Rational design, synthesis and characterization of novel chimeric peptides containing the kappa-opioid agonists CR845 and dynorphin A-derived peptides.

Pharmacological characterization of EN-9, a novel chimeric peptide of endomorphin-2 and neuropeptide FF that produces potent antinociceptive activity and limited tolerance

Thank you for the opportunity to comment on the letter by Smith et al. and to clarify some recurring misconceptions regarding the pharmacology of oxycodone.Oxycodone is a potent opioid, comparable to morphine, when given systemically. However, as Smith et al. also write, oxycodone has a significantly lower affinity for the μ-opioid receptor compared with morphine. This has been shown in several studies.1,2Indeed, the results of our own recent study3further support the findings that oxycodone has a significantly lower potency compared with morphine when administrated directly to the central nervous system in rats.4,5Smith et al. challenge our study and those by many others by arguing that the antinociceptive effect of oxycodone is mediated trough the κ-opioid receptor. The previous in vivo studies clearly demonstrate that oxycodone is a μ-opioid receptor agonist.1,2,6,7In these studies, the affinity of oxycodone for the κ-opioid receptor is remarkably lower than for the μ-opioid receptor.2,6To the best of our knowledge, not a single in vitro study (binding- or G-protein activation) showing κ-opioid receptor agonism of oxycodone has been published.Beardsley et al. 8studied the pharmacology of oxycodone in mice, rats, and rhesus monkeys. In an excellent article, they reported that oxycodone had potent antinociceptive effects in the mouse paraphenylquinone writhing, hot-plate, and tail-flick assays acting as a μ-opioid receptor agonist. The selective opioid receptor antagonists were studied in the mice tail-flick test. The antinociceptive effect of oxycodone (subcutaneous administration) was only blocked by the selective μ-opioid receptor antagonist β-funaltrexamine (intracerebroventricular administration). The selective κ- and δ-opioid receptor antagonists nor-binaltorphimine (administered subcutaneously 2 h before the agonist) and naltrindole (subcutaneous administration) were not able to block the antinociceptive effect of oxycodone.8Beardsley et al. wrote, “The results have shown that oxycodone is a robust antinociceptive agent, with an abuse liability profile consistent with full μ-opioid receptor agonists.”8Oxycodone completely suppressed signs of withdrawal in morphine-dependent rhesus monkeys. In the previous studies, the selective κ-opioid receptor agonists did not suppress signs of morphine withdrawal.9,10Beardsley et al. 8also demonstrated that even very high doses of oxycodone did not induce behavior (salivation or diuresis) indicative of κ-opioid like activity. Therefore, high-quality classic pharmacology experiments clearly show that oxycodone is a μ-opioid receptor agonist, not a κ-opioid receptor agonist.Smith et al. agree that oxymorphone is a potent μ-opioid receptor agonist.11In our recent study,3nor-binaltorphimine (administered 30 min before study drugs) was not able to antagonize the antinociceptive effect of oxymorphone or oxycodone. Should nor-binaltorphimine behave as a μ-opioid receptor antagonist when given 30 min before the study drugs, as suggested by Smith et al. , the antinociceptive effect of oxymorphone should have been significantly attenuated. The antinociceptive effect of oxymorphone, like that of oxycodone, was prevented only by naloxone, not by nor-binaltorphimine.In our recent study,3oxycodone showed weaker activation of the spinal μ-opioid receptors compared with morphine. The mechanisms behind this difference are interesting and will be studied further. In the brain, oxycodone activated the μ-opioid receptors, albeit to a lesser extent than morphine. The reason why oxycodone produces more potent antinociception compared with morphine after systemic administration remains to be clarified. Smith et al. argue that we are suggesting that the analgesic effects of systemic oxycodone are due to oxymorphone. This is not what we concluded. We suggested that the metabolites may have a role in oxycodone-induced analgesia. We agree that the circulating concentrations of oxymorphone after systemic administration of oxycodone are low, as we12–14and others15have shown. Because systemic oxycodone causes potent μ-opioid receptor agonist effects while being a weak μ-opioid receptor agonist with insignificant binding to other opioid receptors, pharmacokinetic explanations must be considered. One possibility is that the access of either oxycodone itself and/or some of its active metabolites to the central nervous system are more effective compared with that of morphine. There are a number of oxycodone metabolites produced in oxidative and reductive reactions that have not been studied in vivo . Oxymorphone, on the other hand, is an interesting spinal analgesic, and it has recently been launched as an oral analgesic, too.Oxycodone binds to the μ-opioid receptor and activates the μ-opioid receptor, whereas it does not bind to the κ-opioid receptor and does not activate the κ-opioid receptor. Importantly, in human beings, oxycodone behaves as a μ-opioid receptor agonist producing analgesia, euphoria, dependence, and other typical μ-opioid effects. Oxycodone does not cause psychotomimetic effects, dysphoria, diuresis, or other effects typical for a κ-opioid agonist. Several aspects of oxycodone pharmacology still need to be studied. However, it is obvious that oxycodone is a μ-opioid receptor agonist, not a κ-opioid receptor agonist.*University of Helsinki, Helsinki, Finland. kim.lemberg@helsinki.fi

Ondansetron is effective for the treatment of intrathecal morphine-induced pruritus. There is evidence that kappa-opioid receptor agonists have antipruritic activity. Pentazocine is an agonist of kappa-opioid receptors and partial agonist at mu-opioid receptors. We therefore performed a randomized, double-blind trial to compare the efficacy of pentazocine and ondansetron for the treatment of pruritus associated with intrathecal injection of morphine in patients undergoing cesarean delivery. Two hundred eight parturients who developed moderate to severe pruritus after the administration of intrathecal morphine were randomly allocated to 2 groups: IV pentazocine 15 mg (n = 104) and IV ondansetron 4 mg (n = 104). The successful treatment of pruritus (no or mild pruritus) and other adverse effects were determined 15 min after study drug administration, and patients were observed for recurrence of pruritus for 4 h. The treatment success rate at 15 min was higher in the pentazocine group (96.1%) than in the ondansetron group (80.8%) (95% confidence interval of difference: 7.0%, 23.8%; P = 0.001). The recurrence rate of moderate to severe pruritus within 4 h after treatment in the pentazocine group (12.0%) was lower than in the ondansetron group (32.1%) (P = 0.001). There were no significant differences between groups in nausea/vomiting, sedation, shivering, pain scores, and pain at injection site. No respiratory depression was observed. Pentazocine 15 mg is superior to ondansetron 4 mg for the treatment of intrathecal morphine-induced pruritus and has a lower recurrence rate. The side effects after treatment are mild.

