Comparative Pharmacological Effects of Lisuride and Lysergic Acid Diethylamide Revisited.

Lisuride is a non-psychedelic serotonin (5-HT) 2A receptor (5-HT2A) agonist and analogue of the psychedelic lysergic acid diethylamide (LSD). Lisuride also acts as an agonist at the serotonin 1A receptor (5-HT1A), a property known to counter psychedelic effects. Here, we tested whether lisuride lacks psychedelic activity due to a dual mechanism: (1) partial agonism at 5-HT2A and (2) potent agonism at 5-HT1A. The in vitro effects of lisuride, LSD, and related analogues on 5-HT2A signaling were characterized by using miniGαq and ß-arrestin 2 recruitment assays. The 5-HT1A- and 5-HT2A-mediated effects of lisuride and LSD were also compared in male C57BL/6J mice. The in vitro results confirmed that LSD is an agonist at 5-HT2A, with high efficacy and potency for recruiting miniGαq and ß-arrestin 2. By contrast, lisuride displayed partial efficacy for both functional end points (6-52% of 5-HT or LSD Emax) and antagonized the effects of LSD. The mouse experiments demonstrated that LSD induces head twitch responses (HTRs)(ED50 = 0.039 mg/kg), while lisuride suppresses HTRs (ED50 = 0.006 mg/kg). Lisuride also produced potent hypothermia and hypolocomotion (ED50 = 0.008-0.023 mg/kg) that was blocked by the 5-HT1A antagonist WAY100635 (3 mg/kg). Blockade of 5-HT1A prior to lisuride restored basal HTRs, but it failed to increase HTRs above baseline levels. HTRs induced by LSD were blocked by lisuride (0.03 mg/kg) or the 5-HT1A agonist 8-OH-DPAT (1 mg/kg). Overall, our findings show that lisuride is an ultrapotent 5-HT1A agonist in C57BL/6J mice, limiting its use as a 5-HT2A ligand in mouse studies examining acute drug effects. Results also indicate that the 5-HT2A partial agonist-antagonist activity of lisuride explains its lack of psychedelic effects.

Psychedelic-like Activity of Norpsilocin Analogues.

Primary metabolites of mushroom tryptamines, psilocybin and baeocystin (i.e., psilocin and norpsilocin), exhibit potent agonist activity at the serotonin 2A receptor (5-HT2A) in vitro but differ in their 5-HT2A-mediated effects in vivo. In particular, psilocin produces centrally mediated psychedelic effects in vivo, whereas norpsilocin, differing only by the loss of an N-methyl group, is devoid of psychedelic-like effects. These observations suggest that the secondary methylamine group in norpsilocin impacts its central nervous system (CNS) bioavailability but not its receptor pharmacodynamics. To test this hypothesis, eight norpsilocin derivatives were synthesized with varied secondary alkyl-, allyl-, and benzylamine groups, primarily aiming to increase their lipophilicity and brain permeability. Structure-activity relationships for the norpsilocin analogues were evaluated using the mouse head-twitch response (HTR) as a proxy for CNS-mediated psychedelic-like effects. HTR studies revealed that extending the N-methyl group of norpsilocin by a single methyl group, to give the corresponding secondary N-ethyl analogue (4-HO-NET), was sufficient to produce psilocin-like activity (median effective dose or ED50 = 1.4 mg/kg). Notably, N-allyl, N-propyl, N-isopropyl, and N-benzyl derivatives also induced psilocin-like HTR activity (ED50 = 1.1-3.2 mg/kg), with variable maximum effects (26-77 total HTR events). By contrast, adding bulkier tert-butyl or cyclohexyl groups in the same position did not elicit psilocin-like HTRs. Pharmacological assessments of the tryptamine series in vitro demonstrated interactions with multiple serotonin receptor subtypes, including 5-HT2A, and other CNS signaling proteins (e.g., sigma receptors). Overall, our data highlight key structural requirements for CNS-mediated psychedelic-like effects of norpsilocin analogues.

Unique pharmacodynamic properties and low abuse liability of the µ-opioid receptor ligand (S)-methadone.

