SR-17018 Stimulates Atypical µ-Opioid Receptor Phosphorylation and Dephosphorylation.

Opioid-associated overdoses and deaths due to respiratory depression are a major public health problem in the US and other Western countries. In the past decade, much research effort has been directed towards the development of G-protein-biased µ-opioid receptor (MOP) agonists as a possible means to circumvent this problem. The bias hypothesis proposes that G-protein signaling mediates analgesia, whereas ß-arrestin signaling mediates respiratory depression. SR-17018 was initially reported as a highly biased µ-opioid with an extremely wide therapeutic window. It was later shown that SR-17018 can also reverse morphine tolerance and prevent withdrawal via a hitherto unknown mechanism of action. Here, we examined the temporal dynamics of SR-17018-induced MOP phosphorylation and dephosphorylation. Exposure of MOP to saturating concentrations of SR-17018 for extended periods of time stimulated a MOP phosphorylation pattern that was indistinguishable from that induced by the full agonist DAMGO. Unlike DAMGO-induced MOP phosphorylation, which is reversible within minutes after agonist washout, SR-17018-induced MOP phosphorylation persisted for hours under otherwise identical conditions. Such delayed MOP dephosphorylation kinetics were also found for the partial agonist buprenorphine. However, buprenorphine, SR-17018-induced MOP phosphorylation was fully reversible when naloxone was included in the washout solution. SR-17018 exhibits a qualitative and temporal MOP phosphorylation profile that is strikingly different from any other known biased, partial, or full MOP agonist. We conclude that detailed analysis of receptor phosphorylation may provide novel insights into previously unappreciated pharmacological properties of newly synthesized MOP ligands.

Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors.

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with ß-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.

Modulation of µ-opioid receptor activation by acidic pH is dependent on ligand structure and an ionizable amino acid residue.

BACKGROUND AND PURPOSE: Adverse side effects of conventional opioids can be avoided if ligands selectively activate peripheral opioid receptors in injured tissue. Injury and inflammation are typically accompanied by acidification. In this study, we examined influences of low pH and mutation of the ionizable amino acid residue H2976.52 on µ-opioid receptor binding and signalling induced by the µ-opioid receptor ligands fentanyl, DAMGO, and naloxone. EXPERIMENTAL APPROACH: HEK 293 cells stably transfected with µ-opioid receptors were used to study opioid ligand binding, [35 S]-GTPγS binding, and cAMP reduction at physiological and acidic pH. We used µ-opioid receptors mutated at H2976.52 to A (MOR-H2976.52 A) to delineate ligand-specific interactions with H2976.52 . KEY RESULTS: Low pH and the mutant receptor MOR-H2976.52 A impaired naloxone binding and antagonism of cAMP reduction. In addition, DAMGO binding and G-protein activation were decreased under these conditions. Fentanyl-induced signalling was not influenced by pH and largely independent of H2976.52 . CONCLUSIONS AND IMPLICATIONS: Our investigations indicate that low pH selectively impairs µ-opioid receptor signalling modulated by ligands capable of forming hydrogen bonds with H2976.52 . We propose that protonation of H2976.52 at acidic pH reduces binding and subsequent signalling of such ligands. Novel agonists targeting opioid receptors in injured tissue might benefit from lack of hydrogen bond formation with H2976.52 .

Antinociceptive activity of thiazole-containing cyclized DAMGO and Leu-(Met) enkephalin analogs.

