The delta opioid receptor agonist KNT-127 relieves innate anxiety-like behavior in mice by suppressing transmission from the prelimbic cortex to basolateral amygdala.

AIM: Excitatory projections from the prelimbic cortex (PL) to the basolateral nucleus of the amygdala (BLA) are implicated in the regulation of anxiety-like behaviors, and we previously demonstrated that anxiolytic-like effects of the selective delta-opioid receptor (DOP) agonist KNT-127 is involved in suppressing glutamate neurotransmission in the PL. Here, we investigated the mechanisms underlying the anxiolytic-like effect of KNT-127 in mice by combining optogenetic stimulation of the PL-BLA pathway with behavioral analyses. METHODS: Four-week-old male C57BL/6J mice received bilateral administration of adeno-associated virus (AAV)2-CaMKIIa-hChR2(H134R)-enhanced yellow fluorescent protein (EYFP) into the PL to induce expression of the light-activated excitatory ionic channel ChR2. Subsequently, an optic fiber cannula connected to a wireless photo-stimulator was implanted into the BLA for optogenetic PL-BLA pathway stimulation. We evaluated innate anxiety using the elevated plus maze (EPM) and open field (OF) tests as well as learned anxiety using the contextual fear conditioning (CFC) test. RESULTS: Optogenetic activation of the PL-BLA pathway enhanced anxiety-like behaviors in the EPM and OF, while prior subcutaneous administration of KNT-127 (10 mg/kg) reduced this anxiogenic effect. In contrast, optogenetic activation of the PL-BLA pathway had no significant effect on conditioned fear. CONCLUSION: Our findings indicate that the PL-BLA circuit contributes to innate anxiety and that the anxiolytic-like effects of KNT-127 are mediated at least in part by suppression of PL-BLA transmission. The PL delta-opioid receptor may thus be an effective therapeutic target for anxiety disorders.

Identification and Pharmacological Characterization of a Low-Liability Antinociceptive Bifunctional MOR/DOR Cyclic Peptide.

Peptide-based opioid ligands are important candidates for the development of novel, safer, and more effective analgesics to treat pain. To develop peptide-based safer analgesics, we synthesized a mixture-based cyclic pentapeptide library containing a total of 24,624 pentapeptides and screened the mixture-based library samples using a 55 °C warm water tail-withdrawal assay. Using this phenotypic screening approach, we deconvoluted the mixture-based samples to identify a novel cyclic peptide Tyr-[D-Lys-Dap(Ant)-Thr-Gly] (CycloAnt), which produced dose- and time-dependent antinociception with an ED50 (and 95% confidence interval) of 0.70 (0.52-0.97) mg/kg i.p. mediated by the mu-opioid receptor (MOR). Additionally, higher doses (≥3 mg/kg, i.p.) of CycloAnt antagonized delta-opioid receptors (DOR) for at least 3 h. Pharmacological characterization of CycloAnt showed the cyclic peptide did not reduce breathing rate in mice at doses up to 15 times the analgesic ED50 value, and produced dramatically less hyperlocomotion than the MOR agonist, morphine. While chronic administration of CycloAnt resulted in antinociceptive tolerance, it was without opioid-induced hyperalgesia and with significantly reduced signs of naloxone-precipitated withdrawal, which suggested reduced physical dependence compared to morphine. Collectively, the results suggest this dual MOR/DOR multifunctional ligand is an excellent lead for the development of peptide-based safer analgesics.

Activation of the Mu-Delta Opioid Receptor Heteromers Blocks Morphine Rewarding Effects.

BACKGROUND: Evidence has accumulated demonstrating the existence of opioid receptor heteromers, and recent data suggest that targeting these heteromers could reduce opioid side effects while retaining therapeutic effects. Indeed, CYM51010 characterized as a MOR (mu opioid receptor)/DOR (delta opioid receptor) heteromer-preferring agonist promoted antinociception comparable with morphine but with less tolerance. In the perspective of developing these new classes of pharmacological agents, data on their putative side effects are mandatory. METHODS: Therefore, in this study, we investigated the effects of CYM51010 in different models related to drug addiction in mice, including behavioral sensitization, conditioned place preference and withdrawal. RESULTS: We found that, like morphine, CYM51010 promoted acute locomotor activity as well as psychomotor sensitization and rewarding effect. However, it induced less physical dependence than morphine. We also investigated the ability of CYM51010 to modulate some morphine-induced behavior. Whereas CYM51010 was unable to block morphine-induced physical dependence, it blocked reinstatement of an extinguished morphine induced-conditioned place preference. CONCLUSIONS: Altogether, our results reveal that targeting MOR-DOR heteromers could represent a promising strategy to block morphine reward.