(R,S)-methadone ((R,S)-MTD) is a µ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers. (S)-MTD is being developed as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. We compared the pharmacology of (R)-MTD and (S)-MTD and found they bind to MORs, but not NMDARs, and induce full analgesia. Unlike (R)-MTD, (S)-MTD was a weak reinforcer that failed to affect extracellular dopamine or induce locomotor stimulation. Furthermore, (S)-MTD antagonized motor and dopamine releasing effects of (R)-MTD. (S)-MTD acted as a partial agonist at MOR, with complete loss of efficacy at the MOR-galanin Gal1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use. One-sentence summary: (S)-MTD, like (R)-MTD, binds to and activates MORs in vitro, but (S)-MTD antagonizes the MOR-Gal1R heteromer, decreasing its abuse liability.

Alkoxy chain length governs the potency of 2-benzylbenzimidazole 'nitazene' opioids associated with human overdose.

RATIONALE: Novel synthetic opioids (NSOs) are emerging in recreational drug markets worldwide. In particular, 2-benzylbenzimidazole 'nitazene' compounds are problematic NSOs associated with serious clinical consequences, including fatal respiratory depression. Evidence from in vitro studies shows that alkoxy chain length can influence the potency of nitazenes at the mu-opioid receptor (MOR). However, structure-activity relationships (SARs) of nitazenes for inducing opioid-like effects in animal models are not well understood compared to relevant opioids contributing to the ongoing opioid crisis (e.g., fentanyl). OBJECTIVES: Here, we examined the in vitro and in vivo effects of nitazene analogues with varying alkoxy chain lengths (i.e., metonitazene, etonitazene, isotonitazene, protonitazene, and butonitazene) as compared to reference opioids (i.e., morphine and fentanyl). METHODS AND RESULTS: Nitazene analogues displayed nanomolar affinities for MOR in rat brain membranes and picomolar potencies to activate MOR in transfected cells. All compounds induced opioid-like effects on locomotor activity, hot plate latency, and body temperature in male mice, and alkoxy chain length markedly influenced potency. Etonitazene, with an ethoxy chain, was the most potent analogue in MOR functional assays (EC50 = 30 pM, Emax = 103%) and across all in vivo endpoints (ED50 = 3-12 µg/kg). In vivo SARs revealed that ethoxy, isopropoxy, and propoxy chains engendered higher potencies than fentanyl, whereas methoxy and butoxy analogues were less potent. MOR functional potencies, but not MOR affinities, were positively correlated with in vivo potencies to induce opioid effects. CONCLUSIONS: Overall, our data show that certain nitazene NSOs are more potent than fentanyl as MOR agonists in mice, highlighting concerns regarding the high potential for overdose in humans who are exposed to these compounds.

Comparative neuropharmacology of structurally distinct non-fentanyl opioids that are appearing on recreational drug markets worldwide.

BACKGROUND: The emergence of novel synthetic opioids (NSOs) is contributing to the opioid overdose crisis. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring. METHODS: Here, we examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human mu-opioid receptor (MOR). Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured. RESULTS: Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cyclic AMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED50s) correlated with in vitro functional potencies (i.e., EC50s) but not binding affinities (i.e., Kis) at MOR. CONCLUSIONS: Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets.

Receptor Binding Profiles for Tryptamine Psychedelics and Effects of 4-Propionoxy-N,N-dimethyltryptamine in Mice.

Analogues of 4-phosphoryloxy-N,N-dimethyltryptamine (psilocybin) are being sold on recreational drug markets and developed as potential medications for psychedelic-assisted therapies. Many of these tryptamine-based psilocybin analogues produce psychedelic-like effects in rodents and humans primarily by agonist activity at serotonin 2A receptors (5-HT2A). However, the comprehensive pharmacological target profiles for these compounds compared to psilocybin and its active metabolite 4-hydroxy-N,N-dimethyltryptamine (psilocin) are unknown. The present study determined the receptor binding profiles of various tryptamine-based psychedelics structurally related to psilocybin across a broad range of potential targets. Specifically, we examined tryptamine psychedelics with different 4-position (hydroxy, acetoxy, propionoxy) and N,N-dialkyl (dimethyl, methyl-ethyl, diethyl, methyl-propyl, ethyl-propyl, diisopropyl, methyl-allyl, diallyl) substitutions. Further, the psilocybin analogue 4-propionoxy-N,N-dimethyltryptamine (4-PrO-DMT) was administered to mice in experiments measuring head twitch response (HTR), locomotor activity, and body temperature. Overall, the present pharmacological profile screening data show that the tryptamine psychedelics target multiple serotonin receptors, including serotonin 1A receptors (5-HT1A). 4-Acetoxy and 4-propionoxy analogues of 4-hydroxy compounds displayed somewhat weaker binding affinities but similar target profiles across 5-HT receptors and other identified targets. Additionally, differential binding screen profiles were observed with N,N-dialkyl position variations across several non-5-HT receptor targets (i.e., alpha receptors, dopamine receptors, histamine receptors, and serotonin transporters), which could impact in vivo pharmacological effects of the compounds. In mouse experiments, 4-PrO-DMT displayed dose-related psilocybin-like effects to produce 5-HT2A-mediated HTR (0.3-3 mg/kg s.c.) as well as 5-HT1A-mediated hypothermia and hypolocomotion (3-30 mg/kg s.c.). Lastly, our data support a growing body of evidence that the 5-HT2A-mediated HTR induced by tryptamine psychedelics is attenuated by 5-HT1A receptor agonist activity at high doses in mice.

