Impact of HIV-1 tat protein on methamphetamine-induced inhibition of vesicular monoamine transporter2-mediated dopamine transport and methamphetamine conditioned place preference in HIV-1 tat transgenic mice.

Perturbation of dopamine transmission has been implicated as a contributing factor in HIV-1 associated neurocognitive disorders with concurrent methamphetamine (METH) abuse. We have demonstrated that the HIV-1 protein, transactivator of transcription (Tat), decreases dopamine transport through inhibition of vesicular monoamine transporter2 (VMAT2). This study determined the effects of Tat protein on METH-inhibited VMAT2 function and METH-conditioned place preference (CPP). In vitro exposure of isolated mouse whole brain vesicles to recombinant Tat1-86 or METH displayed a concentration-dependent inhibition of the vesicular [3H]Dopamine uptake, in which a combination of Tat and METH induced a greater reduction of dopamine uptake compared to Tat or METH alone. In vivo, the maximal velocity (Vmax) of vesicular [3H]Dopamine uptake was decreased in inducible Tat transgenic (iTat-tg) mice harvested after treatment with either 21-day doxycycline (Dox) or 14-day METH (3 mg/kg, i.p., daily), whereas these mice treated with both Dox and METH displayed an additive reduction of the Vmax compared to either Tat or METH alone. Moreover, Dox-induced Tat expression increased METH-CPP in an exposure-dependent manner, with iTat-tg mice demonstrating a 2.3-fold potentiation of METH-CPP compared with Tat null control mice upon administration of Dox for 14 days. Furthermore, a 7-day administration of Dox reinstated extinguished METH-CPP. Collectively, these results suggest a synergistic effect of Tat protein and METH on inhibition of VMAT2-mediated DA transport, potentially contributing to potentiation of METH-CPP in iTat-tg mice.

Signaling Modulation Mediated by Ligand Water Interactions with the Sodium Site at µOR.

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 RO76 where 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 RO76 bound to the µOR-Gi complex confirmed that RO76 interacts with the sodium site residues through a water molecule, unlike C6guano which 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, RO76 was 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.

A µ-opioid receptor modulator that works cooperatively with naloxone.

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.

SRI-30827, a novel allosteric modulator of the dopamine transporter, alleviates HIV-1 Tat-induced potentiation of cocaine conditioned place preference in mice.

Objectives: HIV-1 Tat (transactivator of transcription) protein disrupts dopaminergic transmission and potentiates the rewarding effects of cocaine. Allosteric modulators of the dopamine transporter (DAT) have been shown to reverse Tat-induced DAT dysfunction. We hypothesized that a novel DAT allosteric modulator, SRI-30827, would counteract Tat-induced potentiation of cocaine reward. Methods: Doxycycline (Dox)-inducible Tat transgenic (iTat-tg) mice and their G-tg (Tat-null) counterparts were tested in a cocaine conditioned place preference (CPP) paradigm. Mice were treated 14 days with saline, or Dox (100 mg/kg/day, i.p.) to induce Tat protein. Upon induction, mice were place conditioned two days with cocaine (10 mg/kg/day) after a 1-h daily intracerebroventricular (i.c.v.) pretreatment with SRI-30827 (1 nmol) or a vehicle control, and final place preference assessed as a measure of cocaine reward. Results: Dox-treatment significantly potentiated cocaine-CPP in iTat-tg mice over the response of saline-treated control littermates. SRI-30827 treatment eliminated Tat-induced potentiation without altering normal cocaine-CPP in saline-treated mice. Likewise, SRI-30827 did not alter cocaine-CPP in both saline- and Dox-treated G-tg mice incapable of expressing Tat protein. Conclusions: These findings add to a growing body of evidence that allosteric modulation of DAT could provide a promising therapeutic intervention for patients with comorbid HIV-1 and cocaine use disorder (CUD).

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

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

Tryptophan Substitution in CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) Introduces δ-Opioid Receptor Antagonism, Preventing Antinociceptive Tolerance and Stress-Induced Reinstatement of Extinguished Cocaine-Conditioned Place Preference.

The macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibit kappa opioid receptor (KOR) antagonism which prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference. Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides' opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [35S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2'-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.

Solid-Phase Synthesis of the Bicyclic Peptide OL-CTOP Containing Two Disulfide Bridges, and an Assessment of Its In Vivo µ-Opioid Receptor Antagonism after Nasal Administration.