Numerous studies demonstrate the promise of opioid peptides as analgesics, but poor oral bioavailability has limited their therapeutic development. This study sought to increase the oral bioavailability of opioid peptides by cyclization, using Hantzsch-based macrocyclization strategies to produce two new series of cyclized DAMGO and Leu/Met-enkephalin analogs. Opioid receptor affinity and selectivity for compounds in each series were assessed in vitro with radioligand competition binding assays. Compounds demonstrated modest affinity but high selectivity for the mu, delta, and kappa opioid receptors (MOR, DOR and KOR), while selectivity for mu opioid receptors varied by structure. Antinociceptive activity of each compound was initially screened in vivo following intracerebroventricular (i.c.v.) administration and testing in the mouse 55 °C warm-water tail-withdrawal test. The four most active compounds were then evaluated for dose- and time-dependent antinociception, and opioid receptor selectivity in vivo. Cyclic compounds 1924-10, 1936-1, 1936-7, and 1936-9 produced robust and long- lasting antinociception with ED50 values ranging from 0.32-0.75 nmol following i.c.v. administration mediated primarily by mu- and delta-opioid receptor agonism. Compounds 1924-10, 1936-1 and 1936-9 further displayed significant time-dependent antinociception after oral (10 mg kg-1, p.o.) administration. A higher oral dose (30 mg kg-1. p.o.) of all four cyclic peptides also reduced centrally-mediated respiration, suggesting successful penitration into the CNS. Overall, these data suggest cyclized opioid peptides synthesized by a Hantzsch-based macrocyclization strategy can retain opioid agonist activity to produce potent antinociception in vivo while conveying improved bioavailability following oral administration.

All-Electronic Quantification of Neuropeptide-Receptor Interaction Using a Bias-Free Functionalized Graphene Microelectrode.

Opioid neuropeptides play a significant role in pain perception, appetite regulation, sleep, memory, and learning. Advances in understanding of opioid peptide physiology are held back by the lack of methodologies for real-time quantification of affinities and kinetics of the opioid neuropeptide-receptor interaction at levels typical of endogenous secretion (<50 pM) in biosolutions with physiological ionic strength. To address this challenge, we developed all-electronic opioid-neuropeptide biosensors based on graphene microelectrodes functionalized with a computationally redesigned water-soluble µ-opioid receptor. We used the functionalized microelectrode in a bias-free charge measurement configuration to measure the binding kinetics and equilibrium binding properties of the engineered receptor with [d-Ala2, N-MePhe4, Gly-ol]-enkephalin and ß-endorphin at picomolar levels in real time.

Pharmacokinetic considerations of nanodelivery to the brain: Using modeling and simulations to predict the outcome of liposomal formulations.

The use of nanocarriers is an intriguing solution to increase the brain delivery of novel therapeutics. The aim of this paper was to use pharmacokinetic analysis and simulations to identify key factors that determine the effective drug concentration-time profile at the target site in the brain. Model building and simulations were based on experimental data obtained from the administration of the opioid peptide DAMGO in glutathione tagged PEGylated liposomes to rats. Different pharmacokinetic models were investigated to explore the mechanisms of increased brain delivery. Concentration-time profiles for a set of formulations with varying compound and carrier characteristics were simulated. By controlling the release rate from the liposome, the time profile and the extent of brain delivery can be regulated. The modeling did not support a mechanism of the liposomes passing the brain endothelial cell membrane in an intact form through endocytosis or transcytosis. The most likely process was found to be fusion of the liposome with the endothelial luminal membrane. The simulations revealed that low permeable compounds, independent on efflux, will gain the most from a nanocarrier formulation. The present model based approach is useful to explore and predict possibilities and limitations of carrier-based systems to the brain.

Agonist stimulation at human µ opioid receptors in a [(35)S]GTPγS incorporation assay: observation of "bell-shaped" concentration-response relationships under conditions of strong receptor G protein coupling.