Plasma pharmacokinetics and pharmacodynamic effects of the 2-benzylbenzimidazole synthetic opioid, isotonitazene, in male rats.

RATIONALE: Isotonitazene is an illicit synthetic opioid associated with many intoxications and fatalities. Recent studies show that isotonitazene is a potent µ-opioid receptor (MOR) agonist in vitro, but little information is available about its in vivo effects. OBJECTIVES: The aims of the present study were to investigate the pharmacokinetics of isotonitazene in rats, and relate pharmacokinetic parameters to pharmacodynamic effects. METHODS: Isotonitazene and its metabolites were identified and quantified by liquid chromatography tandem quadrupole mass spectrometry (LC-QQQ-MS). Male Sprague-Dawley rats with jugular catheters and subcutaneous (s.c.) temperature transponders received isotonitazene (3, 10, 30 µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, and 240 min post-injection, and plasma was assayed using LC-QQQ-MS. At each blood draw, body temperature, catalepsy scores, and hot plate latencies were recorded. RESULTS: Maximum plasma concentrations of isotonitazene rose in parallel with increasing dose (range 0.2-9.8 ng/mL) and half-life ranged from 23.4 to 63.3 min. The metabolites 4'-hydroxy nitazene and N-desethyl isotonitazene were detected, and plasma concentrations were below the limit of quantitation (0.5 ng/mL) but above the limit of detection (0.1 ng/mL). Isotonitazene produced antinociception (ED50 = 4.22 µg/kg), catalepsy-like symptoms (ED50 = 8.68 µg/kg), and hypothermia (only at 30 µg/kg) that were significantly correlated with concentrations of isotonitazene. Radioligand binding in rat brain tissue revealed that isotonitazene displays nM affinity for MOR (Ki = 15.8 nM), while the N-desethyl metabolite shows even greater affinity (Ki = 2.2 nM). CONCLUSIONS: In summary, isotonitazene is a potent MOR agonist whose pharmacodynamic effects are related to circulating concentrations of the parent drug. The high potency of isotonitazene portends substantial risk to users who are exposed to the drug.

Structure-Activity Relationships for Psilocybin, Baeocystin, Aeruginascin, and Related Analogues to Produce Pharmacological Effects in Mice.

4-Phosphoryloxy-N,N-dimethyltryptamine (psilocybin) is a naturally occurring tertiary amine found in many mushroom species. Psilocybin is a prodrug for 4-hydroxy-N,N-dimethyltryptamine (psilocin), which induces psychedelic effects via agonist activity at the serotonin (5-HT) 2A receptor (5-HT2A). Several other 4-position ring-substituted tryptamines are present in psilocybin-containing mushrooms, including the secondary amine 4-phosphoryloxy-N-methyltryptamine (baeocystin) and the quaternary ammonium 4-phosphoryloxy-N,N,N-trimethyltryptamine (aeruginascin), but these compounds are not well studied. Here, we investigated the structure-activity relationships for psilocybin, baeocystin, and aeruginascin, as compared to their 4-acetoxy and 4-hydroxy analogues, using in vitro and in vivo methods. Broad receptor screening using radioligand binding assays in transfected cells revealed that secondary and tertiary tryptamines with either 4-acetoxy or 4-hydroxy substitutions display nanomolar affinity for most human 5-HT receptor subtypes tested, including the 5-HT2A and the serotonin 1A receptor (5-HT1A). The same compounds displayed affinity for 5-HT2A and 5-HT1A in mouse brain tissue in vitro and exhibited agonist efficacy in assays examining 5-HT2A-mediated calcium mobilization and ß-arrestin 2 recruitment. In mouse experiments, only the tertiary amines psilocin, psilocybin, and 4-acetoxy-N,N-dimethyltryptamine (psilacetin) induced head twitch responses (ED50 0.11-0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT2A antagonist pretreatment, supporting 5-HT2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, the effects of which were blocked by 5-HT1A antagonist pretreatment. Across all assays, the pharmacological effects of 4-acetoxy and 4-hydroxy compounds were similar, and these compounds were more potent than their 4-phosphoryloxy counterparts. Importantly, psilacetin appears to be a prodrug for psilocin that displays substantial serotonin receptor activities of its own.