Chronic pruritus is frequently refractory to currently available treatments. Studies suggest that pruritus may arise from an imbalance of the mu- and kappa-opioid receptor system activity in either the skin or the central nervous system. Stimulation of kappa-opioid receptors by their agonists inhibits pruritus in both animals and humans. The antipruritic effect of kappa-opioid receptors agonists can currently be assumed to be related to their binding to kappa-opioid receptors on keratinocytes and cutaneous and/or central itch neurones. To date, several case reports and 2 controlled trials have demonstrated a beneficial effect of systemic kappa-opioid receptor agonists in the treatment of uraemic pruritus, prurigo nodularis, paraneoplastic and cholestatic pruritus. Nalfurafine hydrochloride (Remitch(®)), a selective kappa-opioid receptor agonist, is approved for the treatment of chronic pruritus in Japan. The aim of this review is to provide an overview of the promising role of kappa- opioid receptors and their agonist in the pathophysiology and treatment of pruritus.

<p>Chronic pain causes patients to endure prolonged suffering and discomfort, often having profound effects on quality of life. In New Zealand, one in five people currently suffer from chronic pain. To treat chronic pain, patients are typically prescribed drugs that activate the mu opioid receptor (MOPr), such as morphine, codeine and oxycodone. In recent years in the United States of America, there has been a rapid increase in the use of prescription and non-prescription opioid drugs, with opioid overdoses now the leading cause of accidental death. In New Zealand, daily doses of prescription opioids quadrupled in the ten year period from 2001-2011. Clearly, there is a need for the development of more effective and safe medications. This thesis evaluated two classes of non-addictive compounds: bioactive lipids and kappa opioid receptor (KOPr) agonists. N-docosahexaenoyl ethanolamine (DHEA) is an N-acyl ethanolamine class lipid that is structurally similar to the endocannabinoid anandamide. DHEA has previously been shown to have immune-modulatory effects in vitro, however, the in vivo effects have not previously been tested. Using the intraplantar 2% formaldehyde model in mice, DHEA reduced inflammatory and nociceptive pain via both intraperitoneal (i.p.) and local intraplantar (i.pl.) administration. DHEA significantly reduced formaldehyde-induced footpad oedema and reduced the infiltration of neutrophils into the inflamed tissue. The antinociceptive and anti-oedematous effects were not modulated by pre-treatment with either cannabinoid 1- or 2-type receptor antagonists. DHEA did not have any effect in a thermal nociceptive pain model and did not show any motor coordination impairment or changes in thermoregulation. In the search for non-addictive analgesics, KOPr agonists are a promising alternative. In contrast to MOPr agonists, KOPr agonists play a critical role in regulating the reward system. Salvinorin A (SalA) is a selective KOPr agonist that has antinociceptive and anti-inflammatory effects in vivo, with limited abuse potential. However, the short duration of action and aversive side effects limit the clinical usefulness. The present study aimed to investigate the antinociceptive effects of acute administration of novel analogues of SalA. In the dose-response tail withdrawal assay, SalA and the novel analogues 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail withdrawal assay and the hot plate test compared to SalA. In the intraplantar 2% formaldehyde test, SalA, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced nociceptive pain and inflammatory pain, effects which were reversed by the KOPr antagonist nor-binaltorphimine. SalA, 16-Ethynyl SalA and 16-Bromo SalA reduced paw oedema and reduced the infiltration of neutrophils into the inflamed tissue. However, SalA, 16-Ethynyl SalA and 16-Bromo SalA produced motor incoordination effects. However, 16-Ethynyl SalA did not alter thermoregulation. The KOPr agonists were further assessed in a model of paclitaxel-induced neuropathic pain. In the acute dose-response experiment, 16-Ethynyl SalA was significantly more potent at reducing mechanical allodynia compared to morphine in both male and female mice. SalA and 16-Ethynyl SalA were more potent at reducing cold allodynia than morphine. In a chronic administration model over 22 days, for the treatment of cold and mechanical allodynia, all of the opioid treatments reduced pain, however, the traditional KOPr agonist U50,488, was the most potent, by reducing the male mechanical allodynia and cold allodynia in both sexes back to baseline levels. The ultrastructure of the sciatic nerves were studied, however, it was found that U50,488 did not reverse the effects of paclitaxel on myelin degeneration and mitochondrial damage. Overall, this study has identified DHEA as a modest treatment for inflammatory pain, with reduced side effects and a mechanism of action in contrast to other compounds with a similar structure. The novel KOPr agonists had significant effects in acute pain models with longer duration of action than the parent compound SalA. This is the first known study to investigate the effects of KOPr agonists in a paclitaxel-induced neuropathic pain model, showing that KOPr agonists are a potential therapeutic avenue for this debilitating condition.</p>