New strategies facilitate the design of cyclic peptides which can penetrate the brain. We have designed a bicyclic peptide, OL-CTOP, composed of the sequences of a selective µ-opioid receptor antagonist, CTOP (f-cyclo(CYwOTX)T) (X = penicillamine, Pen; O = ornithine) and odorranalectin, OL (YASPK-cyclo(CFRYPNGVLAC)T), optimized its solid-phase synthesis and demonstrated its ability for nose-to-brain delivery and in vivo activity. The differences in reactivity of Cys and Pen thiol groups protected with trityl and/or acetamidomethyl protecting groups toward I2 in different solvents were exploited for selective disulfide bond formation on the solid phase. Both the single step and the sequential strategy applied to macrocyclization reactions generated the desired OL-CTOP, with the sequential strategy yielding a large quantity and better purity of crude OL-CTOP. Importantly, intranasally (i.n.s.) administered OL-CTOP dose-dependently antagonized the analgesic effect of morphine administered to mice through the intracerebroventricular route and prevented morphine-induced respiratory depression. In summary, the results demonstrate the feasibility of our solid-phase synthetic strategy for the preparation of the OL-CTOP bicyclic peptide containing two disulfide bonds and reveal the potential of odorranalectin for further modifications and the targeted delivery to the brain.

Structure-based design of bitopic ligands for the µ-opioid receptor.

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 site2 found in µOR3 and 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 Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy 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, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Bis-Cyclic Guanidine Heterocyclic Peptidomimetics as Opioid Ligands with Mixed µ-, κ- and δ-Opioid Receptor Interactions: A Potential Approach to Novel Analgesics.

The design and development of analgesics with mixed-opioid receptor interactions has been reported to decrease side effects, minimizing respiratory depression and reinforcing properties to generate safer analgesic therapeutics. We synthesized bis-cyclic guanidine heterocyclic peptidomimetics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for the mu-opioid receptor (MOR), delta-opioid receptor (DOR), and kappa-opioid receptor (KOR) across the series, with compound 1968-22 displaying good affinity for all three receptors. Central intracerebroventricular (i.c.v.) administration of 1968-22 produced dose-dependent, opioid receptor-mediated antinociception in the mouse 55 °C warm-water tail-withdrawal assay, and 1968-22 also produced significant antinociception up to 80 min after oral administration (10 mg/kg, p.o.). Compound 1968-22 was detected in the brain 5 min after intravenous administration and was shown to be stable in the blood for at least 30 min. Central administration of 1968-22 did not produce significant respiratory depression, locomotor effects or conditioned place preference or aversion. The data suggest these bis-cyclic guanidine heterocyclic peptidomimetics with multifunctional opioid receptor activity may hold potential as new analgesics with fewer liabilities of use.

SRI-32743, a novel allosteric modulator, attenuates HIV-1 Tat protein-induced inhibition of the dopamine transporter and alleviates the potentiation of cocaine reward in HIV-1 Tat transgenic mice.

Cocaine abuse increases the incidence of HIV-1-associated neurocognitive disorders. We have demonstrated that HIV-1 transactivator of transcription (Tat) allosterically modulates dopamine (DA) reuptake through the human DA transporter (hDAT), potentially contributing to Tat-induced cognitive impairment and potentiation of cocaine conditioned place preference (CPP). This study determined the effects of a novel allosteric modulator of DAT, SRI-32743, on the interactions of HIV-1 Tat, DA, cocaine, and [3H]WIN35,428 with hDAT in vitro. SRI-32743 (50 nM) attenuated Tat-induced inhibition of [3H]DA uptake and decreased the cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting a SRI-32743-mediated allosteric modulation of the Tat-DAT interaction. In further in vivo studies utilizing doxycycline-inducible Tat transgenic (iTat-tg) mice, 14 days of Tat expression significantly reduced the recognition index by 31.7% in the final phase of novel object recognition (NOR) and potentiated cocaine-CPP 2.7-fold compared to responses of vehicle-treated control iTat-tg mice. The Tat-induced NOR deficits and potentiation of cocaine-CPP were not observed in saline-treated iTat-tg or doxycycline-treated G-tg (Tat-null) mice. Systemic administration (i.p.) of SRI-32743 prior to behavioral testing ameliorated Tat-induced impairment of NOR (at a dose of 10 mg/kg) and the Tat-induced potentiation of cocaine-CPP (at doses of 1 or 10 mg/kg). These findings demonstrate that Tat and cocaine interactions with DAT may be regulated by compounds interacting at the DAT allosteric modulatory sites, suggesting a potential therapeutic intervention for HIV-infected patients with concurrent cocaine abuse.

