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The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to ‘steer’ hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2found in µOR3and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50residue in the Na+site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gisubtypes and show strongly reduced arrestin recruitment—one (C6 guano) also shows the lowest Gzefficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Goand Gzsubtypes and arrestins, thus modulating their in vivo pharmacology.

Arginine vasopressin (AVP) and oxytocin (OT) influence social behavior and cognitive processes and may explain some of the variance associated with individual differences in behavior. Although great focus has been placed on the roles of these peptides in learning and memory, less attention has been given to the receptors to which they bind. The authors exposed male prairie voles (Microtus ochrogaster) to novel females in a multitrial social recognition test to investigate whether individual differences in vasopressin receptor (VlaR) or oxytocin receptor (OTR) related to social recognition. The authors also explored differences in OTR and VlaR throughout the brain to determine whether patterns of receptor coexpression emerged in functionally related structures. Male investigation of females was highly variable, and those that investigated females the most did not habituate to repeated female presentation. Moreover, high investigators expressed significantly more VlaR and less OTR in the lateral septum. Coexpression patterns of these receptors emphasize the role of OT and AVP in neural circuits involved in social behavior, reward, learning, and memory. Understanding individual differences in the laboratory may provide insight into evolved natural behavior. Keywords: individual differences, lateral septum, prairie vole, social behavior network, social recognition

The leaves of Mitragyna speciosa(kratom), a plant native to Southeast Asia, are increasingly used as a pain reliever and for attenuation of opioid withdrawal symptoms. Using the tools of natural products chemistry, chemical synthesis, and pharmacology, we provide a detailed in vitroand in vivopharmacological characterization of the alkaloids in kratom. We report that metabolism of kratom’s major alkaloid, mitragynine, in mice leads to formation of (a) a potent mu opioid receptor agonist antinociceptive agent, 7-hydroxymitragynine, through a CYP3A-mediated pathway, which exhibits reinforcing properties, inhibition of gastrointestinal (GI) transit and reduced hyperlocomotion, (b) a multifunctional mu agonist/delta-kappa antagonist, mitragynine pseudoindoxyl, through a CYP3A-mediated skeletal rearrangement, displaying reduced hyperlocomotion, inhibition of GI transit and reinforcing properties, and (c) a potentially toxic metabolite, 3-dehydromitragynine, through a non-CYP oxidation pathway. Our results indicate that the oxidative metabolism of the mitragynine template beyond 7-hydroxymitragynine may have implications in its overall pharmacology in vivo.

Mitragynine and 7-hydroxymitragynine (7OH) are the major alkaloids mediating the biological actions of the psychoactive plant kratom. To investigate the structure–activity relationships of mitragynine/7OH templates, we diversified the aromatic ring of the indole at the C9, C10, and C12 positions and investigated their G-protein and arrestin signaling mediated by mu opioid receptors (MOR). Three synthesized lead C9 analogs replacing the 9-OCH3group with phenyl (4), methyl (5), or 3′-furanyl [6(SC13)] substituents demonstrated partial agonism with a lower efficacy than DAMGO or morphine in heterologous G-protein assays and synaptic physiology. In assays limiting MOR reserve, the G-protein efficacy of all three was comparable to buprenorphine. 6(SC13) showed MOR-dependent analgesia with potency similar to morphine without respiratory depression, hyperlocomotion, constipation, or place conditioning in mice. These results suggest the possibility of activating MOR minimally (G-protein Emax≈ 10%) in cell lines while yet attaining maximal antinociception in vivowith reduced opioid liabilities.

Abstract ID 130158

Abstract ID 100583

Abstract ID 95191

Innovative discovery strategies are essential to address the ongoing opioid epidemic in the United States. Misuse of prescription and illegal opioids (e.g., morphine, heroin) has led to major problems with addiction and overdose. We used vincamine, an indole alkaloid, as a synthetic starting point for dramatic structural alterations of its complex, fused ring system to synthesize 80 diverse compounds with intricate molecular architectures. A select series of vincamine-derived compounds were screened for both agonistic and antagonistic activities against a panel of 168 G protein-coupled receptor (GPCR) drug targets. Although vincamine was without an effect, the novel compound 4(V2a) demonstrated antagonistic activities against hypocretin (orexin) receptor 2. When advanced to animal studies, 4(V2a) significantly prevented acute morphine-conditioned place preference (CPP) and stress-induced reinstatement of extinguished morphine-CPP in mouse models of opioid reward and relapse. These results demonstrate that the ring distortion of vincamine offers a promising way to explore new chemical space of relevance to opioid addiction.

The mu opioid receptor (μOR) is a target for clinically used analgesics. However, adverse effects, such as respiratory depression and physical dependence, necessitate the development of alternative treatments. Recently we reported a novel strategy to design functionally selective opioids by targeting the sodium binding allosteric site in μOR with a supraspinally active analgesic named C6guano. Presently, to improve systemic activity of this ligand, we used structure-based design, identifying a new ligand named RO76where the flexible alkyl linker and polar guanidine guano group is swapped with a benzyl alcohol, and the sodium site is targeted indirectly through waters. A cryoEM structure of RO76bound to the μOR-Gicomplex confirmed that RO76interacts with the sodium site residues through a water molecule, unlike C6guanowhich engages the sodium site directly. Signaling assays coupled with APEX based proximity labeling show binding in the sodium pocket modulates receptor efficacy and trafficking. In mice, RO76was systemically active in tail withdrawal assays and showed reduced liabilities compared to those of morphine. In summary, we show that targeting water molecules in the sodium binding pocket may be an avenue to modulate signaling properties of opioids, and which may potentially be extended to other G-protein coupled receptors where this site is conserved.