<p>Chronic pain causes patients to endure prolonged suffering and discomfort, often having profound effects on quality of life. In New Zealand, one in five people currently suffer from chronic pain. To treat chronic pain, patients are typically prescribed drugs that activate the mu opioid receptor (MOPr), such as morphine, codeine and oxycodone. In recent years in the United States of America, there has been a rapid increase in the use of prescription and non-prescription opioid drugs, with opioid overdoses now the leading cause of accidental death. In New Zealand, daily doses of prescription opioids quadrupled in the ten year period from 2001-2011. Clearly, there is a need for the development of more effective and safe medications. This thesis evaluated two classes of non-addictive compounds: bioactive lipids and kappa opioid receptor (KOPr) agonists. N-docosahexaenoyl ethanolamine (DHEA) is an N-acyl ethanolamine class lipid that is structurally similar to the endocannabinoid anandamide. DHEA has previously been shown to have immune-modulatory effects in vitro, however, the in vivo effects have not previously been tested. Using the intraplantar 2% formaldehyde model in mice, DHEA reduced inflammatory and nociceptive pain via both intraperitoneal (i.p.) and local intraplantar (i.pl.) administration. DHEA significantly reduced formaldehyde-induced footpad oedema and reduced the infiltration of neutrophils into the inflamed tissue. The antinociceptive and anti-oedematous effects were not modulated by pre-treatment with either cannabinoid 1- or 2-type receptor antagonists. DHEA did not have any effect in a thermal nociceptive pain model and did not show any motor coordination impairment or changes in thermoregulation. In the search for non-addictive analgesics, KOPr agonists are a promising alternative. In contrast to MOPr agonists, KOPr agonists play a critical role in regulating the reward system. Salvinorin A (SalA) is a selective KOPr agonist that has antinociceptive and anti-inflammatory effects in vivo, with limited abuse potential. However, the short duration of action and aversive side effects limit the clinical usefulness. The present study aimed to investigate the antinociceptive effects of acute administration of novel analogues of SalA. In the dose-response tail withdrawal assay, SalA and the novel analogues 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail withdrawal assay and the hot plate test compared to SalA. In the intraplantar 2% formaldehyde test, SalA, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced nociceptive pain and inflammatory pain, effects which were reversed by the KOPr antagonist nor-binaltorphimine. SalA, 16-Ethynyl SalA and 16-Bromo SalA reduced paw oedema and reduced the infiltration of neutrophils into the inflamed tissue. However, SalA, 16-Ethynyl SalA and 16-Bromo SalA produced motor incoordination effects. However, 16-Ethynyl SalA did not alter thermoregulation. The KOPr agonists were further assessed in a model of paclitaxel-induced neuropathic pain. In the acute dose-response experiment, 16-Ethynyl SalA was significantly more potent at reducing mechanical allodynia compared to morphine in both male and female mice. SalA and 16-Ethynyl SalA were more potent at reducing cold allodynia than morphine. In a chronic administration model over 22 days, for the treatment of cold and mechanical allodynia, all of the opioid treatments reduced pain, however, the traditional KOPr agonist U50,488, was the most potent, by reducing the male mechanical allodynia and cold allodynia in both sexes back to baseline levels. The ultrastructure of the sciatic nerves were studied, however, it was found that U50,488 did not reverse the effects of paclitaxel on myelin degeneration and mitochondrial damage. Overall, this study has identified DHEA as a modest treatment for inflammatory pain, with reduced side effects and a mechanism of action in contrast to other compounds with a similar structure. The novel KOPr agonists had significant effects in acute pain models with longer duration of action than the parent compound SalA. This is the first known study to investigate the effects of KOPr agonists in a paclitaxel-induced neuropathic pain model, showing that KOPr agonists are a potential therapeutic avenue for this debilitating condition.</p>

ABSTRACTNalfurafine is the only clinically approved kappa opioid receptor (KOPr) agonist that can cross the blood–brain barrier and exert CNS effects. Because its clinical use is not associated with dysphoria, it is widely believed to have an atypical pharmacological profile. Nalfurafine's atypical properties are proposed to result from its G‐protein‐biased KOPr agonist property, leading to the widespread use of nalfurafine as a nonaversive KOPr agonist in preclinical research. The validity of nonaversive claims for nalfurafine was investigated in mice by comparing its antinociceptive and aversive effects with those of the typical, nonbiased KOPr agonist U50,488 in tail withdrawal and conditioned place aversion (CPA) tests. Dose responses for tail withdrawal with nalfurafine and U50,488 were determined in warm (52°C) water in adult male and female C57BL/6J mice. Doses of U50,488 produced antinociception from 5 mg/kg, and doses of nalfurafine from 0.06 mg/kg. Four‐fold lower doses of either KOPr agonist (U50,488: 1.25 mg/kg; nalfurafine: 0.015 mg/kg) were subthreshold for antinociception. No sex differences were seen. Antinociceptive effects were fully blocked by the KOPr antagonist norBNI (10 mg/kg). Antinociceptive doses of nalfurafine (0.06 mg/kg) and U50,488 (5.0 mg/kg) both induced CPA. Subantinociceptive doses of nalfurafine (0.015 mg/kg) and U50,488 (1.25 mg/kg) were nonaversive in CPA. Thus, in mice, at doses that are antinociceptive, CPA was evident for both KOPr agonists. Neither nalfurafine nor U50,488 showed a separation between their antinociceptive and aversive effects, contradicting the hypothesis that nalfurafine is a nonaversive analgesic in mice. The findings caution against assuming nalfurafine is a nonaversive KOPr agonist for use in preclinical research.

