Interactive Responses of Enkephalin, δ-opioid Receptor and Dopamine Receptor D1/D2 to Hypoxia and/or MPP+ Stress in PC12 Cells

Hypoxic/ischemic brain injury is a potential etiology of Parkinson’s disease (PD). There is evidence suggesting that the up-regulation of enkephalin, an endogenous opioid, in the midbrain may have a compensatory effect against Parkinson’s disease (PD) related motor symptoms. To explore the potential mechanism underlying this action, we investigated the effects of hypoxia and MPP+, a pathological inducer PD, on enkephalin, δ-opioid receptor (DOR, an enkephalin receptor), and prohormone convertases 1 and 2 (PC1/PC2) on in- vitro PD model of PC12 cells. We found that (1) short-term hypoxia could inducing cell protection by up-regulating the level of enkephalin, accompanied by the synergistic up-regulation of δ-opioid receptor (DOR) ; (2) a longer period of hypoxia or MPP+ insult accelerated the proteolysis of proenkephalin by up-regulating PC1/PC2 which might produce more active enkephalin and thus activating DOR for cell protection; (3) The levels of enkephalin and DOR decreased significantly after a prolonged hypoxia or MPP+ insult; and (4) a certain degree of hypoxia improved cell viability and enhance the transcription of dopamine D1/D2 receptorby increasing their mRNA level. Our findings suggest that hypoxia may induce an interactive reaction of enkephalin, DOR and dopamine receptor D1/D2, which is potentially beneficial for cell surviving to severe/prolonged hypoxia and PD condition.

Hydrogen Sulfide Inhibits Inflammatory Pain and Enhances the Analgesic Properties of Delta Opioid Receptors

Chronic inflammatory pain is present in many pathologies and diminishes the patient’s quality of life. Moreover, most current treatments have a low efficacy and significant side effects. Recent studies demonstrate the analgesic properties of slow-releasing hydrogen sulfide (H2S) donors in animals with osteoarthritis or neuropathic pain, but their effects in inflammatory pain and related pathways are not completely understood. Several treatments potentiate the analgesic actions of δ-opioid receptor (DOR) agonists, but the role of H2S in modulating their effects and expression during inflammatory pain remains untested. In C57BL/6J male mice with inflammatory pain provoked by subplantar injection of complete Freund’s adjuvant, we evaluated: (1) the antiallodynic and antihyperalgesic effects of different doses of two slow-releasing H2S donors, i.e., diallyl disulfide (DADS) and phenyl isothiocyanate (P-ITC) and their mechanism of action; (2) the pain-relieving effects of DOR agonists co-administered with H2S donors; (3) the effects of DADS and P-ITC on the oxidative stress and molecular changes caused by peripheral inflammation. Results demonstrate that both H2S donors inhibited allodynia and hyperalgesia in a dose-dependent manner, potentiated the analgesic effects and expression of DOR, activated the antioxidant system, and reduced the nociceptive and apoptotic pathways. The data further demonstrate the possible participation of potassium channels and the Nrf2 transcription factor signaling pathway in the pain-relieving activities of DADS and P-ITC. This study suggests that the systemic administration of DADS and P-ITC and local application of DOR agonists in combination with slow-releasing H2S donors are two new strategies for the treatment of inflammatory pain.

Identification of a Novel Delta Opioid Receptor Agonist Chemotype with Potential Negative Allosteric Modulator Capabilities

The δ-opioid receptor (δOR) holds great potential as a therapeutic target. Yet, clinical drug development, which has focused on δOR agonists that mimic the potent and selective tool compound SNC80 have largely failed. It has increasingly become apparent that the SNC80 scaffold carries with it potent and efficacious β-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit β-arrestin, as it has been suggested that compounds that efficaciously recruit β-arrestin are proconvulsant. The screen identified a hit compound and further characterization using cellular binding and signaling assays revealed that this molecule (R995045, compound 1) exhibited ten-fold selectivity over µ- and κ-opioid receptors. Compound 1 represents a novel chemotype at the δOR. A subsequent characterization of fourteen analogs of compound 1, however did not identify a more potent δOR agonist. Computational modeling and in vitro characterization of compound 1 in the presence of the endogenous agonist leu-enkephalin suggest compound 1 may also bind allosterically and negatively modulate the potency of Leu-enkephalin to inhibit cAMP, acting as a ‘NAM-agonist’ in this assay. The potential physiological utility of such a class of compounds will need to be assessed in future in vivo assays.

In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

Delta Opioid Receptor Agonists Ameliorate Colonic Inflammation by Modulating Immune Responses

The opioid receptors play important roles in the regulation of sense and emotions. Although it is recently revealed that opioid receptors are also expressed in various cells, but not restricted in the central nervous system, the effects of opioids on peripheral immune cells are largely unknown. In the current study, we evaluated the effect of opioids on immune system by using selective agonists for δ opioid receptor. Systemic administration of KNT-127 or intraperitoneal injection of YNT-2715 (a KNT-127-related compound that cannot pass through the blood-brain barrier) significantly alleviated the pathology of dextran sodium sulfate-induced colitis. In KNT-127-treated mice, the levels of an inflammatory cytokine IL-6 in the serum, and macrophages in the mesenteric lymph nodes (MLNs) were decreased in the progression stage, and those of regulatory T cells (Tregs) in the MLN were increased in the recovery stage. In vitro experiments revealed that KNT-127 inhibited the release of IL-6 and another inflammatory cytokine TNF-α from macrophages and accelerated the development of Tregs. Our study suggests that δ opioid agonists act directly on immune cells to improve the pathology of the colitis and can be candidates of immunomodulatory drugs.

Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional μ/δ-Opioid Agonist–Neurokinin Antagonist Peptidomimetics

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic μ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) and the dual μ/δ opioid agonist H-Dmt-d-Arg-Aba-βAla-NH2 (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range μ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.

β-Arrestin–dependent ERK signaling reduces anxiety-like and conditioned fear–related behaviors in mice

G protein–coupled receptors (GPCRs) are implicated in the regulation of fear and anxiety. GPCR signaling involves canonical G protein pathways but can also engage downstream kinases and effectors through scaffolding interactions mediated by β-arrestin. Here, we investigated whether β-arrestin signaling regulates anxiety-like and fear-related behavior in mice in response to activation of the GPCR δ-opioid receptor (δOR or DOR). Administration of β-arrestin–biased δOR agonists to male C57BL/6 mice revealed β-arrestin 2–dependent activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2) in the dorsal hippocampus and amygdala and β-arrestin 1–dependent activation of ERK1/2 in the nucleus accumbens. In mice, β-arrestin–biased agonist treatment was associated with reduced anxiety-like and fear-related behaviors, with some overlapping and isoform-specific input. In contrast, applying a G protein–biased δOR agonist decreased ERK1/2 activity in all three regions as well as the dorsal striatum and was associated with increased fear-related behavior without effects on baseline anxiety. Our results indicate a complex picture of δOR neuromodulation in which β-arrestin 1– and 2–dependent ERK signaling in specific brain subregions suppresses behaviors associated with anxiety and fear and opposes the effects of G protein–biased signaling. Overall, our findings highlight the importance of noncanonical β-arrestin–dependent GPCR signaling in the regulation of these interrelated emotions.