is deeply affected by the nature of their -substituents. Here, our efforts were focused on the synthesis and pharmacological evaluation of novel derivatives of the lead LP1, a multitarget opioid analgesic compound featuring an -phenylpropanamido substituent. LP1 derivatives - and - were characterized by flexible groups at the -substituent that allow them to reposition themselves relative to -(-)--normetazocine nucleus, thus producing different pharmacological profiles at the mu, delta and kappa opioid receptors (MOR, DOR and KOR) in in vitro and in vivo assays.

Development of novel LP1-based analogues with enhanced delta opioid receptor profile.

Pain relief achieved by co-administration of drugs acting at different targets is more effective than that obtained with conventional MOR selective agonists usually associated to relevant side effects. It has been demonstrated that simultaneously targeting different opioid receptors is a more effective therapeutic strategy. Giving the promising role for DOR in pain management, novel LP1-based analogues with different N-substituents were designed and synthesized with the aim to improve DOR profile.

A novel regulatory role of RGS4 in STAT5B activation, neurite outgrowth and neuronal differentiation.

The Regulator of G protein Signalling 4 (RGS4) is a multitask protein that interacts with and negatively modulates opioid receptor signalling. Previously, we showed that the δ-opioid receptor (δ-OR) forms a multiprotein signalling complex consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription 5B (STAT5B) that leads to neuronal differentiation and neurite outgrowth upon δ-ΟR activation. Here, we investigated whether RGS4 could participate in signalling pathways to regulate neurotropic events.

Prognostic significance of signal transducer and activator of transcription 5 and 5b expression in Epstein-Barr virus-positive patients with chronic lymphocytic leukemia.

Signal transducer and activator of transcription (STAT) proteins have been intensively studied in hematologic malignancies, and the efficacy of agents against STATs in lymphomas is already under research. We investigated the expression of total STAT5 and STAT5b in peripheral blood samples of patients with chronic lymphocytic leukemia (CLL) in correlation with the presence of Epstein-Barr Virus (EBV) and its major oncoprotein (latent membrane protein 1, LMP1). The EBV load was measured in the peripheral blood by real-time PCR for the BXLF1 gene and the levels of LMP1 by PCR and ELISA.

Evaluation of N-substituent structural variations in opioid receptor profile of LP1.

The benzomorphan scaffold has great potential as lead structure and the nature of the N-substituent is able to influence affinity, potency, and efficacy at all three opioid receptors. Building upon these considerations, we synthesized a new series of LP1 analogues by introducing naphthyl or heteroaromatic rings in propanamide side chain of its N-substituent (9-15). In vitro competition-binding assays in HEK293 cells stably expressing MOR, DOR or KOR showed that in compound 9 the 1-naphthyl ring led to the retention of MOR affinity (Ki(MOR)=38±4nM) displaying good selectivity versus DOR and KOR.

RGS2 and RGS4 proteins: New modulators of the κ-opioid receptor signaling.

Previous studies have shown that RGS4 associates with the C-termini of μ- and δ-opioid receptors in living cells and plays a key role in Gi/Go protein coupling selectivity and signalling of these receptors [12,20]. To deduce whether similar effects also occur for the κ-opioid receptor (κ-ΟR) and define the ability of members of the Regulators of G protein Signaling (RGS) of the B/R4 subfamily to interact with κ-ΟR subdomains we generated glutathione S-transferase fusion peptides encompassing the carboxyl-termini of κ-OR (κ-CT). Results from pull down experiments indicated that RGS2 and RGS4 directly interact within different domains of the κ-CT.

δ-opioid receptor activation leads to neurite outgrowth and neuronal differentiation via a STAT5B-Gαi/o pathway.

It remains unclear how opioid receptors (δ, μ, κ) are implicated in mechanisms controlling differentiation, cell proliferation, and survival. Opioid receptors are coupled to Gi/Go proteins and recent findings have shown that opioid receptors can form a multicomponent signaling complex, consisting of members of G protein and the signal transducer and activator of transcription (STAT)5B. We thus wondered whether activation of the opioid receptors could direct differentiation and neurite outgrowth through a molecular pathway involving STAT5B and other signaling intermediates.

Selective interactions of spinophilin with the C-terminal domains of the δ- and μ-opioid receptors and G proteins differentially modulate opioid receptor signaling.

Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners (Leontiadis et al., 2009, Cell Signal. 21, 1218-1228; Georganta et al.

The benzomorphan-based LP1 ligand is a suitable MOR/DOR agonist for chronic pain treatment.

Aims: Powerful analgesics relieve pain primarily through activating mu opioid receptor (MOR), but the long-term use of MOR agonists, such as morphine, is limited by the rapid development of tolerance. Recently, it has been observed that simultaneous stimulation of the delta opioid receptor (DOR) and MOR limits the incidence of tolerance induced by MOR agonists. 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) is a centrally acting agent with antinociceptive activity comparable to morphine and is able to bind and activate MOR and DOR.