Receptor expression and signaling properties in the brain, and structural ligand motifs that contribute to delta opioid receptor agonist-induced seizures.

The δ opioid receptor (δOR) is a therapeutic target for the treatment of various neurological disorders, such as migraines, chronic pain, alcohol use, and mood disorders. Relative to µ opioid receptor agonists, δOR agonists show lower abuse liability and may be potentially safer analgesic alternatives. However, currently no δOR agonists are approved for clinical use. A small number of δOR agonists reached Phase II trials, but ultimately failed to progress due to lack of efficacy. One side effect of δOR agonism that remains poorly understood is the ability of δOR agonists to produce seizures. The lack of a clear mechanism of action is partly driven by the fact that δOR agonists range in their propensity to induce seizure behavior, with multiple δOR agonists reportedly not causing seizures. There is a significant gap in our current understanding of why certain δOR agonists are more likely to induce seizures, and what signal-transduction pathway and/or brain area is engaged to produce these seizures. In this review we provide a comprehensive overview of the current state of knowledge of δOR agonist-mediated seizures. The review was structured to highlight which agonists produce seizures, which brain regions have been implicated and which signaling mediators have been examined in this behavior. Our hope is that this review will spur future studies that are carefully designed and aimed to solve the question why certain δOR agonists are seizurogenic. Obtaining such insight may expedite the development of novel δOR clinical candidates without the risk of inducing seizures. This article is part of the Special Issue on "Opioid-induced changes in addiction and pain circuits".

KNT-127, a selective delta opioid receptor agonist, shows beneficial effects in the hippocampal dentate gyrus of a chronic vicarious social defeat stress mouse model.

Delta opioid receptors (DOPs) play an important role in depression and other mood disorders. However, little is known about the underlying physiological mechanisms. The hypothalamic-pituitary-adrenal axis, adult hippocampal neurogenesis, and neuroinflammation are regarded as key pathophysiological factors in depression. In this study, we investigated the influence of DOP activation on those factors in a valid animal model of depression, chronic vicarious social defeat stress (cVSDS) mice. cVSDS mice (male C57BL/6J mice) were produced following a 10-day exposure to witness of social defeat stress, and each evaluation was performed more than 28 days after the stress period. Repeated administrations to cVSDS mice with a selective DOP agonist, KNT-127, both during (10 days) and after (28 days) the stress period respectively improved their decreased social interaction behaviors and increased serum corticosterone levels. When administered during the stress period, KNT-127 suppressed decreases in the hippocampal newborn neuron survival rate in cVSDS mice. Moreover, in both administration paradigms, KNT-127 reduced the number of Iba-1- and CD11b-positive cells in the subgranular zone and the granule cell layer of the hippocampal dentate gyrus, indicating a suppression of cVSDS-induced microglial overactivation. These results suggest that KNT-127 acts over the hypothalamic-pituitary-adrenal axis and regulates neurogenesis and neuroinflammation resulting in anti-stress effects, and the antidepressant-like effects of the DOP agonist are implicated in the suppression of the neuroinflammation. This study presents a new finding on the effects of repeated DOP activations on the pathophysiological states of depression.

CYX-5, a G-protein biassed MOP receptor agonist, DOP receptor antagonist and KOP receptor agonist, evokes constipation but not respiratory depression relative to morphine in rats.