Sequence Dependent Efficiency of Cross-Presentation in MHC Class I Requires Rational Design of Long Synthetic Peptides for Vaccination or Ex Vivo Activation

Discovery of N-methyltetrahydroprotoberberines with κ-opioid receptor agonists-opioid receptor agonist activities from corydalis yanhusuo W. T. Wang by using two-dimensional liquid chromatography

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric ‘bias’ into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists. eLife digest About a third of all drugs work by targeting a group of proteins known as G-protein coupled receptors, or GPCRs for short. These receptors are found on the surface of cells and transmit messages across the cell’s outer barrier. When a signaling molecule, like a hormone, is released in the body, it binds to a GPCR and changes the receptor’s shape. The change in structure affects how the GPCR interacts and binds to other proteins on the inside of the cell, triggering a series of reactions that alter the cell’s activity. Scientists have previously seen that a GPCR can trigger different responses depending on which signaling molecule is binding on the surface of the cell. However, the mechanism for this is unknown. One hypothesis is that different signaling molecules change the GPCR’s preference for binding to different proteins on the inside of the cell. The challenge has been to observe this happening without interfering with the process. Stoeber et al. have now tested this idea by attaching fluorescent tags to proteins that bind to activated GPCRs directly and without binding other signaling proteins. This meant these proteins could be tracked under a microscope as they made their way to bind to the GPCRs. Stoeber et al. focused on one particular GPCR, known as the opioid receptor, and tested the binding of two different opioid signaling molecules, etorphine and Dynorphin A. The experiments revealed that the different opioids did affect which of the engineered proteins would preferentially bind to the opioid receptor. This was followed by a similar experiment, where the engineered proteins were replaced with another protein called GRK2, which binds to the opioid receptor under normal conditions in the cell. This showed that GRK2 binds much more strongly to the opioid receptor when Dynorphin A is added compared to adding etorphine. These findings show that GPCRs can not only communicate that a signaling molecule is binding but can respond differently to convey what molecule it is more specifically. This could be important in developing drugs, particularly to specifically trigger the desired response and reduce side effects. Stoeber et al. suggest that an important next step for research is to understand how the GPCRs preferentially bind to different proteins. Introduction G protein-coupled receptors (GPCRs) comprise nature’s largest family of signaling receptors and an important class of therapeutic drug targets. GPCRs signal by allostery, and were considered for many years to operate as binary switches that bind to cognate transducer and regulator proteins in a single agonist-induced activated state. Over the past decade an expanded view has taken hold, supported by accumulating in vitro evidence that GPCRs are conformationally flexible (Lohse and Hofmann, 2015; Mahoney and Sunahara, 2016; Nygaard et al., 2013; Weis and Kobilka, 2018; Wingler et al., 2019) and a confluence of cell biological and in vivo evidence supporting the existence of functionally selective agonist effects (Smith et al., 2018; Urban et al., 2007; Williams et al., 2013). According to this still-evolving view, agonists have the potential to promote GPCRs to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at a receptor-proximal level that is either propagated downstream or eliminated during intermediate transduction steps (Lau et al., 2011; Tsvetanova et al., 2017). Opioid receptors provide a representative example. Interest in selective agonist effects at these GPCRs dates back to the initial demonstration that opioid receptors can be activated by diverse peptide and non-peptide agonists (Kosterlitz and Hughes, 1977). Early experimental evidence for such selectivity among ligands emerged from the observation of an agonist-induced state of opioid receptors in neuroblastoma cells that discriminates between opioid peptides and opiate alkaloids (Von Zastrow et al., 1993). This was followed by the demonstration of agonist-selective control of opioid receptor endocytosis, leading to the identification of functional selectivity among agonists defined by differences in relative ability to drive receptor engagement of G protein relative to beta-arrestin-dependent cellular pathways (Keith et al., 1998; Keith et al., 1996; Whistler et al., 1999; Whistler and von Zastrow, 1998). This concept further evolved to the present view of biased receptor recruitment of G proteins relative to beta-arrestins, with receptor-proximal selectivity calculated by fitting quantitative measures of downstream pathway or protein response to operational models of receptor-effector coupling (Schmid et al., 2017). Two key gaps persist in our present understanding. First, selective protein recruitment by GPCRs in intact cells remains largely calculated rather than directly observed. Accordingly, the understanding of receptor-proximal agonist bias is inherently limited by assumptions of the model used to calculate it (Kenakin, 2018; Klein Herenbrink et al., 2016). Indeed, and despite intense efforts motivated by interest in the therapeutic impact of biased agonist effects at opioid receptors (Johnson et al., 2017; Schmid et al., 2017; Whistler et al., 1999), significant challenges remain in reliably assessing selectivity of receptor-proximal protein recruitment based on downstream cell-based readouts (Conibear and Kelly, 2019). Second, challenges can arise even using cell-based assays that are direct. For example, multiple methods have been developed to detect GPCR interaction with beta-arrestins in intact cells (Chen et al., 2012; Kim et al., 2017). However, this binding involves multiple biochemical steps and, in particular, it typically requires the receptor to undergo prior agonist-induced phosphorylation (Eichel et al., 2018; Gurevich et al., 1995; Thomsen et al., 2016). This has been clearly established for opioid receptors (Whistler and von Zastrow, 1998; Zhang et al., 1998), for which full interaction with beta-arrestin requires the receptor to be phosphorylated at multiple sites in the cytoplasmic tail through a defined sequence of agonist-dependent reactions which are catalyzed by distinct GPCR kinase (GRK) isoforms (Chiu et al., 2017; Just et al., 2013; Lau et al., 2011; Miess et al., 2018). Accordingly, beta-arrestin recruitment measured in such assays clearly reflects a process that is considerably more complex than allosteric selection by the receptor. Here we describe an alternative approach to address these knowledge gaps. We delineate a cell-based method to simply assess selective protein recruitment by opioid receptors at the receptor-proximal level, taking advantage of two engineered protein folds established to bind agonist-activated GPCRs in intact cells without requiring or engaging other known cellular proteins (Stoeber et al., 2018; Wan et al., 2018). Using these engineered proteins comparatively as orthogonal receptor-interaction biosensors, we directly demonstrate selectivity in receptor-proximal protein recruitment elicited by various opioid agonists in living cells. We then show how the principle of receptor-proximal protein selection applies in a more complex manner to GRK2, a biologically relevant regulator. Results Comparative detection of direct protein recruitment by opioid receptors in living cells Two agonist-activated opioid receptor complexes have been described in structural detail (Figure 1A), one bound to a nucleotide-free G protein heterotrimer and another to an active state-stabilizing nanobody (Nb) (Huang et al., 2015; Koehl et al., 2018). The receptor conformation resolved in each complex is similar but not identical, with Nb and G protein interactions involving distinct molecular contacts on cytoplasmic domains of the receptor. Nbs are inherently orthogonal to intracellular biochemistry but heterotrimeric G proteins engage multiple cellular proteins in addition to activated receptors. Thus we focused on mini-G (mG) proteins, engineered versions of the Ras-like domain of G protein alpha subunits which bind directly to activated GPCRs but are not known to engage other cellular proteins (Nehmé et al., 2017; Wan et al., 2018). We assessed binding to receptors in intact cells by redistribution of fluorescently labeled Nb or mG fusion proteins from the cytoplasm to the plasma membrane (Figure 1B). Figure 1 Download asset Open asset Comparative detection of direct probe recruitment by opioid receptors in living cells. (A) Crystal structures of the DAMGO-bound MOR (red) - Gi (green/blue) complex (PDB: 6DDF) and the BU27-bound MOR (red) – nanobody (green) complex (PDB: 5C1M). Ligands are shown in blue. (B) Schematic of nanobody (Nb)/miniGsi (mGsi) and OR localization in cells and expected probe re-localization upon agonist addition. (C) Scheme of a cell imaged by total internal reflection fluorescence microscopy (TIR-FM). The evanescent excitation field selectively illuminates fluorophores close to the plasma membrane. (D) TIR-FM images of a time series of a HEK293 cell, expressing Venus-mGsi and FLAG-KOR (not shown). Medium was exchanged to DynA (agonist, 100 nM) and to 5’GNTI (antagonist, 100 μM) by bath application. The scale bar represents 10 μm. Intensity of mGsi and KOR (labeled with anti-FLAG M1-AF647) during the TIR-FM time-lapse. 