Characterization of CM-398, a Novel Selective Sigma-2 Receptor Ligand, as a Potential Therapeutic for Neuropathic Pain.

Sigma receptors modulate nociception, offering a potential therapeutic target to treat pain, but relatively little is known regarding the role of sigma-2 receptors (S2R) in nociception. The purpose of this study was to investigate the in vivo analgesic and anti-allodynic activity and liabilities of a novel S2R selective ligand, 1-[4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)butyl]-3-methyl-1,3-dihydro-1,3-benzimidazol-2-one (CM-398). The inhibition of thermal, induced chemical, or inflammatory pain as well as the allodynia resulting from chronic nerve constriction injury (CCI) model of neuropathic pain were assessed in male mice. CM-398 dose-dependently (10-45 mg/kg i.p.) reduced mechanical allodynia in the CCI neuropathic pain model, equivalent at the higher dose to the effect of the control analgesic gabapentin (50 mg/kg i.p.). Likewise, pretreatment (i.p.) with CM-398 dose-dependently produced antinociception in the acetic acid writhing test (ED50 (and 95% C.I.) = 14.7 (10.6-20) mg/kg, i.p.) and the formalin assay (ED50 (and 95% C.I.) = 0.86 (0.44-1.81) mg/kg, i.p.) but was without effect in the 55 °C warm-water tail-withdrawal assay. A high dose of CM-398 (45 mg/kg, i.p.) exhibited modest locomotor impairment in a rotarod assay and conditioned place aversion, potentially complicating the interpretation of nociceptive testing. However, in an operant pain model resistant to these confounds, mice experiencing CCI and treated with CM-398 demonstrated robust conditioned place preference. Overall, these results demonstrate the S2R selective antagonist CM-398 produces antinociception and anti-allodynia with fewer liabilities than established therapeutics, adding to emerging data suggesting possible mediation of nociception by S2R, and the development of S2R ligands as potential treatments for chronic pain.

An analog of [d-Trp]CJ-15,208 exhibits kappa opioid receptor antagonism following oral administration and prevents stress-induced reinstatement of extinguished morphine conditioned place preference.

Opioid use disorder (OUD) relapse rates are discouragingly high, underscoring the need for new treatment options. The macrocyclic tetrapeptide natural product CJ-15,208 and its stereoisomer [d-Trp]CJ-15,208 demonstrate kappa opioid receptor (KOR) antagonist activity upon oral administration which prevents stress-induced reinstatement of cocaine-seeking behavior. In order to further explore the structure-activity relationships and expand the potential therapeutic applications of KOR antagonism for the treatment of OUD, we screened a series of 24 analogs of [d-Trp]CJ-15,208 with the goal of enhancing KOR antagonist activity. From this screening, analog 22 arose as a compound of interest, demonstrating dose-dependent KOR antagonism after central and oral administration lasting at least 2.5 h. In further oral testing, analog 22 lacked respiratory, locomotor, or reinforcing effects, consistent with the absence of opioid agonism. Pretreatment with analog 22 (30 mg/kg, p.o.) prevented stress-induced reinstatement of extinguished morphine conditioned place preference and reduced some signs of naloxone-precipitated withdrawal in mice physically dependent on morphine. Collectively, these data support the therapeutic potential of KOR antagonists to support abstinence in OUD and ameliorate opioid withdrawal.

Examination of the Novel Sigma-1 Receptor Antagonist, SI 1/28, for Antinociceptive and Anti-allodynic Efficacy against Multiple Types of Nociception with Fewer Liabilities of Use.