BACKGROUND: Strong opioid analgesics such as morphine alleviate moderate to severe acute nociceptive pain (e.g. post-surgical or post-trauma pain) as well as chronic cancer pain. However, they evoke many adverse effects and so there is an unmet need for opioid analgesics with improved tolerability. Recently, a prominent hypothesis has been that opioid-related adverse effects are mediated by ß-arrestin2 recruitment at the µ-opioid (MOP) receptor and this stimulated research on discovery of G-protein biassed opioid analgesics. In other efforts, opioids with MOP agonist and δ-opioid (DOP) receptor antagonist profiles are promising for reducing side effects c.f. morphine. Herein, we report on the in vivo pharmacology of a novel opioid peptide (CYX-5) that is a G-protein biassed MOP receptor agonist, DOP receptor antagonist and kappa opioid (KOP) receptor agonist. METHODS: Male Sprague-Dawley received intracerebroventricular bolus doses of CYX-5 (3, 10, 20 nmol), morphine (100 nmol) or vehicle, and antinociception (tail flick) was assessed relative to constipation (charcoal meal and castor oil-induced diarrhoea tests) and respiratory depression (whole body plethysmography). RESULTS: CYX-5 evoked naloxone-sensitive, moderate antinociception, at the highest dose tested. Although CYX-5 did not inhibit gastrointestinal motility, it reduced stool output markedly in the castor oil-induced diarrhoea test. In contrast to morphine that evoked respiratory depression, CYX-5 increased tidal volume, thereby stimulating respiration. CONCLUSION: Despite its lack of recruitment of ß-arrestin2 at MOP, DOP and KOP receptors, CYX-5 evoked constipation, implicating a mechanism other than ß-arrestin2 recruitment at MOP, DOP and KOP receptors, mediating constipation evoked by CYX-5 and potentially other opioid ligands.

The Delta-Opioid Receptor Bidirectionally Modulates Itch.

Opioid signaling has been shown to be critically important in the neuromodulation of sensory circuits in the superficial spinal cord. Agonists of the mu-opioid receptor (MOR) elicit itch, whereas agonists of the kappa-opioid receptor (KOR) have been shown to inhibit itch. Despite the clear roles of MOR and KOR for the modulation itch, whether the delta-opioid receptor (DOR) is involved in the regulation of itch remained unknown. Here, we show that intrathecal administration of DOR agonists suppresses chemical itch and that intrathecal application of DOR antagonists is sufficient to evoke itch. We identify that spinal enkephalin neurons co-express neuropeptide Y (NPY), a peptide previously implicated in the inhibition of itch. In the spinal cord, DOR overlapped with both the NPY receptor (NPY1R) and KOR, suggesting that DOR neurons represent a site for convergent itch information in the dorsal horn. Lastly, we found that neurons co-expressing DOR and KOR showed significant Fos induction following pruritogen-evoked itch. These results uncover a role for DOR in the modulation of itch in the superficial dorsal horn. PERSPECTIVE: This article reveals the role of the delta-opioid receptor in itch. Intrathecal administration of delta agonists suppresses itch whereas the administration of delta antagonists is sufficient to induce itch. These studies highlight the importance of delta-opioid signaling for the modulation of itch behaviors, which may represent new targets for the management of itch disorders.

To probe the activation mechanism of the Delta opioid receptor by an agonist ADL5859 started from inactive conformation using molecular dynamic simulations.

The δ-opioid receptor (DOR) is a critical pharmaceutical target for pain management. Although the high-resolution crystal structures of the DOR with both agonist and antagonist have recently been solved, the activation mechanism remains to be elusive. In this study, a DOR agonist ADL5859 was docked to the inactive DOR and multiple microsecond molecular dynamic (MD) simulations were conducted to probe the activation mechanism. While the receptor with the crystal ligand (i.e. antagonist naltrindole) maintained the inactive conformation in all three independent simulations, the receptor with ADL5859 was adopting toward the active conformation in three out of six independent simulations. Major conformational differences were located on transmembrane (TM) 5 and 6, as well as intracellular loop 3. Compared to naltrindole, ADL5859 exhibited high conformational flexibility and strong interaction with the transmission switch. The putative key residues (W274, D95, V267, L139, V263, M142, T260, R146, R258 and others) involving in the activation pathway were identified through the conventional molecular switch analysis and a pairwise distance analysis, which provides a short list for experimental mutagenesis study. These insights will facilitate further development of therapeutic agents targeting the DOR.Communicated by Ramaswamy H. Sarma.