5 s between frames is shown. F0, average fluorescence intensity before agonist. (E) Same as in (D) but with HEK293 cell expressing EGFP-Nb33 instead of mGsi. Intensity of Nb33 and KOR during TIR-FM time-lapse with 5 s between frames is shown. (F) Intensity of mGs and KOR (labeled with anti-FLAG M1-AF647) during the TIR-FM time-lapse, adding increasing concentrations of DynA (1 nM - 10 μM). 5 s between frames is shown. F0, average fluorescence intensity before agonist. (G) mGsi intensity during TIR-FM time-lapse series of a HEK293 cell, co-expressing Venus-mGsi and KOR, adding increasing concentrations of U69 (1 nM - 10 μM) followed by reference compound DynA (10 μM). 5 s between frames is shown. Intensity is normalized between 0 (no agonist) and 1 (reference compound DynA). (H) Same as in (F) with HEK293 cell expressing EGFP-Nb33 instead of mGsi. (I–K) Concentration-dependent recruitment of mGsi and Nb33 probes to KOR, measured by TIR-FM upon different agonists. Normalization of intensity values is shown (range [0–1]). Regression curves with Hill slope of 1 are shown. (I) DynA concentration response (n = 3; average ± SEM). (J) U69 concentration response, normalized to DynA (n = 3; average ± SEM). (K) U50 concentration response, normalized to DynA (n = 4; average ± SEM). Figure 1—source data 1 Concentration-dependent recruitment of mGsi and Nb33 probes to KOR in response to DynA, U69, and U50 (Figure 1I-K). https://cdn.elifesciences.org/articles/54208/elife-54208-fig1-data1-v1.csv Download elife-54208-fig1-data1-v1.csv For a mG probe we chose mGsi, derived from the Ras-like domain of Gs alpha but with nine residues at the distal C-terminus replaced by the corresponding residues from Gi alpha1. These C-terminal residues form a major determinant of G protein coupling specificity (Conklin et al., 1993) by folding into a helical structure (alpha-5 helix) that occupies the agonist-activated GPCR core (Carpenter and Tate, 2017; Koehl et al., 2018). Because Gs couples poorly to opioid receptors, we reasoned that a sensor derived from mGsi would primarily detect this interaction. For a Nb probe we selected Nb33, previously used to detect activated mu (MOR) and delta (DOR) opioid receptors in living cells (Stoeber et al., 2018). Nb33 shares receptor contact residues with Nb39, a close analog that has been resolved at high resolution in complex with activated MOR (Huang et al., 2015) and in a similar complex with activated kappa opioid receptor (KOR) (Che et al., 2018). Because cytoplasmic residues contacted by the Nb in these structures are largely distinct from those engaged by the G protein alpha-5 helix, we reasoned that the Nb-derived sensor has the potential to provide different allosteric information. Fluorescent protein fusions of mGsi or Nb33 localized diffusely when expressed in the cytoplasm of HEK293 cells, and recruitment by receptors was monitored using total internal reflection fluorescence microscopy (TIR-FM) in cells co-expressing Flag-tagged KOR (Figure 1C). Importantly, HEK293 cells do not express endogenous opioid receptors or other opioid ligand binding sites, thereby providing a null genetic background on which to directly examine protein probe recruitment mediated specifically by the co-expressed receptor. We observed rapid and robust recruitment of mGsi by KOR upon application of the kappa-selective peptide agonist Dynorphin A (DynA, Dynorphin 1–17). Recruitment of mGsi was reversible because application of the high-affinity competitive KOR antagonist 5’GNTI resulted in rapid redistribution of the biosensor back to the cytoplasm (Figure 1D). In contrast, mGs was not detectably recruited in response to KOR activation by DynA using the same assay (Figure 1F), verifying assay specificity and that mGsi recruitment is driven primarily by the Gi-derived distal C-terminus. Further, we verified that agonist-induced recruitment of mGsi occurred separately from a change in surface expression of KOR, which was monitored in parallel using anti-Flag antibody (Figure 1D). Nb33 was also rapidly recruited in response to KOR activation by DynA using the same experimental protocol, and this recruitment was also reversible upon antagonist application and occurred without a detectable change in surface receptor expression (Figure 1E). Accordingly, both mGsi and Nb33 can be used as biosensors of ligand-dependent recruitment by KOR in living cells using the TIR-FM assay, and both sensors produce a reversible recruitment signal that is sufficiently robust and fast (t1/2< 30 s) to enable reliable detection of protein recruitment without possible complications of later receptor trafficking. We next tested two non-peptide KOR full agonists, U69593 (U69) and U50488 (U50). We generated concentration-response curves by increasing agonist concentration in a stepwise manner and then adding DynA in excess (10 μM) at the end of each series as an internal reference (Figure 1G and H). Both Nb33 and mGsi were robustly recruited in a concentration-dependent manner in response to DynA and both of the non-peptide full agonist drugs (Figure 1I–K), consistent with the previously established pharmacology of these compounds (DiMattio et al., 2015), but we also noted that the concentration-response relationship for mGsi recruitment was consistently left-shifted relative to Nb33. These results demonstrate that both Nb33 and mGsi are robustly recruited by KOR after activation by peptide and non-peptide full agonists in living cells, but with a potency shift indicating that the interactions are not identical. Agonist-selective recruitment of engineered protein probes We then applied the same approach to investigate the effect of the alkaloid agonist etorphine (ET) on mGsi and Nb33 recruitment by KOR. ET is an opiate alkaloid drug that is structurally distinct from opioid peptides as well as from U50 and U69. ET efficaciously promotes G protein activation and signaling but has long been recognized to drive KOR internalization and phosphorylation poorly, supporting its classification as a G protein-biased agonist by operational criteria (Chu et al., 1997; DiMattio et al., 2015; Jordan et al., 2000). ET behaved as a potent but partial agonist in the mGsi recruitment assay, producing a maximum biosensor recruitment response reaching 67% of that produced by DynA (Figure 2A and D). Remarkably, ET produced little or no recruitment of Nb33 despite a robust response to DynA verified in each assay and in the same cells (Figure 2B and E). This lack of Nb33 recruitment was evident even at very high concentrations of ET (Figure 2B and C), in contrast to mGsi that was potently recruited (Figure 2C–E). Further verifying this difference, selective recruitment of mGsi relative to Nb33 was observed when the biosensors were tagged with distinct fluorophores, co-expressed, and imaged in parallel in the same cells (Figure 2F). Again, mGsi was potently recruited in response to ET but Nb33 was not, despite DynA producing strong recruitment of both probes and in the same cells (Figure 2G). These results indicate that mG and Nb probes can distinguish receptor-proximal agonist effects in intact cells. Figure 2 Download asset Open asset Selective recruitment of protein probes by KOR upon activation by etorphine. (A) mGsi intensity during TIR-FM time-lapse series of a HEK293 cell, co-expressing Venus-mGsi and KOR, adding increasing concentrations of etorphine (1 nM - 10 μM), followed by reference compound DynA (10 μM). 