Neuropathic pain is a significant problem with few effective treatments lacking adverse effects. The sigma-1 receptor (S1R) is a potential therapeutic target for neuropathic pain, as antagonists for this receptor effectively ameliorate pain in both preclinical and clinical studies. The current research examines the antinociceptive and anti-allodynic efficacy of SI 1/28, a recently reported benzylpiperazine derivative and analog of the S1R antagonist SI 1/13, that was 423-fold more selective for S1R over the sigma-2 receptor (S2R). In addition, possible liabilities of respiration, sedation, and drug reinforcement caused by SI 1/28 have been evaluated. Inflammatory and chemical nociception, chronic nerve constriction injury (CCI) induced mechanical allodynia, and adverse effects of sedation in a rotarod assay, conditioned place preference (CPP), and changes in breath rate and locomotor activity were assessed after i.p. administration of SI 1/28. Pretreatment with SI 1/28 produced dose-dependent antinociception in the formalin test, with an ED50 (and 95% C.I.) value of 13.2 (7.42-28.3) mg/kg, i.p. Likewise, SI 1/28 produced dose-dependent antinociception against visceral nociception and anti-allodynia against CCI-induced neuropathic pain. SI 1/28 demonstrated no impairment of locomotor activity, conditioned place preference, or respiratory depression. In summary, SI 1/28 proved efficacious in the treatment of acute inflammatory pain and chronic neuropathy without liabilities at therapeutic doses, supporting the development of S1R antagonists as therapeutics for chronic pain.

Expression of Human Immunodeficiency Virus Transactivator of Transcription (HIV-Tat1-86) Protein Alters Nociceptive Processing that is Sensitive to Anti-Oxidant and Anti-Inflammatory Interventions.

Despite the success of combined antiretroviral therapy (cART) in reducing viral load, a substantial portion of Human Immunodeficiency Virus (HIV)+ patients report chronic pain. The exact mechanism underlying this co-morbidity even with undetectable viral load remains unknown, but the transactivator of transcription (HIV-Tat) protein is of particular interest. Functional HIV-Tat protein is observed even in cerebrospinal fluid of patients who have an undetectable viral load. It is hypothesized that Tat protein exposure is sufficient to induce neuropathic pain-like manifestations via both activation of microglia and generation of oxidative stress. iTat mice conditionally expressed Tat(1-86) protein in the central nervous system upon daily administration of doxycycline (100 mg/kg/d, i.p., up to 14 days). The effect of HIV-Tat protein exposure on the well-being of the animal was assessed using sucrose-evoked grooming and acute nesting behavior for pain-depressed behaviors, and the development of hyperalgesia assessed with warm-water tail-withdrawal and von Frey assays for thermal hyperalgesia and mechanical allodynia, respectively. Tissue harvested at select time points was used to assess ex vivo alterations in oxidative stress, astrocytosis and microgliosis, and blood-brain barrier integrity with assays utilizing fluorescence-based indicators. Tat protein induced mild thermal hyperalgesia but robust mechanical allodynia starting after 4 days of exposure, reaching a nadir after 7 days. Changes in nociceptive processing were associated with reduced sucrose-evoked grooming behavior without altering acute nesting behavior, and in spinal cord dysregulated free radical generation as measured by DCF fluorescence intensity, altered immunohistochemical expression of the gliotic markers, Iba-1 and GFAP, and increased permeability of the blood-brain barrier to the small molecule fluorescent tracer, sodium fluorescein, in a time-dependent manner. Pretreatment with the anti-inflammatory, indomethacin (1 mg/kg/d, i.p.), the antioxidant, methylsulfonylmethane (100 mg/kg/d i.p.), or the immunomodulatory agent, dimethylfumarate (100 mg/kg/d p.o.) thirty minutes prior to daily injections of doxycycline (100 mg/kg/d i.p.) over 7 days significantly attenuated the development of Tat-induced mechanical allodynia. Collectively, the data suggests that even acute exposure to HIV-1 Tat protein at pathologically relevant levels is sufficient to produce select neurophysiological and behavioral manifestations of chronic pain consistent with that reported by HIV-positive patients.

Oxidative Metabolism as a Modulator of Kratom's Biological Actions.

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 vitro and in vivo pharmacological 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.

A Novel Mitragynine Analog with Low-Efficacy Mu Opioid Receptor Agonism Displays Antinociception with Attenuated Adverse Effects.

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-OCH3 group 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 vivo with reduced opioid liabilities.

Kratom Alkaloids as Probes for Opioid Receptor Function: Pharmacological Characterization of Minor Indole and Oxindole Alkaloids from Kratom.