[Neural mechanism underlying the regulation of emotional behavior via δ-opioid receptors].

The delta opioid receptor (DOP) belongs to the G protein-coupled receptor family and is abundant in the limbic system. In recent years, consistent with their distribution, they have been suggested to be involved in the regulation of emotional behavior. In particular, DOP agonists have been shown to exhibit antidepressant and anxiolytic-like effects, and clinical trials are underway as targets for the development of new psychotropic drugs with mechanisms of action different from those of existing monoamine drugs. In this article, we review the roles and mechanisms of DOP in emotion regulation that are being elucidated in basic studies using rodents, and also introduce the current status of its clinical application.

Age-related changes in peripheral nociceptor function.

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4-5 months) and aged (26-27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E2 (PGE2). Intraplantar injection of TRP channel activators, capsaicin (TRPV1), mustard oil (TRPA1) and menthol (TRPM8) each resulted in greater mechanical allodynia in aged versus young rats and capsaicin-induced heat allodynia was also greater in aged rats. A treatment-induced allodynia that was greater in young rats was never observed. The anti-allodynic effects of intraplantar injection of kappa and delta opioid receptor agonists, salvinorin-A and D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, were greater in aged than young rats, whereas mu opioid receptor agonists, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine, were not effective in aged rats. Consistent with these observations, in primary cultures of peripheral sensory neurons, inhibition of cAMP signaling in response to delta and kappa receptor agonists was greater in cultures derived from aged rats. By contrast, mu receptor agonists did not inhibit cAMP signaling in aged rats. Thus, age-related changes in nociceptors generally favor increased pain signaling in aged versus young rats, suggesting that changes in nociceptor sensitivity may play a role in the increased incidence of pain in the elderly population. These results also suggest that development of peripherally-restricted kappa or delta opioid receptor agonists may provide safer and effective pain relief for the elderly.

Designer Benzodiazepines' Activity on Opioid Receptors: A Docking Study.

BACKGROUND: Previous studies have reported that benzodiazepines (BZDs) seem to enhance euphoric and reinforcing properties of opioids in opioid users so that a direct effect on opioid receptors has been postulated, together with a possible synergistic induction of severe side effects due to co use of BDZs and opioids. This is particularly worrisome given the appearance on the market of designer benzodiazepines (DBZDs), whose activity/toxicity profiles are scarcely known. OBJECTIVES: This study aimed to evaluate, through computational studies, the binding affinity (or lack thereof) of 101 DBZDs identified online on the kappa, mu, and delta opioid receptors (K, M, DOR); and to assess whether their mechanism of action could include activation of the latter. METHODS: MOE® was used for the computational studies. Pharmacophore mapping based on strong opioids agonist binders' 3D chemical features was used to filter the DBZDs. Resultant DBZDs were docked into the crystallised 3D active conformation of KOR (PDB6B73), DOR (PDB6PT3) and MOR (PDB5C1M). Co-crystallised ligands and four strong agonists were used as reference compounds. A score (S, Kcal/mol) representative of the predicted binding affinity, and a description of ligand interactions were obtained from MOE®. RESULTS: The docking results, filtered for S < -8.0 and the interaction with the Asp residue, identified five DBZDs as putative binders of the three ORs : ciclotizolam, fluloprazolam, JQ1, Ro 48-6791, and Ro 48-8684. CONCLUSION: It may be inferred that at least some DBZDs may have the potential to activate opioid receptors. This could mediate/increase their anxiolytic, analgesic, and addiction potentials, as well as worsen the side effects associated with opioid co-use.

Enhanced antidepressant-like effects of a delta opioid receptor agonist, SNC80, in rats under inflammatory pain.