5 s between frames is shown. Intensity is normalized between 0 (no agonist) and 1 (reference compound DynA). (B) Nb33 intensity during TIR-FM time-lapse series of a HEK293 cell, co-expressing EGFP-Nb33 and KOR, treated, imaged, and normalized as in (A). (C) Concentration-dependent recruitment of mGsi and Nb33 probes to KOR upon etorphine (ET) addition, measured by TIR-FM and using DynA as reference. Normalization of intensity values is shown (range [0–1]). Regression curves with Hill slope of 1 are shown. n = 5; average ± SEM. (D) TIR-FM images of a time series of a HEK293 cell, expressing Venus-mGsi and KOR (not shown). Increasing concentrations of etorphine were added, followed by DynA. Venus-mGsi is pseudocolored, low to high intensity. The scale bar represents 10 μm. (E) Same as in (D) but with HEK293 cell expressing EGFP-Nb33 instead of mGsi. EGFP-Nb33 is pseudocolored, low to high intensity. The scale bar represents 10 μm. (F) Experimental set up for measuring agonist-dependent recruitment of both mGsi and Nb33 to KOR in same cell. (G) mGsi and Nb33 intensity during TIR-FM time-lapse series of a HEK293 cell, co-expressing Venus-mGsi, mCherry-Nb33, and FLAG-KOR. Cell was treated with increasing concentrations of etorphine, followed by DynA, and antagonist 5’GNTI. 5 s between frames is shown. Intensity is normalized between 0 (no agonist) and 1 (reference DynA). Lower panel: 10 min kymograph traced inside the cell, depicting intensities of Venus-mGsi, mCherry-Nb33, and FLAG-KOR (labeled with anti-FLAG M1-AF647), all pseudocolored, low to high intensity. (H) Schematic of the C-tail domain of KOR, indicating the known agonist-dependent phosphorylation sites that are mutated to alanine in KOR-TPD. (I) Same as in (G) but with HEK293 cell, co-expressing Venus-mGsi, mCherry-Nb33, and FLAG-KOR-TPD. (J) Concentration-dependent recruitment of mGsi and Nb33 probes to KOR-TPD upon etorphine addition. Experimental setup and analysis as in (C). n = 5; average ± SEM. (K) Concentration-dependent recruitment of mGsi and Nb33 probes to KOR upon etorphine addition, in cells pre-treated with GRK2/3 inhibitor Cmpd101 (30 μM). Experimental setup and analysis as in (C). n = 3; average ± SEM. Figure 2—source data 1 ET concentration-dependent recruitment of mGsi and Nb33 probes to KOR, or to KOR-TPD, or to KOR in the presence of Cmpd101. https://cdn.elifesciences.org/articles/54208/elife-54208-fig2-data1-v1.csv Download elife-54208-fig2-data1-v1.csv A simple interpretation of these results is that differential probe recruitment reflects a primary allosteric effect at the level of receptor-proximal protein engagement by the agonist-activated opioid receptor. An alternative possibility is that agonists produce differential probe recruitment as a secondary consequence of agonist-selective post-translational modifications of the receptor. In particular, because agonist-induced internalization of KOR requires multi-site phosphorylation on its cytoplasmic tail, and ET is known to stimulate this phosphorylation less strongly than DynA (Chen et al., we considered the possibility that differential biosensor recruitment to differential we measured biosensor recruitment by a KOR all relevant phosphorylation sites in the cytoplasmic tail for phosphorylation Figure The in mGsi relative to Nb33 recruitment was observed (Figure and verifying selective probe recruitment by KOR was not detectably in the presence of (Figure a inhibitor of GRK2/3 known to strongly reduce KOR phosphorylation in HEK293 cells (Chiu et al., 2017). these results the hypothesis that selective recruitment of mG relative to Nb probes as a primary consequence of allosteric protein selection at the receptor, rather than a secondary effect of differential Agonist-selective probe recruitment is not to KOR We next our experimental can also detect differential protein recruitment by Nb33 is known to be recruited by agonist-activated (Stoeber et al., and we verified that this is also the for mGsi. a peptide full agonist of produced rapid and robust recruitment of mGsi that was rapidly by the competitive antagonist (Figure and to what was observed for recruitment of the engineered protein probes by KOR, the concentration-response relationship for recruitment of mGsi by was left-shifted relative to Nb33 (Figure ET an agonist of promoted recruitment of both probes by and to the same maximum when compared to the peptide full agonist (Figure This with partial recruitment of mGsi and no detectable recruitment of Nb33 by KOR (Figure indicating that differential recruitment of the engineered protein probes by opioid receptors is both agonist-dependent and receptor Figure Download asset Open asset Agonist-selective protein probe recruitment by (A) min kymograph traced inside a cell expressing Venus-mGsi, mCherry-Nb33, and (labeled with anti-FLAG M1-AF647) and treated with increasing concentrations of followed by addition of intensities are pseudocolored, low to high intensity. (B) mGsi and Nb33 intensity during TIR-FM time-lapse series of a HEK293 cell, co-expressing Venus-mGsi, mCherry-Nb33, and adding increasing concentrations of followed by 5 s between frames is shown. Intensity is normalized between 0 (no agonist) and 1 (10 (C) Concentration-dependent recruitment of mGsi and Nb33 to MOR upon and etorphine addition, measured by Normalization of intensity values is shown (range with as reference. Regression curves with Hill slope of 1 are shown. n = etorphine n = average ± SEM. (D) mGsi and Nb33 intensity during TIR-FM time-lapse series of a HEK293 cell, co-expressing Venus-mGsi, mCherry-Nb33, and adding increasing concentrations of or followed by reference compound (10 μM). 5 s between frames is shown. Intensity is normalized between 0 (no agonist) and 1 (10 (E) Concentration-dependent recruitment of mGsi and Nb33 probes to MOR upon or setup and analysis as in (C). n = n = average ± SEM. (F) mGsi and Nb33 intensity during TIR-FM time-lapse series of a HEK293 cell, co-expressing Venus-mGsi, mCherry-Nb33, and adding increasing concentrations of followed by using bath application. 5 s between frames is shown. (G) mGsi and Nb33 intensity during TIR-FM time-lapse series of a cell, co-expressing Venus-mGsi, mCherry-Nb33, and adding 10 of reference followed by agonist using in and addition of 10 5 s between frames is shown. Intensity is normalized between 0 (no agonist) and 1 (10 (H) Concentration-dependent recruitment of mGsi and Nb33 to MOR upon addition, measured by TIR-FM with as reference. n = 4; average ± SEM. Figure data 1 Concentration-dependent recruitment of mGsi and Nb33 probes to MOR in response to or Download our and taking into the that and ET are both as full agonists at we next and Both of these non-peptide drugs are partial agonists with to assays of G protein activation or but each is derived from a different and in of bias using a beta-arrestin recruitment assay et al., 2016). Using the same experimental protocol, and recruitment promoted by the ligand relative to the peptide full agonist both and produced partial recruitment of mGsi as well as Nb33 (Figure and E). and were similar in the of mGsi recruitment that they produced at was found to be more than in Nb33. these results a of selective protein recruitment effects among chemically diverse MOR partial agonists. The experimental used to agonist effects relative to the peptide reference was robust in in it could differences relative to the reference peptide the agonist or has an much than the peptide reference. We found evidence for this when another chemically distinct MOR partial the et al., 2016). Using the agonist addition protocol, to be to in recruitment of mGsi because no further was elicited by addition of in contrast, to produce detectable recruitment of Nb33. However, we noted that also to promote recruitment of Nb33 in cells that were previously to (Figure despite a strong Nb33 recruitment response in cells not previously to (Figure a in to excess agonist between this this was verified to promote mGsi recruitment by but to a relative to and without detectable recruitment of Nb33 (Figure and H). These results further the of differential protein recruitment effects among chemically diverse MOR agonists. protein recruitment can be elicited by diverse opioid agonists across agonists and receptors, we defined the maximum recruitment response elicited by each agonist compared to the corresponding peptide full agonist reference for KOR and for as a relative for each agonist (Figure We then these relative values for each biosensor (Figure non-peptide agonists were from the reference peptide by this both protein probes to a similar to an of 1 for both but from the This is not consistent with the concept of partial based on a agonist-induced receptor which would the recruitment responses elicited by all agonists to the the present results the view that opioid receptors are more to selectively recruit one interaction probe over another in living cells. further suggest that the ability to promote selective protein recruitment is among chemically diverse opioid agonists (Figure Figure Download asset Open asset probe recruitment across agonists and receptors. (A) mGsi and Nb33 recruitment to KOR and MOR upon different agonists.

