Salvinorin A is a potent and selective agonist of kappa opioid receptors in the brain. Recent studies in several animal models have revealed that Salvinorin A has anti-addiction, anti-depression properties and exhibits pronounced neuroprotective effects against hypoxia/ischemia induced brain damage, and have raised interest in potential clinical applications in several acute pathologies involving oxygen deficiency in the brain. This review focuses on the chemical and physical properties of Salvinorin A and their impact on development of a rational formulation to enable its translation from a research compound to a novel therapeutic agent.

With the recently solved crystal structure of the murine mu opioid receptor, the elucidation of the structure function relationships of the human mu receptor becomes feasible. In this study, we analyzed the available structural information along with ligand binding and G protein activation of human mu receptor. Affinity determinations were performed in a HEK293 cell line stably transfected with the human mu opioid receptor for 6 different agonists (morphine, DMAGO, and herkinorn) and antagonists (naloxone, beta-Funaltrexamine, and Norbinaltorphimine). G protein activation was investigated in membrane preparations containing human mu receptors treated with the agonist, partial agonist, or antagonist compounds. 4DKL.pdb was utilized for structural analysis and docking calculations for 28 mu receptor ligands. The predicted affinities from docking were compared with those experimentally determined. While all known ligands bind to the receptor through the same binding site that is large enough to accommodate molecules of various sizes, interaction with D147 (D149 in human mu receptor) is essential for binding. No distinguishable interaction pattern in the binding site for agonist, partial agonist, or antagonist to predict pharmacological activities was found. The failure to reconcile the predicted affinities from docking with experimental values indicates that the receptor might undergo significant conformational changes from one state to the other states upon different ligand binding. A simplified model to understand the complicated system is proposed and further study on these multiple conformations using high resolution structural approaches is suggested.

Early brain injury (EBI) refers to the direct injury to the brain during the first 72 h after subarachnoid hemorrhage (SAH), which is one of the major causes for the poor clinical outcome after SAH. In this study, we investigated the effect and the related mechanism of Salvinorin A (SA), a selective kappa opioid receptor agonist, on EBI after SAH. SA was administered by intraperitoneal injection at 24 h, 48 h and 72 h after SAH. The volume of lateral ventricle was measured by magnetic resonance imaging (MRI). The neuronal morphological changes and the apoptotic level in CA1 area of hippocampus were observed by Nissl and TUNEL staining respectively. Protein expression of p-PI3K, p-Akt, p-IKKα/β, p-NF-κB, FoxO1, Bim, Bax and Cleaved-caspase-3 was measured to explore the potential mechanism. We found that SA alleviated the neuronal morphological changes and apoptosis in CA1 area of hippocampus. The mechanism might be related to the increased protein expression of p-PI3K/p-Akt, which accompanied by decreased expression of p-IKKα/β, p-NF-κB, FoxO1, Bim, Bax and Cleaved-caspase-3 in the hippocampus. Thus, therapeutic interventions of SA targeting the PI3K/Akt pathway might be a novel approach to ameliorate EBI via reducing the apoptosis and inflammation after SAH.

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Barbiturates potentiate GABA actions at the GABA(A) receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10-500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.

Perioperative infections, particularly surgical site infections pose significant morbidity and mortality. Phagocytosis is a critical step for microbial eradication. We examined the effect of commonly used anesthetics on macrophage phagocytosis and its mechanism.

Hypoxia induces reversible κ-opioid receptor (KOR) internalization similar to the internalization that is induced by KOR agonists. In the current study, we demonstrate that this KOR internalization is a protective mechanism via the β-arrestin specific pathway in an oxygen-glucose deprivation (OGD) model. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse KOR-tdTomato fusion protein (N2A-mKOR-tdT cells). Various concentrations of salvinorin A (SA), a highly selective KOR agonist, were given in the presence and absence of norbinaltorphimine (norBNI), which is a KOR antagonist, or Dyngo-4a (internalization inhibitor) or API-2 (Akt/Protein kinase B signaling inhibitor-2). Various concentrations of SA and RB-64 (22-thiocyanatosalvinorin A, selective for the G protein signaling pathway) were administered both in normoxic and hypoxic conditions. Autophagosomes and ultrastructural components of cells were observed using transmission electron microscopy (TEM). Cell viability, severity of cell injury, and levels of proteins related to the Akt signaling pathway were evaluated using live cell counting (by Cell Counting Kit-8), the lactic acid dehydrogenase (LDH) release rate, and Western blot analysis, respectively. SA promoted cell survival and attenuated OGD-induced cell injury. The Akt signaling pathway is activated by SA. KOR internalization, when blocked by norBNI or Dyngo-4a, increased LDH release and decreased cell viability under OGD. Treatment with SA significantly inhibited autophagy, and the effects of SA on autophagy were reversed by API-2 pretreatment. RB-64 in a low concentration without β-arrestin recruitment did not reduce LDH release and increase cell viability as observed with SA. KOR internalization through β-arrestin activation is a protective mechanism against OGD. The Akt pathway might play a critical role in modulating these protective effects by inhibiting autophagy.