Depression is a debilitating mental disorder that affects a large population worldwide. Depression and pain comorbidity is well recognized in both clinical and preclinical settings. Research studies suggest delta opioid receptor (DOR) may be involved in modulating pain as well as depression. DOR agonists produce antidepressant-like effects in animal models and their antihyperalgesic effects are enhanced in rats under inflammatory pain. However, it is unclear whether the antidepressant-like effects of DOR agonists may change in the models of pain. In this study, the antidepressant-like effects of a DOR agonist, SNC80, and a tricyclic antidepressant, amitriptyline, were compared following intracerebroventricular (i.c.v.) administration in rats under normal or inflammatory pain state elicited by injection of complete Freund's adjuvant. The forced swim test was used to determine the antidepressant-like effects. Results showed that i.c.v. SNC80 and amitriptyline dose-dependently produced antidepressant-like effects in rats under normal state. The potency of SNC80-induced antidepressant-like effects, but not amitriptyline, was enhanced in rats under inflammatory pain. This study provides functional evidence of the state-dependent effects of DOR agonists and suggests that DOR agonists may be more effective as potential antidepressants for patients experiencing both depression and pain.

Analgesic Opioid Ligand Discovery Based on Nonmorphinan Scaffolds Derived from Natural Sources.

Strong opioid analgesics, including morphine, are the mainstays for treating moderate to severe acute pain and alleviating chronic cancer pain. However, opioid-related adverse effects, including nausea or vomiting, sedation, respiratory depression, constipation, pruritus (itch), analgesic tolerance, and addiction and abuse liability, are problematic. In addition, the use of opioids to relieve chronic noncancer pain is controversial due to the "opioid crisis" characterized by opioid misuse or abuse and escalating unintentional death rates due to respiratory depression. Hence, considerable research internationally has been aimed at the "Holy Grail" of the opioid analgesic field, namely the discovery of novel and safer opioid analgesics with improved opioid-related adverse effects. In this Perspective, medicinal chemistry strategies are addressed, where structurally diverse nonmorphinan-based opioid ligands derived from natural sources were deployed as lead molecules. The current state of play, clinical or experimental status, and novel opioid ligand discovery approaches are elaborated in the context of retaining analgesia with improved safety and reduced adverse effects, especially addiction liability.

Positive allosteric modulation of endogenous delta opioid receptor signaling in the enteric nervous system is a potential treatment for gastrointestinal motility disorders.

Allosteric modulators (AMs) are molecules that can fine-tune signaling by G protein-coupled receptors (GPCRs). Although they are a promising therapeutic approach for treating a range of disorders, allosteric modulation of GPCRs in the context of the enteric nervous system (ENS) and digestive dysfunction remains largely unexplored. This study examined allosteric modulation of the delta opioid receptor (DOR) in the ENS and assessed the suitability of DOR AMs for the treatment of irritable bowel syndrome (IBS) symptoms using mouse models. The effects of the positive allosteric modulator (PAM) of DOR, BMS-986187, on neurogenic contractions of the mouse colon and on DOR internalization in enteric neurons were quantified. The ability of BMS-986187 to influence colonic motility was assessed both in vitro and in vivo. BMS-986187 displayed DOR-selective PAM-agonist activity and orthosteric agonist probe dependence in the mouse colon. BMS-986187 augmented the inhibitory effects of DOR agonists on neurogenic contractions and enhanced reflex-evoked DOR internalization in myenteric neurons. BMS-986187 significantly increased DOR endocytosis in myenteric neurons in response to the weakly internalizing agonist ARM390. BMS-986187 reduced the generation of complex motor patterns in the isolated intact colon. BMS-986187 reduced fecal output and diarrhea onset in the novel environment stress and castor oil models of IBS symptoms, respectively. DOR PAMs enhance DOR-mediated signaling in the ENS and have potential benefit for the treatment of dysmotility. This study provides proof of concept to support the use of GPCR AMs for the treatment of gastrointestinal motility disorders.NEW & NOTEWORTHY This study assesses the use of positive allosteric modulation as a pharmacological approach to enhance opioid receptor signaling in the enteric nervous system. We demonstrate that selective modulation of endogenous delta opioid receptor signaling can suppress colonic motility without causing constipation. We propose that allosteric modulation of opioid receptor signaling may be a therapeutic strategy to normalize gastrointestinal motility in conditions such as irritable bowel syndrome.

Morphinan derivatives with an oxabicyclo[3.2.1]octane structure as dual agonists toward δ and κ opioid receptors.