The aims of the present study were to investigate the protective effect of a κ-opioid receptor (KOR) agonist on intestinal barrier dysfunction in rats during cardiopulmonary bypass (CPB), as well as to examine the role of NF-κB and the transcription factor hypoxia-inducible factor-1α (HIF-1α) signaling pathway in the regulatory mechanism. A total of 50 rats were randomly divided into five groups, with 10 rats in each group: Sham surgery group (group Sham), CPB surgery group (group CPB), KOR agonist + CPB (group K), KOR agonist + specific KOR antagonist + CBP (group NK) and KOR agonist + NF-κB pathway specific inhibitor + CPB (group NF). Intestinal microcirculation was evaluated to determine intestinal barrier dysfunction in rats following CPB surgery. Hematoxylin and eosin (H&E) staining was used to observe intestinal tissue injury in the rats. ELISA was used to detect the inflammatory factors interleukin (IL)-1β, IL-6, IL10 and tumor necrosis factor-α, and the oxidative stress factors superoxidase dismutase, malondialdehyde and nitric oxide in serum. In addition, ELISA was used to investigate the serum levels of the intestinal damage markers D-lactic acid, diamine oxidase and intestinal fatty acid-binding protein. Western blotting was used to investigate the protein expression levels of tight junction proteins zonula occludens-1 and claudin-1. Furthermore, immunohistochemistry was used to examine intestinal injuries and western blotting was used to detect expression levels of NF-κB/HIF-1α signaling pathway-related proteins. H&E staining results suggested that the KOR agonist alleviated intestinal damage in the CPB model rats. This effect was reversed by the addition of a KOR antagonist. Further investigation of inflammatory and oxidative stress factors using ELISA revealed that the KOR agonist reduced the inflammatory and oxidative stress responses in the intestinal tissues of the CPB model rats. The ELISA results of intestinal damage markers and western blotting results of tight junction protein expression suggested that KOR agonist treatment may alleviate intestinal injury in CPB model rats. In addition, the western blotting and immunohistochemistry results suggested that KOR agonists may decrease the expression levels of NF-κB, p65 and HIF-1α in CPB. Collectively, the present results suggested that KOR agonists are able to ameliorate the intestinal barrier dysfunction in rats undergoing CPB by inhibiting the expression levels of NF-κB/HIF-1α signaling pathway-related proteins.