Methods to Assess Melatonin Receptor-Mediated Phase-Shift and Re-entrainment of Rhythmic Behaviors in Mouse Models.

The neurohormone melatonin facilitates entrainment of biological rhythms to environmental light-dark conditions as well as phase-shifts of circadian rhythms in constant conditions via activation of the MT1 and/or MT2 receptors expressed within the suprachiasmatic nucleus of the hypothalamus. The efficacy of melatonin and related agonists to modulate biological rhythms can be assessed using two well-validated mouse models of rhythmic behaviors. These models serve as predictive measures of therapeutic efficacy for treatment of circadian phase disorders caused by internal (e.g., clock gene mutations, blindness, depression, seasonal affective disorder) or external (e.g., shift work, travel across time zones) causes in humans. Here we provide background and detailed protocols for quantitative assessment of the magnitude and efficacy of melatonin receptor ligands in mouse circadian phase-shift and re-entrainment paradigms. The utility of these models in the discovery of novel therapeutics acting on melatonin receptors will also be discussed.

Synthesis, Structural Characterization, and Pharmacological Activity of Novel Quaternary Salts of 4-Substituted Tryptamines.

Aeruginascin (4-phosphoryloxy-N,N,N-trimethyltryptammonium) is an analogue of psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) that has been identified in several species of psilocybin-containing mushrooms. Our team previously reported the synthesis, structural characterization, and biological activity of the putative metabolite of aeruginascin (4-hydroxy-N,N,N-trimethyltryptammonium; 4-HO-TMT) and its potential prodrug (4-acetoxy-N,N,N-trimethyltryptammonium; 4-AcO-TMT). Here, we report the synthesis, structural characterization, and pharmacological activity of several quaternary tryptammonium analogues of 4-HO-TMT and 4-AcO-TMT, namely, 4-hydroxy-N,N-dimethyl-N-ethyltryptammonium (4-HO-DMET), 4-hydroxy-N,N-dimethyl-N-n-propyltryptammonium (4-HO-DMPT), and 4-hydroxy-N,N-dimethyl-N-isopropyltryptammonium (4-HO-DMiPT), as well as their hypothesized prodrugs 4-acetoxy-N,N-dimethyl-N-ethyltryptammonium (4-AcO-DMET), 4-acetoxy-N,N-dimethyl-N-n-propyltryptammonium (4-AcO-DMPT), and 4-acetoxy-N,N-dimethyl-N-isopropyltryptammonium (4-AcO-DMiPT). Compounds were synthesized using established methods, and structures were characterized by single-crystal X-ray diffraction. Test compounds were screened for in vitro pharmacological activity at a variety of receptors and transporters to determine potential targets of action. None of the compounds exhibited measurable affinity for the serotonin 2A receptor (5-HT2A), but several analogues had low micromolar affinity (K i) for the serotonin 1D receptor (5-HT1D) and serotonin 2B receptor (5-HT2B), where they appeared to be weak partial agonists with low micromolar potencies. Importantly, 4-HO-DMET, 4-HO-DMPT, and 4-HO-DMiPT displayed sub-micromolar affinity for the serotonin transporter (SERT; 370-890 nM). The same 4-hydroxy analogues had low to sub-micromolar potencies (IC50) for inhibition of 5-HT uptake at SERT in transfected cells (3.3-12.3 µM) and rat brain tissue (0.31-3.5 µM). Overall, our results show that quaternary tryptammonium analogues do not target 5-HT2A sites, suggesting the compounds lack psychedelic-like subjective effects. However, certain 4-hydroxy quaternary tryptammonium analogues may provide novel templates for exploring structure-activity relationships for selective actions at SERT.