CONTEXT: The appearance of "bell"- (or "inverted U"-) shaped agonist concentration-response curves (CRCs) in in vitro pharmacological experiments is a frequently observed but poorly communicated phenomenon. In the context of G protein coupled receptor research, it is commonly attributed to the recruitment of secondary targets or to desensitization or feedback processes, but the concrete background of these observations often remains intriguing. OBJECTIVE: Here, we addressed the subject of bell-shaped agonist CRCs at the µ opioid receptor (µOR) by testing the impact of experimental conditions favoring G protein coupling. METHODS: G protein activation by recombinant human µORs heterologously expressed in CHO cells was assessed in [(35)S]GTPγS binding assays using the opioid ligands DAMGO, morphine, fentanyl and naloxone. Experimental conditions were varied by changing the NaCl (10-300 mM) and the GDP concentration (0.3-30 µM). RESULTS: Both the sodium and the GDP concentration were inversely related to G protein coupling, as evident by an increase in basal [(35)S]GTPγS incorporation at low sodium and low GDP levels and by the concomitant appearance of the partial agonist activity of the µOR antagonist, naloxone. Bell-shaped CRCs were observed for the efficacious agonists DAMGO, fentanyl and morphine, and this phenomenon was promoted by low sodium as well as by low GDP concentrations. CONCLUSION: µOR agonist CRCs show a non-monotonic behavior with a decline of maximal stimulation under conditions of strong receptor-G protein coupling, and this behavior is visible at the level of G protein activation itself.

Activation and Allosteric Modulation of Human µ Opioid Receptor in Molecular Dynamics.

Allosteric protein modulation has gained increasing attention in drug design. Its application as a mechanism of action could bring forth safer and more effective medicines. Targeting opioid receptors with allosteric modulators can result in better treatment of pain, depression, and respiratory and immune disorders. In this work we use recent reports on negative modulators of µ opioid receptor as a starting point for identification of allosteric sites and mechanisms of opioid receptor modulation using homology modeling and docking and molecular dynamics studies. An allosteric binding site description is presented. Results suggest a shared binding region for lipophilic allosteric ligands, reveal possible differences in the modulation mechanism between cannabinoids and salvinorin A, and show ambiguous properties of the latter. Also, they emphasize the importance of native-like environment in molecular dynamics simulations and uncover relationships between modulator and orthosteric ligand binding and receptor behavior. Relationships between ligands, transmission switch, and hydrophobic lock are analyzed.

In vitro and in vivo efficacy of a potent opioid receptor agonist, biphalin, compared to subtype-selective opioid receptor agonists for stroke treatment.

To meet the challenge of identification of new treatments for stroke, this study was designed to evaluate a potent, nonselective opioid receptor (OR) agonist, biphalin, in comparison to subtype selective OR agonists, as a potential neuroprotective drug candidate using in vitro and in vivo models of ischemic stroke. Our in vitro approach included mouse primary neuronal cells that were challenged with glutamate and hypoxic/aglycemic (H/A) conditions. We observed that 10nM biphalin, exerted a statistically significant neuroprotective effect after glutamate challenge, compared to all selective opioid agonists, according to lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, 10nM biphalin provided superior neuroprotection after H/A-reoxygenation compared to selective opioid agonists in all cases. Our in vitro investigations were supported by in vivo studies which indicate that the nonselective opioid agonist, biphalin, achieves enhanced neuroprotective potency compared to any of the selective opioid agonists, evidenced by reduced edema and infarct ratios. Reduction of edema and infarction was accompanied by neurological improvement of the animals in two independent behavioral tests. Collectively these data strongly suggest that concurrent agonist stimulation of mu, kappa and delta ORs with biphalin is neuroprotective and superior to neuroprotection by activation of any single OR subtype.

Solubilization and reconstitution of the mu-opioid receptor expressed in human neuronal SH-SY5Y and CHO cells.

The zwitterionic detergent CHAPS was used to solubilize the human mu-opioid receptor (hMOR) from SH-SY5Y neuroblastoma cells and recombinant hMOR-CHO (CHO-T7-hMOR) and hMOR-SH-SY5Y (SH-SY5Y-T7-hMOR) cell membranes. Agonist stimulation and G-protein activation by the mu-selective opioid agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) were recovered after removing of CHAPS after polyethylene glycol (PEG) precipitation. Binding assays show that hMOR solubilized and reconstituted this way was functional and able to interact with both agonist peptides and with G-protein. The effective solubilization and reconstitution of hMOR from mammalian cells, without truncation and extensive modification, represent an essential step toward the purification of a receptor bearing important post-translational modifications.