The G-protein biased kappa opioid agonists, triazole 1.1 and nalfurafine, produce non-uniform behavioral effects in male rhesus monkeys

Kappa opioid receptor (KOR) agonists have been promising therapeutic candidates, owing to their potential for relieving pain and treating intractable pruritus. Although lacking morphine-like central nervous system (CNS) effects, KOR agonists do elicit sedation, dysphoria and diuresis which seriously impede their development. Peripherally-restricted KOR agonists have a poor ability to penetrate into the CNS system, so that CNS-related adverse effects can be ameliorated or even abolished. However, the only approved peripherally-restricted KOR agonist CR845 remains some frequent CNS adverse events. In the present study, we aim to address pharmacological profiles of HSK21542, with an expectation to provide a safe and effective alternative for patients who are suffering from pain and pruritus. The in vitro experimental results showed that HSK21542 was a selective and potent KOR agonist with higher potency than CR845, and had a brain/plasma concentration ratio of 0.001, indicating its peripheral selectivity. In animal models of pain, HSK21542 significantly inhibited acetic acid-, hindpaw incision- or chronic constriction injury-induced pain-related behaviors, and the efficacy was comparable to CR845 at 15 min post-dosing. HSK21542 had a long-lasting analgesic potency with a median effective dose of 1.48 mg/kg at 24 h post-drug in writhing test. Meanwhile, the antinociceptive activity of HSK21542 was effectively reversed by a KOR antagonist nor-binaltorphimine. In addition, HSK21542 had powerful antipruritic activities in compound 48/80-induced itch model. On the other hand, HSK21542 had a weak ability to produce central antinociceptive effects in a hot-plate test and fewer effects on the locomotor activity of mice. HSK21542 didn’t affect the respiratory rate of mice. Therefore, HSK21542 might be a safe and effective KOR agonist and promising candidate for treating pain and pruritus.

Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

It has been demonstrated that κ-opioid receptor agonists can reduce hypoxia-ischemia brain injury in animal models. However, it is unclear how the κ-opioid receptor responds to hypoxia-ischemia. In the current study, the authors used an in vitro model of oxygen-glucose deprivation and reoxygenation to explore how κ-opioid receptors respond to hypoxia and reoxygenation. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse κ-opioid receptor-tdTomato fusion protein or Flag-tagged mouse κ-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the κ-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of κ-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. Hypoxic conditions caused the κ-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific κ-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific κ-opioid receptor. κ-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular κ-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen-glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced κ-opioid receptor internalization. Hypoxia induces reversible κ-opioid receptor internalization, which was inhibited by selective κ-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between κ-opioid receptor and hypoxia via κ-opioid receptor activation and the dynamin-dependent mechanism.