Stroke is the leading cause of death and disability in China. The median time of stroke pre-hospital delay is more than 15 hours, mainly due to the lack of awareness on stroke symptoms and calling emergency services. We developed Stroke 1-2-0 recognition tool in China, by adapting Face, Arm, Speech and Time. Our preliminary findings suggested that Stroke 1-2-0 can improve public's knowledge of the stroke symptoms, but its impact on the prehospital delay is still unclear. Furthermore, these findings were mainly obtained from Shanghai, one of the largest metropolises in China. However, more than half of population in China lives in the rural area. Given the striking disparities in socioeconomic status and quality of stroke care across the nation, a multicentre trial is warranted.

Epinephrine, a non-specific adrenergic agonist, is one of the most commonly used inotropes perioperatively. Recent studies have shown that inflammatory response in cardiac surgery could result in hypoperfusion, dysrhythmias, myocardial ischemia, and other pathophysiological alterations in the postoperative period. These alterations might be contributing to the adverse clinical outcome. Although epinephrine has been shown to have effects on the immune system, how epinephrine affects inflammatory response is unclear. We hypothesized that epinephrine exposure may alter the inflammatory response which may potentially contribute to the adverse clinical outcomes. We used cultured rat cardiomyocytes (H9C2) with epinephrine exposure in this study. The expression of mRNA for inflammation-related genes was quantitated for the comparison of experimental group (with epinephrine) and control group (without epinephrine). The results demonstrated significant changes of inflammation-related gene expressions in cardiomyocytes after epinephrine administration. The clinical implications of the gene expression changes in cardiomyocytes are unclear.

Respiratory failure significantly increases mortality in critically ill patients. While opioids are often used during the perioperative period and in critically ill situations, little is known about how opioids are involved in pulmonary immune function and the inflammatory response. There is currently no clear information on the role of the kappa opioid receptor (KOR) in pulmonary inflammation. Here we evaluate whether KORs are involved in the modulation of lung macrophages by the use of selective KOR agonists in lipopolysaccharide (LPS) activated alveolar macrophages.

Dezocine is an opioid that was used in clinical practice for acute pain management in the US (1986 to 2011) and is currently in use in China. It is not listed as a controlled substance in the US due to no reported cases of addiction. Dezocine is a partial agonist at the mu opioid receptor (MOR); however, it is unclear whether dezocine can activate both the G protein pathway and the beta-arrestin pathway. In this study we hypothesized that dezocine does not activate the beta-arrestin pathway, which could be the potential molecular mechanism by which dezocine is not addictive or at least less addictive than other classic opioids. Both morphine, a MOR full agonist and buprenorphine, a partial MOR agonist similar to dezocine, were used for comparison purposes. The major side effects of dezocine in clinical usage are its gastrointestinal side effects and first pass effects; therefore, we explored the possibility of administering dezocine intranasally in rodents to demonstrate the feasibility of intranasal administration for new clinical usage purposes. With proper formulation it is possible to administer dezocine intranasally to achieve a high concentration in the brain in the rodent model. The results indicate that dezocine does not activate the beta-arrestin pathway in MOR. Intranasal delivery of dezocine achieves a much higher medication concentration in the blood and brain as compared to intraperitoneal injection. It also persists a longer time before it falls below detection in the blood. This study provides a possible explanation of why dezocine is not addictive or at least less addictive than other commonly used opioids. This study also demonstrates that intranasal administration offers an alternative strategy for its potential clinical applications.

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An optimal therapeutics to manage opioid withdrawal syndrome is desired for opioid addiction treatment. Down-regulation of endogenous endomorphin-2 (EM2) level in the central nervous system after continuous morphine exposure was observed, which suggested that increase of EM2 could be an alternative novel method for opioid dependence. As a short peptide, the short half-life of EM2 limits its clinical usage through conventional administration. In the present study, we engineered an EM2 gene using a signal peptide of mouse growth factor for an out-secretory expression of EM2 and an adenovirus as a vector, which ultimately sustained the release of EM-2. After administration of the adenovirus in central nervous system, a sustained increase of EM2 level in the cerebral spinal fluid (CSF) was observed along with a reduction of morphine withdrawal syndrome. These findings suggest that the engineered EM2 gene delivered to the central nervous system could be a novel therapeutics for withdrawal syndrome in opioid dependent subjects.

Volatile anesthetics have been in clinical use for a long period of time and are considered to be promiscuous by presumably interacting with several ion channels in the central nervous system to produce anesthesia. Because ion channels and their existing evolutionary analogues, ion transporters, are very important in various organisms, it is possible that volatile anesthetics may affect some bacteria. In this study, we hypothesized that volatile anesthetics could affect bacterial behaviors. We evaluated the impact of anesthetics on bacterial growth, motility (swimming and gliding) and biofilm formation of four common bacterial pathogens in vitro. We found that commonly used volatile anesthetics isoflurane and sevoflurane affected bacterial motility and biofilm formation without any effect on growth of the common bacterial pathogens studied here. Using available Escherichia coli gene deletion mutants of ion transporters and in silico molecular docking, we suggested that these altered behaviors might be at least partly via the interaction of volatile anesthetics with ion transporters.