The prevalence of major depression in people with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome is substantially higher than in the general population. Mechanisms underlying this comorbidity are poorly understood. HIV transactivator of transcription (Tat) protein, produced and excreted by HIV, could be involved. We determined whether conditional Tat protein expression in mice is sufficient to induce depression-like behaviors and oxidative stress. Further, as oxidative stress is associated with depression, we determined whether decreasing or increasing oxidative stress by administering methylsulfonylmethane (MSM) or diethylmaleate, respectively, altered depression-like behavior.

Abstract ID 52769

Abstract ID 17740

Exposure to HIV-1 trans-activator of transcription (Tat) protein potentiates the psychostimulant effects of cocaine, but the functional consequences of the interaction between HIV-1 Tat and other abused drugs is poorly understood. We hypothesized that exposure to HIV-1 Tat would potentiate the rewarding effects of ethanol. GT-tg transgenic mice, where Tat protein is conditionally expressed in brain by a doxycycline-dependent GFAP-linked promoter, were used to test the effects of Tat on ethanol-conditioned place preference (CPP). Compared to uninduced littermates or doxycycline-treated C57BL/6J mice, Tat-induced GT-tg mice demonstrated a 3-fold increase in ethanol-CPP. The potentiation of ethanol-CPP was dependent on the dose and duration of doxycycline treatment used to express Tat protein. Moreover, induction of Tat protein after the extinction of CPP produced reinstatement without additional exposure to ethanol. Together, these data suggest that CNS exposure to HIV-1 Tat protein potentiates the rewarding effects of ethanol in mice.

Maladaptive behavioral responses characteristic of post-traumatic stress disorders are notably resistant to treatment. We hypothesized that the pharmacological disruption of memories activated during reconsolidation might reverse established stress-induced increases in depression-like behaviors and cocaine reward. C57BL6J mice were subjected to repeated social defeat stress (SDS), and examined for time spent immobile in a subsequent forced swim test (FST). An additional set of SDS-exposed mice were place-conditioned with cocaine, and tested for cocaine-conditioned place preference (CPP). All stress-exposed mice were then subjected to a single additional trial of SDS while under the influence of propranolol or cycloheximide to disrupt memory reconsolidation, then given one additional FST or CPP test the next day. Mice subjected to repeated SDS subsequently demonstrated increases in time spent immobile in the FST or in the cocaine-paired chamber. Vehicle-treatment followed by additional SDS exposure did not alter these behaviors, but propranolol or cycloheximide treatment reversed each of the potentiated responses in a dose-dependent manner. Overall, these results demonstrate that while repeated exposure to a social defeat stressor subsequently increased depression-like behavior and cocaine-CPP, disruption of traumatic memories made labile by re-exposure to SDS during reconsolidation may have therapeutic value in the treatment of established post-traumatic stress disorder-related behaviors.

The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) exhibited both dose-dependent antinociception and kappa opioid receptor (KOR) antagonist activity after oral administration. CJ-15,208 antagonized a centrally administered KOR selective agonist, providing strong evidence it crosses the blood–brain barrier to reach KOR in the CNS. Orally administered CJ-15,208 also prevented both cocaine- and stress-induced reinstatement of extinguished cocaine-seeking behavior in the conditioned place preference assay in a time- and dose-dependent manner. Thus, CJ-15,208 is a promising lead compound with a unique activity profile for potential development, particularly as a therapeutic to prevent relapse to drug-seeking behavior in abstinent subjects.

A letter to the editor is presented in response to the article "Wal-Mart's Rx for Health Care," in the April 17, 2006 issue.

The article discusses Drenge band from England performance at the different locations in the U.S. including Austin, Texas and New York.

Mosquitoes play an important role as vectors for a variety of pathogens. Insecticide use for control means results in selection of tolerant/resistant mosquitoes. An esterase cDNA was isolated from Culex nigripalpus Theobald (Diptera: Culicidae), and its spatial expression was characterized. GenBank tblastx and VectorBase searches with the translation product of the longest cDNA fragment (397 bp, CN Temsha est-1) of the Cx. nigripalpus esterase revealed similarity (95%) to the estalpha2 esterase gene product of Culex pipiens quinquefasciatus Say, suggesting it is a member of the esterase family. Transcripts encoding Cx. nigripalpus esterase were detected in midgut tissue, thoraces, and abdomens of female Cx. nigripalpus, findings that may support possible roles in feeding, digestion, or reproduction. Gene expression studies to determine the role of Cx. nigripalpus esterase in the mosquito are being conducted.

The potential risk of infection among newborn infants raised under open radiant heaters (RH) Is uncertain. In a prospective, controlled study, rates of bacterial colonization of the anterior nares, umbilicus and groin were determined during the first 3 days of life in 15 infants raised under RH and in 27 Infants raised in isolette incubators (I). None of the infants received systemic antibiotics during the study.Decreased bacterial colonization was observed at all sites on each day of the study in the RH group. This was due to the almost complete absence of colonization with S. aureus in these infants. In contrast, colonization with S. aureus at the 3 sites ranged from 3.7 to 40.7% among the I Infants. S. epidermidis, the most commonly encounted organism, was seen with comparable frequency in both groups. There was also no intergroup difference in the prevalence of E. coli and other enterobacteriaceae. Systemic and topical infections were not observed in either group.These results suggest that RH inhibits colonization of the skin and nares with S. aureus, and may play a beneficial role in reducing the incidence of staphylococcal infection.