Hybrid peptides endomorphin-2/DAMGO: design, synthesis and biological evaluation.

Endomorphin-2 [Tyr-Pro-Phe-Phe-NH2] and DAMGO [Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol] are natural (EM2) and synthetic (DAMGO) opioid peptides both selective for µ opioid receptor with high analgesic activity. In this work we report synthesis, in vitro and in vivo biological evaluation of five new hybrid EM2/DAMGO analogues, with the aim to obtain compounds with high affinity at µ-opioid receptor, high activity in animal nociception tests (hot plate and tail flick) and improved enzymatic stability. Double N-methylation on both Phe residues and C-terminal ethanolamide led to analogue 6e, which possesses a good in vitro µ affinity (Kiµ=34 nM), combined with a remarkable in vivo antinociceptive activity.

Conformational analysis of endomorphin-2 analogs with phenylalanine mimics by NMR and molecular modeling.

We investigated a series of conformations of endomorphin-2 (EM-2) analogs substituted by phenylglycine (Phg) and homophenylalanine (Hfe) in the position 3 or 4 by two-dimensional (1)H NMR spectroscopy and molecular modeling. Evaluating the aromatic interactions and the dihedral angles in these phenylalanine mimics, we have observed that the conformations in trans isomer have varied from extended to folded as bioactivity decreases. It is suggested that the flexibility of aromatic side chain affects the backbone of EM-2 to adopt folded structures, which may block the ligands in binding to micro-opioid receptor.

Distribution of (3)H-dopamine and (3)H-DAGO binding sites in the central part of rat sinoatrial node.

Distribution of (3)H-dopamine and (3)H-DAGO binding sites was studied by autoradiography on semithin sections of total preparations of rat sinoatrial node. The relative density of (3)H-dopamine and (3)H-DAGO binding sites in the functional nucleus of the sinoatrial node was minimum and increased in the cranial and caudal directions. The level of (3)H-dopamine binding in the perinodal atrial myocardium was appreciably lower (22+/-6%), while binding of (3)H-DAGO was similar (76+/-16%) to that in the periarterial zone of the sinoatrial node.

Stereochemical requirements for receptor recognition of the mu-opioid peptide endomorphin-1.

A series of diastereoisomers of endomorphin-1 (EM1, Tyr(1)-Pro(2)-Trp(3)-Phe(4)-NH(2)) have been synthesized and their potency measured using the guinea pig ileum assay. [D-Phe(4)]EM1 possessed 1/10 the potency of EM1, while potencies of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 were 50- and 100-fold lower, respectively. Drastic loss of activity occurred in the [D-Pro(2)]EM1 peptide. The structural determinants for the inactivity and reduced potency of the diastereoisomers were investigated using NMR spectroscopy and conformational analysis. Simulations of trans-[D-Pro(2)]EM1 using NOE-derived distance constraints afforded well-defined structures in which Tyr and Trp side chains stack against the proline ring. The inactivity of [D-Pro(2)]EM1 was explained by structural comparison with EM1 (, FEBS Lett. 439:13-20). The two peptides showed an opposite orientation of the Trp(3) residue with respect to Tyr(1), thus suggesting a role of Pro(2) as a stereochemical spacer in orienting Trp(3) and Phe(4) toward regions suitable for mu-receptor interaction. The agonist activity of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 was attributed to their ability to adopt low-energy conformations that mimic those of EM1. The requirements for mu-receptor activation were examined further by comparing EM1 with the mu-peptide [D-Ala(2), MePhe(4), Gly-ol]-enkephalin (DAMGO). Conformations of DAMGO with a Tyr(1)-MePhe(4) phenyl ring separation of approximately 12 A were found to mimic Tyr(1)-Phe(4) of EM1, thus suggesting overlapping binding modes between these two peptides.