Since herkinorin is the first non-opioid mu agonist derived from salvinorin A that has the ability to induce cerebral vascular dilatation, we hypothesized that herkinorin could have similar vascular dilatation effect via the mu and kappa opioid receptors and the cAMP pathway. The binding affinities of herkinorin to kappa and mu opioid receptors were determined by in-vitro competition binding assays. The cerebral arteries were monitored in piglets equipped with a closed cranial window and the artery responses were recorded before and every 30s after injection of artificial cerebrospinal fluid (CSF) in the presence or absence of the investigated drugs: herkinorion, norbinaltorphimine (NTP), a kappa opioid receptor antagonist, β-funaltrexamine (β-FNA), a mu opioid receptor antagonist, or Rp-8-Br-cAMPS (Rp-cAMPS), an inhibitor of protein kinase A (PKA). CSF samples were collected before and 10 min after herkinorin and NTP administration for the measurement of cAMP levels. Data were analyzed by repeated-measures analysis of variance. Our results show that herkinorin binds to both kappa and mu opioid receptors. Its vasodilation effect is totally abolished by NTP, but is not affected by β-FNA. The levels of cAMP in the CSF elevate after herkinorin administration, but are abolished with NTP administration. The cerebral vasodilative effect of herkinorin is also blunted by Rp-cAMPS. In conclusion, as a non-opioid kappa and mu opioid receptor agonist, herkinorin exhibits cerebral vascular dilatation effect. The dilatation is mediated though the kappa opioid receptor rather than the mu opioid receptor. cAMP signaling also plays an important role in this process.

Artificial cells are constructed from synthetic materials to imitate the biological functions of natural cells. By virtue of nanoengineering techniques, artificial cells with designed biomimetic functions provide alternatives to natural cells, showing vast potential for biomedical applications. Especially in cancer treatment, the deficiency of immunoactive macrophages results in tumor progression and immune resistance. To overcome the limitation, a BaSO4@ZIF-8/transferrin (TRF) nanomacrophage (NMΦ) is herein constructed as an alternative to immunoactive macrophages. Alike to natural immunoactive macrophages, NMΦ is stably retained in tumors through the specific affinity of TRF to tumor cells. Zn2+ as an "artificial cytokine" is then released from the ZIF-8 layer of NMΦ under tumor microenvironment. Similar as proinflammatory cytokines, Zn2+ can trigger cell anoikis to expose tumor antigens, which are selectively captured by the BaSO4 cavities. Therefore, the hierarchical nanostructure of NMΦs allows them to mediate immunogenic death of tumor cells and subsequent antigen capture for T cell activation to fabricate long-term antitumor immunity. As a proof-of-concept, the NMΦ mimics the biological functions of macrophage, including tumor residence, cytokine release, antigen capture and immune activation, which is hopeful to provide a paradigm for the design and biomedical applications of artificial cells.

The treatment of neuropathic pain (NP) currently remains clinically challenging. In an attempt to identify novel targets of known opioids, we found that dezocine, a non-addictive opioid, inhibits norepinephrine and serotonin reuptake through their transporter proteins which open the potential for dezocine to manage NP. In the present study, the effect of dezocine on NP was observed in a rat model of chronic constriction injury (CCI). The paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were used to evaluate thermal hyperalgesia and mechanic allodynia for nociceptive response. PWL and PWT tests were performed at 11:00 AM starting from 1 day before CCI surgery and 1, 3, 7, 10 days after right sciatic nerve ligation in the presence or absence of daily intraperitoneal injection of dezocine. The results demonstrated that the CCI-induced thermal and mechanical pain hypersensitivity was attenuated by dezocine significantly and persistently without sign of tolerance, indicating that dezocine could be an alternative medication for the treatment of NP. Clinical trial to confirm such discovery is warranted.

Models of the human μ opioid receptor were constructed using available G-protein-coupled receptor (GPCR) structures using homology (comparative) modeling techniques. The recent publication of a high-resolution crystal structure of a construct based on the murine μ opioid receptor offers a unique opportunity to evaluate the reliability of the homology models and test the relevance of introducing more templates (known structures) to increase the accuracy of the comparative models. In the first model two templates were used: the β2 adrenergic and bovine rhodopsin receptors. For the second model, four templates were utilized: the β2 adrenergic, bovine rhodopsin, β1 adrenergic, and A2A adenosine receptors. Including additional templates improved the accuracy of structural motifs and other features of the model when the same sequence alignment was used. The predicted structures were especially relevant in the case of important receptor regions such as the DRY motif, which has been associated with receptor activation. Additionally, this study showed that receptor sequence similarity is crucial in homology modeling, as indicated in the case of the highly diverse EC2 loop. This study demonstrates the reliability of the homology modeling technique in the case of the μ opioid receptor, a member of the rhodopsin-like family class of GPCRs. The addition of more templates improved the accuracy of the model. The findings regarding the modeling has significant implication to other GPCRs where the crystal structure is still unknown and suggest that homology modeling techniques can provide high quality structural models for interpreting experimental findings and formulating structurally based hypotheses regarding the activity of these important receptors.

G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (Kd = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.