Automated Computer Software Assessment of 5-Hydroxytryptamine 2A Receptor-Mediated Head Twitch Responses from Video Recordings of Mice.

Psychedelics are a class of drugs that produce unique subjective effects via agonist actions at the 5-hydroxytryptamine 2A receptor (5-HT2A). The 5-HT2A-mediated head twitch response (HTR) in rodents is used as a reliable proxy for psychedelic drug activity in humans, but existing methods for measuring HTRs require surgery or time-consuming visual scoring. In the present work, we validated a simple noninvasive method for quantitating HTRs using computer-based analysis of experimental video recordings. Male C57BL/6J mice received injections of the 5-HT2 receptor agonist (±)2,5-dimethoxy-4-iodoamphetamine (DOI; 0.03-3 mg/kg, s.c.) and were placed into cylindrical arenas. High frame rate videos were recorded via cameras mounted above the arenas. Antagonist experiments, which entailed pretreatment with the 5-HT2A antagonist M100907 (0.01 or 0.1 mg/kg s.c.) prior to DOI (1 mg/kg s.c.), were also recorded. The experimental videos were analyzed for HTRs using a newly developed feature of a commercial software package and compared to visual scoring carried out by trained observers. As expected, DOI produced dose-related increases in HTRs, which were blocked by M100907. Computer scoring was positively correlated with visual scoring, and no statistical difference between the two methods was found. The software captured nearly all visually observed HTRs, false positives induced by other behaviors (e.g., grooming) were rare and easily identified, and results were improved by optimizing lighting conditions. Our findings demonstrate the utility of combining high frame rate video recordings with commercial software analyses to measure HTRs, validating an additional reliable method to study psychedelic-like drug activity in mice.

First identification, chemical analysis and pharmacological characterization of N-piperidinyl etonitazene (etonitazepipne), a recent addition to the 2-benzylbenzimidazole opioid subclass.

N-Piperidinyl etonitazene ('etonitazepipne') represents a recent addition to the rapidly expanding class of 2-benzylbenzimidazole 'nitazene' opioids. Following its first identification in an online-sourced powder and in biological samples from a patient seeking help for detoxification, this report details its in-depth chemical analysis and pharmacological characterization. Analysis of the powder via different techniques (LC-HRMS, GC-MS, UHPLC-DAD, FT-IR) led to the unequivocal identification of N-piperidinyl etonitazene. Furthermore, we report the first activity-based detection and analytical identification of N-piperidinyl etonitazene in authentic samples. LC-HRMS analysis revealed concentrations of 1.21 ng/mL in serum and 0.51 ng/mL in urine, whereas molecular networking enabled the tentative identification of various (potentially active) urinary metabolites. In addition, we determined that the extent of opioid activity present in the patient's serum was equivalent to the in vitro opioid activity exerted by 2.5-10 ng/mL fentanyl or 10-25 ng/mL hydromorphone in serum. Radioligand binding assays in rat brain tissue revealed that the drug binds with high affinity (Ki = 14.3 nM) to the µ-opioid receptor (MOR). Using a MOR-ß-arrestin2 activation assay, we found that N-piperidinyl etonitazene is highly potent (EC50 = 2.49 nM) and efficacious (Emax = 183% versus hydromorphone) in vitro. Pharmacodynamic evaluation in male Sprague Dawley rats showed that N-piperidinyl etonitazene induces opioid-like antinociceptive, cataleptic, and thermic effects, its potency in the hot plate assay (ED50 = 0.0205 mg/kg) being comparable to that of fentanyl (ED50 = 0.0209 mg/kg), and > 190 times higher than that of morphine (ED50 = 3.940 mg/kg). Taken together, our findings indicate that N-piperidinyl etonitazene is a potent opioid with the potential to cause harm in users.

Pharmacological evaluation and forensic case series of N-pyrrolidino etonitazene (etonitazepyne), a newly emerging 2-benzylbenzimidazole 'nitazene' synthetic opioid.

Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles ('nitazenes'), are being exploited to create new µ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligand binding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09 nM) over δ-opioid (Ki = 959 nM) and κ-opioid (Ki = 980 nM) receptors. In a MOR-ß-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348 nM), similar to etonitazene (EC50 = 0.360 nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9 nM) and morphine (EC50 = 290 nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50 = 0.0017 mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209 mg/kg) and morphine (ED50 = 3.940 mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2 ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.

(2-Aminopropyl)benzo[ß]thiophenes (APBTs) are novel monoamine transporter ligands that lack stimulant effects but display psychedelic-like activity in mice.

Derivatives of (2-aminopropyl)indole (API) and (2-aminopropyl)benzofuran (APB) are new psychoactive substances which produce stimulant effects in vivo. (2-Aminopropyl)benzo[ß]thiophene (APBT) is a novel sulfur-based analog of API and APB that has not been pharmacologically characterized. In the current study, we assessed the pharmacological effects of six APBT positional isomers in vitro, and three of these isomers (3-APBT, 5-APBT, and 6-APBT) were subjected to further investigations in vivo. Uptake inhibition and efflux assays in human transporter-transfected HEK293 cells and in rat brain synaptosomes revealed that APBTs inhibit monoamine reuptake and induce transporter-mediated substrate release. Despite being nonselective transporter releasers like MDMA, the APBT compounds failed to produce locomotor stimulation in C57BL/6J mice. Interestingly, 3-APBT, 5-APBT, and 6-APBT were full agonists at 5-HT2 receptor subtypes as determined by calcium mobilization assays and induced the head-twitch response in C57BL/6J mice, suggesting psychedelic-like activity. Compared to their APB counterparts, ABPT compounds demonstrated that replacing the oxygen atom with sulfur results in enhanced releasing potency at the serotonin transporter and more potent and efficacious activity at 5-HT2 receptors, which fundamentally changed the in vitro and in vivo profile of APBT isomers in the present studies. Overall, our data suggest that APBT isomers may exhibit psychedelic and/or entactogenic effects in humans, with minimal psychomotor stimulation. Whether this unique pharmacological profile of APBT isomers translates into potential therapeutic potential, for instance as candidates for drug-assisted psychotherapy, warrants further investigation.

Structure-activity relationships for 5F-MDMB-PICA and its 5F-pentylindole analogs to induce cannabinoid-like effects in mice.

Synthetic cannabinoid receptor agonists (SCRAs) are an evolving class of new psychoactive substances found on recreational drug markets worldwide. The indole-containing compound, 5F-MDMB-PICA, is a popular SCRA associated with serious medical consequences, including overdose and hospitalizations. In vitro studies reveal that 5F-MDMB-PICA is a potent agonist at cannabinoid type 1 receptors (CB1), but little information exists regarding in vivo pharmacology of the drug. To this end, we examined the in vitro and in vivo cannabinoid-like effects produced by 5F-MDMB-PICA and related 5F-pentylindole analogs with differing composition of the head group moiety (i.e., 5F-NNEI, 5F-SDB-006, 5F-CUMYL-PICA, 5F-MMB-PICA). In mouse brain membranes, 5F-MDMB-PICA and its analogs inhibited binding to [3H]rimonabant-labeled CB1 and displayed agonist actions in [35S]GTPγS functional assays. 5F-MDMB-PICA exhibited the highest CB1 affinity (Ki = 1.24 nM) and functional potency (EC50 = 1.46 nM), but head group composition markedly influenced activity in both assays. For example, the 3,3-dimethylbutanoate (5F-MDMB-PICA) and cumyl (5F-CUMYL-PICA) head groups engendered high CB1 affinity and potency, whereas a benzyl (5F-SDB-006) head group did not. In C57BL/6J mice, all 5F-pentylindole SCRAs produced dose- and time-dependent hypothermia, catalepsy, and analgesia that were reversed by rimonabant, indicating CB1 involvement. In vitro Ki and EC50 values were positively correlated with in vivo ED50 potency estimates. Our findings demonstrate that 5F-MDMB-PICA is a potent SCRA, and subtle alterations to head group composition can have profound influence on pharmacological effects at CB1. Importantly, measures of CB1 binding and efficacy in mouse brain tissue seem to accurately predict in vivo drug potency in this species.

Eutylone and Its Structural Isomers Interact with Monoamine Transporters and Induce Locomotor Stimulation.