The κ opioid receptor (KOR) is one of the promising targets to develop analgesics lacking morphine like side effects. To seek a novel KOR agonist we designed 6-amide derivatives with an oxabicyclo[3.2.1]octane structure based on a proposed active conformation of a selective KOR agonist nalfurafine. All the synthesized compounds strongly bound to the KOR and some compound showed KOR selectivities. 6R-Amides were more potent and efficacious KOR agonists than the corresponding 6S-isomers. However, most 6-amide derivatives were partial KOR agonist. Conformational analyses of 6R- and 6S-amide derivatives and nalfurafine well accounted for the difference of KOR agonistic activities between two diastereomers. Surprisingly, the tested N-H amides were full δ opioid receptor (DOR) agonists. Among the tested compounds 7a with benzamide moiety was the most potent dual DOR/KOR agonist. On the other hand, 6S-phenylacetamide 8b was potent full DOR agonist with less efficacious agonist activity for the µ receptor and KOR. 6-Amide derivatives with an oxabicyclo[3.2.1]octane structure were expected to be a promising fundamental skeleton for the dual DOR/KOR agonists and/or selective DOR agonists.

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 ß-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit ß-arrestin, as it has been suggested that compounds that efficaciously recruit ß-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.

Characterization of the Synergistic Effect between Ligands of Opioid and Free Fatty Acid Receptors in the Mouse Model of Colitis.

BACKGROUND: Recent studies suggest that lipids, including free fatty acids (FFAs), are necessary for proper µ opioid receptor (MOR) binding and that activation of opioid receptors (ORs) improves intestinal inflammation. The objective of the study was to investigate a possible interaction between the ORs and FFA receptors (FFARs) ligands in the colitis. METHODS: The potential synergistic effect of ORs and FFARs ligands was evaluated using mouse model of acute colitis induced by dextran sulfate sodium (DSS, 4%). Compounds were injected intraperitoneally (i.p.) once or twice daily at the doses of 0.01 or 0.02 mg/kg body weight (BW) (DAMGO-an MOR agonist), 0.3 mg/kg BW (DPDPE-a δ OR (DOR) agonist) and 1 mg/kg BW (naloxone-a non-selective OR antagonist, GLPG 0974-a FFAR2 antagonist, GSK 137647-a FFAR4 agonist and AH 7614-a FFAR4 antagonist) for 4 days. RESULTS: Myeloperoxidase (MPO) activity was significantly decreased after DAMGO (0.02 mg/kg BW) and GSK 137647 (1 mg/kg BW) administration and co-administration as compared to DSS group. CONCLUSIONS: Treatment with ligands of ORs and FFARs may affect the immune cells in the inflammation; however, no significant influence on the severity of colitis and no synergistic effect were observed.

Discovery of Novel Delta Opioid Receptor (DOR) Inverse Agonist and Irreversible (Non-Competitive) Antagonists.

The delta opioid receptor (DOR) is a crucial receptor system that regulates pain, mood, anxiety, and similar mental states. DOR agonists, such as SNC80, and DOR-neutral antagonists, such as naltrindole, were developed to investigate the DOR in vivo and as potential therapeutics for pain and depression. However, few inverse agonists and non-competitive/irreversible antagonists have been developed, and none are widely available. This leaves a gap in our pharmacological toolbox and limits our ability to investigate the biology of this receptor. Thus, we designed and synthesized the novel compounds SRI-9342 as an irreversible antagonist and SRI-45128 as an inverse agonist. These compounds were then evaluated in vitro for their binding affinity by radioligand binding, their functional activity by 35S-GTPγS coupling, and their cAMP accumulation in cells expressing the human DOR. Both compounds demonstrated high binding affinity and selectivity at the DOR, and both displayed their hypothesized molecular pharmacology of irreversible antagonism (SRI-9342) or inverse agonism (SRI-45128). Together, these results demonstrate that we have successfully designed new inverse agonists and irreversible antagonists of the DOR based on a novel chemical scaffold. These new compounds will provide new tools to investigate the biology of the DOR or even new potential therapeutics.

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.

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

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the ß-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® ß-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® ß-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.