Superpotent [Dmt¹] dermorphin tetrapeptides containing the 4-aminotetrahydro-2-benzazepin-3-one scaffold with mixed μ/δ opioid receptor agonistic properties.

Novel dermorphin tetrapeptides are described in which Tyr(1) is replaced by Dmt(1), where d-Ala(2) and Gly(4) are N-methylated, and where Phe(3)-Gly(4) residue is substituted by the constrained Aba(3)-Gly(4) peptidomimetic. Most of these peptidic ligands displayed binding affinities in the nanomolar range for both μ- and δ-opioid receptors but no detectable affinity for the κ-opioid receptor. Measurements of cAMP accumulation, phosphorylation of extracellular signal-regulated kinase (ERK1/2) in HEK293 cells stably expressing each of these receptors individually, and functional screening in primary neuronal cultures confirmed the potent agonistic properties of these peptides.

The other side of opioid receptor signalling: regulation by protein-protein interaction.

Opiate drugs mediate their analgesic, euphoriant, and rewarding effects by activating opioid receptors. Pharmacological and molecular studies have demonstrated the existence of three opioid receptor subtypes, μ, δ, and κ- that couple predominantly to Gi/Go types of G proteins to regulate the activity of a diverse array of effector systems. Ample experimental evidence has demonstrated that these receptors can physically interact with a variety of accessory proteins, confirming that signal transduction of the opioid receptors is not restricted to heterotrimeric G protein activation.

Expression and membrane topology of Anopheles gambiae odorant receptors in lepidopteran insect cells.

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel 'topology screen' assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell.

Evaluation of N-substitution in 6,7-benzomorphan compounds.

6,7-benzomorphan derivatives, exhibiting different mu, delta, and kappa receptor selectivity profiles depending on the N-substituent, represent a useful skeleton for the synthesis of new and better analgesic agents. In this work, an aromatic ring and/or alkyl residues have been used with an N-propanamide or N-acetamide spacer for the synthesis of a new series of 5,9-dimethyl-2'-hydroxy-6,7-benzomorphan derivatives (12-22). Data obtained by competition binding assays showed that the mu opioid receptor seems to prefer an interaction with the 6,7-benzomorphan ligands having an N-substituent with a propanamide spacer and less hindered amide.

Multi-component signaling complexes of the delta-opioid receptor with STAT5B and G proteins.

Besides mediating opioid responses in the nervous system and the peripheral tissues, opioid receptors are implicated in signaling mechanisms shared by cytokine receptors. Recent observations have shown that the Signal Transducer and Activator of Transcription 5A (STAT5A) interacts with the mu-opioid receptor (mu-OR) and is phosphorylated upon mu-OR stimulation (Mazarakou and Georgoussi, 2005). In the present study we demonstrate that another member of the STAT family, STAT5B, associates constitutively with the C-terminal tail of the delta-opioid receptor (delta-CT).

Regulator of G protein signaling 4 confers selectivity to specific G proteins to modulate mu- and delta-opioid receptor signaling.

In vitro studies have shown that the Regulator of G protein Signaling 4 (RGS4) interacts with the C-termini of mu- and delta-opioid receptors (mu-OR, delta-OR) (Georgoussi et al., 2006, Cell. Signal.

Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery.

Insect cell-based expression systems are prominent amongst current expression platforms for their ability to express virtually all types of heterologous recombinant proteins. Stably transformed insect cell lines represent an attractive alternative to the baculovirus expression system, particularly for the production of secreted and membrane-anchored proteins. For this reason, transformed insect cell systems are receiving increased attention from the research community and the biotechnology industry.

Expression of the third intracellular loop of the delta-opioid receptor inhibits signaling by opioid receptors and other G protein-coupled receptors.

To explore the feasibility of developing inhibitors of signaling by opioid receptors and other G protein-coupled receptors (GPCRs) that use the same G protein pool, we investigated the capacity of a minigene encoding the third intracellular loop of the delta-opioid receptor (delta-i3L) to act as competitive antagonist of the receptor-G protein interface interaction. In delta-i3L-expressing cells, the peptide blocked high-affinity agonist binding to both the delta- and the mu-opioid (delta-OR and mu-OR) and attenuated opioid and alpha2-adrenergic receptor (alpha2AR)-dependent [35S]guanosine-5'-O-(3-thio)triphosphate binding. Furthermore, delta-i3L expression resulted in inhibition of delta-, mu-OR-, and alpha2AR-receptor-mediated cAMP accumulation, whereas the cAMP response produced by activation of the beta2-adrenergic receptor was unaffected, suggesting that the inhibitory effects of delta-i3L expression were selective for Gi/Go proteins.