Synthetic cathinones are a class of new psychoactive substances that induce psychostimulant effects and pose risk for hospitalizations, overdose, and death. At the present time, derivatives of the synthetic cathinone, methylone, are being confiscated in nonmedical (i.e., recreational) drug markets worldwide. In particular, eutylone is a newly emerging methylone analog that possesses ethyl groups at the α-carbon and amine positions. Little information is available about the pharmacological effects of eutylone, but based on its structure, we surmised that the compound interacts with monoamine transporters in the brain. To test this hypothesis, we compared the effects of eutylone and its structural isomers, dibutylone and pentylone, using in vitro transporter assays in rat brain synaptosomes and in vivo locomotor activity assessments in mice. All drugs displayed dose-related inhibition of [3H]neurotransmitter uptake at dopamine transporters (DAT) and norepinephrine transporters (NET), but effects at DAT were 10-fold more potent (IC50 = 120 nM). Eutylone and pentylone inhibited uptake at serotonin transporters (SERT), while dibutylone did not. Additionally, eutylone and pentylone displayed weak partial releasing actions at SERT which achieved 50% of maximal response. All drugs stimulated dose-related locomotion in mice, and eutylone was most potent and efficacious in this regard (ED50 = 2 mg/kg, sc). Our results demonstrate that eutylone is a hybrid transporter compound with uptake inhibition properties at DAT and NET but substrate activity at SERT. The effects of eutylone are similar to those produced by pentylone, which suggests that eutylone will exhibit abuse liability and pose risks for psychostimulant side-effects in human users.

Pharmacological Actions of Carbamate Insecticides at Mammalian Melatonin Receptors.

Integrated in silico chemical clustering and melatonin receptor molecular modeling combined with in vitro 2-[125I]-iodomelatonin competition binding were used to identify carbamate insecticides with affinity for human melatonin receptor 1 (hMT1) and human melatonin receptor 2 (hMT2). Saturation and kinetic binding studies with 2-[125I]-iodomelatonin revealed lead carbamates (carbaryl, fenobucarb, bendiocarb, carbofuran) to be orthosteric ligands with antagonist apparent efficacy at hMT1 and agonist apparent efficacy at hMT2 Furthermore, using quantitative receptor autoradiography in coronal brain slices from C3H/HeN mice, carbaryl, fenobucarb, and bendiocarb competed for 2-[125I]-iodomelatonin binding in the suprachiasmatic nucleus (SCN), paraventricular nucleus of the thalamus (PVT), and pars tuberalis (PT) with affinities similar to those determined for the hMT1 receptor. Carbaryl (10 mg/kg i.p.) administered in vivo also competed ex vivo for 2-[125I]-iodomelatonin binding to the SCN, PVT, and PT, demonstrating the ability to reach brain melatonin receptors in C3H/HeN mice. Furthermore, the same dose of carbaryl given to C3H/HeN mice in constant dark for three consecutive days at subjective dusk (circadian time 10) phase-advanced circadian activity rhythms (mean = 0.91 hours) similar to melatonin (mean = 1.12 hours) when compared with vehicle (mean = 0.04 hours). Carbaryl-mediated phase shift of overt circadian activity rhythm onset is likely mediated via interactions with SCN melatonin receptors. Based on the pharmacological actions of carbaryl and other carbamate insecticides at melatonin receptors, exposure may modulate time-of-day information conveyed to the master biologic clock relevant to adverse health outcomes. SIGNIFICANCE STATEMENT: In silico chemical clustering and molecular modeling in conjunction with in vitro bioassays identified several carbamate insecticides (i.e., carbaryl, carbofuran, fenobucarb, bendiocarb) as pharmacologically active orthosteric melatonin receptor 1 and 2 ligands. This work further demonstrated that carbaryl competes for melatonin receptor binding in the master biological clock (suprachiasmatic nucleus) and phase-advances overt circadian activity rhythms in C3H/HeN mice, supporting the relevance of circadian effects when interpreting toxicological findings related to carbamate insecticide exposure.

Virtual discovery of melatonin receptor ligands to modulate circadian rhythms.

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT1 and MT2. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle1-4. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep5,6 and depression1-4,7-9. Despite their importance, few in vivo active MT1-selective ligands have been reported2,8,10-12, hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT1 inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT1-selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT1- but not in MT2-knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT1-selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.