Dry leaves of kratom (mitragyna speciosa) are anecdotally consumed as pain relievers and antidotes against opioid withdrawal and alcohol use disorders. There are at least 54 alkaloids in kratom; however, investigations to date have focused around mitragynine, 7-hydroxy mitragynine (7OH), and mitragynine pseudoindoxyl (MP). Herein, we probe a few minor indole and oxindole based alkaloids, reporting the receptor affinity, G-protein activity, and ßarrestin-2 signaling of corynantheidine, corynoxine, corynoxine B, mitraciliatine, and isopaynantheine at mouse and human opioid receptors. We identify corynantheidine as a mu opioid receptor (MOR) partial agonist, whereas its oxindole derivative corynoxine was an MOR full agonist. Similarly, another alkaloid mitraciliatine was found to be an MOR partial agonist, while isopaynantheine was a KOR agonist which showed reduced ßarrestin-2 recruitment. Corynantheidine, corynoxine, and mitraciliatine showed MOR dependent antinociception in mice, but mitraciliatine and corynoxine displayed attenuated respiratory depression and hyperlocomotion compared to the prototypic MOR agonist morphine in vivo when administered supraspinally. Isopaynantheine on the other hand was identified as the first kratom derived KOR agonist in vivo. While these minor alkaloids are unlikely to play the majority role in the biological actions of kratom, they represent excellent starting points for further diversification as well as distinct efficacy and signaling profiles with which to probe opioid actions in vivo.

Binge ethanol consumption-associated behavioral impairments in male mice using a gelatin-based drinking-in-the dark model.

BACKGROUND: Acute intoxication caused by binge ethanol drinking is linked to widespread impairments in brain functions. Various alcohol administration paradigms have been used in animals to model the heterogeneous clinical manifestation of intoxication in people. It is challenging to model a procedure that produces "visible intoxication" in rodents; however, manipulation of variables such as route of alcohol administration, time of availability, frequency, and duration and amount of ethanol exposure has achieved some success. In the current study, we employed a modified drinking-in-the-dark model to assess the validity of this model in producing "post-ethanol consumption intoxication" impairments following prolonged repeated daily voluntary "binge" ethanol consumption. METHODS: Adult male C57BL/6J mice were allowed a daily 3-h access to non-alcoholic plain or ethanol-containing gel during the dark cycle for a total of 83 days. After the initial 2-month daily DID, ethanol intake patterns were intensely characterized during the next 3 weeks. Immediately following the last DID session (day 83), plain and ethanol gel-consuming mice were then subjected to behavioral tests of locomotor ability and/or anxiety (cylinder, wire grip, open field) followed by blood ethanol concentration measurement. RESULT: Mice exhibited a relatively consistent ethanol consumption pattern during and across daily access periods. Ethanol intake of individual mice positively correlated with blood ethanol concentration that averaged 61.64 ± 2.84 mg/dL (n = 12). Compared to the plain gel-consuming control mice, ethanol gel mice exhibited significant locomotor impairment as well as anxiety-like behavior, with the magnitude of impairments of key indices well correlated with blood ethanol levels. CONCLUSION: The gelatin vehicle-based voluntary ethanol drinking-in-the-dark model reliably produced post consumption acute movement impairments as well as anxiety-like behaviors even after 2 months of daily binge ethanol consumption in male mice. Taken together, this mouse binge ethanol model should facilitate the investigation of mechanisms of binge drinking in subjects chronically abusing ethanol and the search for effective novel treatment strategies.

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.

Controlling opioid receptor functional selectivity by targeting distinct subpockets of the orthosteric site.

Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here, we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex with MP1104, to rationally design novel mixed mu (MOR) and kappa (KOR) opioid receptor agonists with reduced arrestin signaling. Analysis of structure-activity relationships for new MP1104 analogs points to a region between transmembrane 5 (TM5) and extracellular loop (ECL2) as key for modulation of arrestin recruitment to both MOR and KOR. The lead compounds, MP1207 and MP1208, displayed MOR/KOR Gi-partial agonism with diminished arrestin signaling, showed efficient analgesia with attenuated liabilities, including respiratory depression and conditioned place preference and aversion in mice. The findings validate a novel structure-inspired paradigm for achieving beneficial in vivo profiles for analgesia through different mechanisms that include bias, partial agonism, and dual MOR/KOR agonism.