Positive allosteric modulation of the mu-opioid receptor produces analgesia with reduced side effects.

Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than ß-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

The Buprenorphine Analogue BU10119 Attenuates Drug-Primed and Stress-Induced Cocaine Reinstatement in Mice.

There are no Food and Drug Administration-approved medications for cocaine use disorder, including relapse. The µ-opioid receptor (MOPr) partial agonist buprenorphine alone or in combination with naltrexone has been shown to reduce cocaine-positive urine tests and cocaine seeking in rodents. However, there are concerns over the abuse liability of buprenorphine. Buprenorphine's partial agonist and antagonist activity at the nociception receptor (NOPr) and κ-opioid receptor (KOPr), respectively, may contribute to its ability to inhibit cocaine seeking. Thus, we hypothesized that a buprenorphine derivative that exhibits antagonist activity at MOPr and KOPr with enhanced agonist activity at the NOPr could provide a more effective treatment. Here we compare the pharmacology of buprenorphine and two analogs, BU10119 and BU12004, in assays for antinociception and for cocaine- and stress-primed reinstatement in the conditioned place preference paradigm. In vitro and in vivo assays showed that BU10119 acts as an antagonist at MOPr, KOPr, and δ-opioid receptor (DOPr) and a partial agonist at NOPr, whereas BU12004 showed MOPr partial agonist activity and DOPr, KOPr, and NOPr antagonism. BU10119 and buprenorphine but not BU12004 lessened cocaine-primed reinstatement. In contrast, BU10119, BU12004, and buprenorphine blocked stress-primed reinstatement. The selective NOPr agonist SCH221510 but not naloxone decreased cocaine-primed reinstatement. Together, these findings are consistent with the concept that NOPr agonism contributes to the ability of BU10119 and buprenorphine to attenuate reinstatement of cocaine-conditioned place preference in mice. The findings support the development of buprenorphine analogs lacking MOPr agonism with increased NOPr agonism for relapse prevention to cocaine addiction. SIGNIFICANCE STATEMENT: There are no Food and Drug Administration-approved medications for cocaine use disorder. Buprenorphine has shown promise as a treatment for cocaine relapse prevention; however, there are concerns over the abuse liability of buprenorphine. Here we show a buprenorphine analogue, BU10119, which lacks µ-opioid receptor agonism and inhibits cocaine-primed and stress-primed reinstatement in a conditioned place-preference paradigm. The results suggest the development of BU10119 for the management of relapse to cocaine seeking.

Regulators of G-Protein Signaling (RGS) Proteins Promote Receptor Coupling to G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) Channels.

Regulators of G-protein signaling (RGS) proteins negatively modulate presynaptic µ-opioid receptor inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). Paradoxically, we find that G-protein-coupled receptor (GPCR) activation of G-protein-gated inwardly rectifying K+ channels (GIRKs) in the vlPAG is reduced in an agonist- and receptor-dependent manner in transgenic knock-in mice of either sex expressing mutant RGS-insensitive Gαo proteins. µ-Opioid receptor agonist activation of GIRK currents was reduced for DAMGO and fentanyl but not for [Met5]-enkephalin acetate salt hydrate (ME) in the RGS-insensitive heterozygous (Het) mice compared with wild-type mice. The GABAB agonist baclofen-induced GIRK currents were also reduced in the Het mice. We confirmed the role of Gαo proteins in µ-opioid receptor and GABAB receptor signaling pathways in wild-type mice using myristoylated peptide inhibitors of Gαo1 and Gαi1-3 The results using these inhibitors indicate that receptor activation of GIRK channels is dependent on the preference of the agonist-stimulated receptor for Gαo versus that for Gαi. DAMGO and fentanyl-mediated GIRK currents were reduced in the presence of the Gαo1 inhibitor, but not the Gαi1-3 inhibitors. In contrast, the Gαo1 peptide inhibitor did not affect ME activation of GIRK currents, which is consistent with results in the Het mice, but the Gαi1-3 inhibitors significantly reduced ME-mediated GIRK currents. Finally, the reduction in GIRK activation in the Het mice plays a role in opioid- and baclofen-mediated spinal antinociception, but not supraspinal antinociception. Thus, our studies indicate that RGS proteins have multiple mechanisms of modulating GPCR signaling that produce negative and positive regulation of signaling depending on the effector.SIGNIFICANCE STATEMENT Regulators of G-protein signaling (RGS) proteins positively modulate GPCR coupling to GIRKs, and this coupling is critical for opioid- and baclofen-mediated spinal antinociception, whereas µ-opioid receptor-mediated supraspinal antinociception depends on presynaptic inhibition that is negatively regulated by RGS proteins. The identification of these opposite roles for RGS proteins has implications for signaling via other GPCRs.

Identification and characterization of two novel alternatively spliced E2F1 transcripts in the rat CNS.

E2F1 is a transcription factor classically known to regulate G0/G1 to S phase progression in the cell cycle. In addition, E2F1 also regulates a wide range of apoptotic genes and thus has been well studied in the context of neuronal death and neurodegenerative diseases. However, its function and regulation in the mature central nervous system are not well understood. Alternative splicing is a well-conserved post-transcriptional mechanism common in cells of the CNS and is necessary to generate diverse functional modifications to RNA or protein products from genes. Heretofore, physiologically significant alternatively spliced E2F1 transcripts have not been reported. In the present study, we report the identification of two novel alternatively spliced E2F1 transcripts: E2F1b, an E2F1 transcript retaining intron 5, and E2F1c, an E2F1 transcript excluding exon 6. These alternatively spliced transcripts are observed in the brain and neural cell types including neurons, astrocytes, and undifferentiated oligodendrocytes. The expression of these E2F1 transcripts is distinct during maturation of primary hippocampal neuroglial cells. Pharmacologically-induced global translation inhibition with cycloheximide, anisomycin or thapsigargin lead to significantly reduced expression of E2F1a, E2F1b and E2F1c. Conversely, increasing neuronal activity by elevating the concentration of potassium chloride selectively increased the expression of E2F1b. Furthermore, experiments expressing these variants in vitro show the transcripts can be translated to generate a protein product. Taken together, our data suggest that the alternatively spliced E2F1 transcript behave differently than the E2F1a transcript, and our results provide a foundation for future investigation of the function of E2F1 splice variants in the CNS.

Effects of adolescent Δ9-tetrahydrocannabinol exposure on the behavioral effects of cocaine in adult Sprague-Dawley rats.

Cannabis is the most popular, illegal drug used by adolescents in the United States. Exposure to cannabis and its main psychoactive ingredient, Δ9-tetrahydrocannabinol (THC), during adolescence may have long-lasting effects on the development of behavioral and neurobiological processes. This study investigated the effects of chronic adolescent exposure to THC on sensitization to the psychomotor-stimulating effects of cocaine and on the reinforcing effects of cocaine in adult male Sprague-Dawley rats. During adolescence (P28-P45), rats were given once-daily intraperitoneal injections of either vehicle or 1 mg/kg THC. On P90, the acute locomotor-stimulating effects of cocaine and sensitization to cocaine were evaluated. Also, cocaine-maintained behavior was evaluated by determining within-session cocaine dose-effect curves, acquisition of behavior maintained by a small cocaine dose (0.1 mg/kg/infusion), and breakpoints on a progressive ratio schedule of reinforcement. In general, there was no effect of adolescent THC exposure on the locomotor-stimulating effects of cocaine following acute or repeated administration. However, the reinforcing effects of cocaine were potentiated in rats treated with THC during adolescence, but this effect was only observed with small doses of cocaine. Rats exposed to THC during adolescence also more rapidly acquired self-administration behavior when a small cocaine dose was available. Together, these results demonstrate that exposure to THC during adolescence may enhance sensitivity to cocaine and/or enhance the reinforcing effects of cocaine even into adulthood under certain conditions. In conclusion, frequent exposure to THC during adolescence may produce long-lasting changes in behavior, possibly increasing susceptibility to addiction. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Selectively promiscuous opioid ligands: discovery of high affinity/low efficacy opioid ligands with substantial nociceptin opioid peptide receptor affinity.

Emerging clinical and preclinical evidence suggests that a compound displaying high affinity for µ, κ, and δ opioid (MOP, KOP, and DOP) receptors and antagonist activity at each, coupled with moderate affinity and efficacy at nociceptin opioid peptide (NOP) receptors will have utility as a relapse prevention agent for multiple types of drug abuse. Members of the orvinol family of opioid ligands have the desired affinity profile but have typically displayed substantial efficacy at MOP and or KOP receptors. In this study it is shown that a phenyl ring analogue (1d) of buprenorphine displays the desired profile in vitro with high, nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo.