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2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on μ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (β-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts., (© 2024. The Author(s).)

Background: Since late 2019, fortification of 'regular' cannabis plant material with synthetic cannabinoid receptor agonists (SCRAs) has become a notable phenomenon on the drug market. As many SCRAs pose a higher health risk than genuine cannabis, recognizing SCRA-adulterated cannabis is important from a harm reduction perspective. However, this is not always an easy task as adulterated cannabis may only be distinguished from genuine cannabis by dedicated, often expensive and time-consuming analytical techniques. In addition, the dynamic nature of the SCRA market renders identification of fortified samples a challenging task. Therefore, we established and applied an in vitro cannabinoid receptor 1 (CB 1 ) activity-based procedure to screen plant material for the presence of SCRAs., Methods: The assay principle relies on the functional complementation of a split-nanoluciferase following recruitment of β-arrestin 2 to activated CB 1 . A straightforward sample preparation, encompassing methanolic extraction and dilution, was optimized for plant matrices, including cannabis, spiked with 5 µg/mg of the SCRA CP55,940., Results: The bioassay successfully detected all samples of a set (n = 24) of analytically confirmed authentic Spice products, additionally providing relevant information on the 'strength' of a preparation and whether different samples may have originated from separate batches or possibly the same production batch. Finally, the methodology was applied to assess the occurrence of SCRA adulteration in a large set (n = 252) of herbal materials collected at an international dance festival. This did not reveal any positives, i.e. there were no samples that yielded a relevant CB 1 activation., Conclusion: In summary, we established SCRA screening of herbal materials as a new application for the activity-based CB 1 bioassay. The simplicity of the sample preparation, the rapid results and the universal character of the bioassay render it an effective and future-proof tool for evaluating herbal materials for the presence of SCRAs, which is relevant in the context of harm reduction., (© 2024. The Author(s).)

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A 3 adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2-substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds 9 - 11 , 13 , 14 , 19 , 22 , 23 , 27 , 29 , 31 , and 34 , lacking a spacer, had human (h) A 3 AR K i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A 3 AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues ( 7 , 8 > 3c , 3d > 3b ). However, 2-aryl-4'-truncated derivatives had greatly reduced hA 3 AR affinity, even containing affinity-enhancing N 6 -dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A 3 AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA 3 AR miniGα i recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a β-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy ( E max ) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, A 3 AR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues 3a - 3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A 3 AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A 3 AR agonist SAR for (N)-methanocarba adenosines., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

Lisuride is a non-psychedelic serotonin (5-HT) 2A receptor (5-HT 2A ) agonist and analogue of the psychedelic lysergic acid diethylamide (LSD). Lisuride also acts as an agonist at the serotonin 1A receptor (5-HT 1A ), 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-HT 2A and (2) potent agonism at 5-HT 1A . The in vitro effects of lisuride, LSD, and related analogues on 5-HT 2A signaling were characterized by using miniGα q and β-arrestin 2 recruitment assays. The 5-HT 1A - and 5-HT 2A -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-HT 2A , with high efficacy and potency for recruiting miniGα q and β-arrestin 2. By contrast, lisuride displayed partial efficacy for both functional end points (6-52% of 5-HT or LSD E max ) and antagonized the effects of LSD. The mouse experiments demonstrated that LSD induces head twitch responses (HTRs)(ED 50 = 0.039 mg/kg), while lisuride suppresses HTRs (ED 50 = 0.006 mg/kg). Lisuride also produced potent hypothermia and hypolocomotion (ED 50 = 0.008-0.023 mg/kg) that was blocked by the 5-HT 1A antagonist WAY100635 (3 mg/kg). Blockade of 5-HT 1A 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-HT 1A agonist 8-OH-DPAT (1 mg/kg). Overall, our findings show that lisuride is an ultrapotent 5-HT 1A agonist in C57BL/6J mice, limiting its use as a 5-HT 2A ligand in mouse studies examining acute drug effects. Results also indicate that the 5-HT 2A partial agonist-antagonist activity of lisuride explains its lack of psychedelic effects., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine ( 1 ), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1 . We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine- N -oxide. In mice, the two diastereomers of pellotine -O -glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT) 1D , 5-HT 6 , and 5-HT 7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT 6 and 5-HT 7 , where it was found to be an agonist at the former (EC 50 = 94 nM, E max = 32%) and an inverse agonist at the latter (EC 50 = 291 nM, E max = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

The A 3 adenosine receptor (A 3 AR) is implicated in a variety of (patho)physiological conditions. While most research has focused on agonists and antagonists, inverse agonism at A 3 AR has been scarcely studied. Therefore, this study aimed at exploring inverse agonism, using two previously engineered cell lines (hA 3 ARLgBiT-SmBiTβarr2 and hA 3 ARLgBiT-SmBiTminiGα i ), both employing the NanoBiT technology. The previously established inverse agonist PSB-10 showed a decrease in basal signal in the β-arrestin 2 (βarr2) but not the miniGα i recruitment assay, indicative of inverse agonism in the former assay. Control experiments confirmed the specificity and reversibility of this observation. Evaluation of a set of presumed neutral antagonists (MRS7907, MRS7799, XAC, and MRS1220) revealed that all displayed concentration-dependent signal decreases when tested in the A 3 AR-βarr2 recruitment assay, yielding EC 50 and E max values for inverse agonism. Conversely, in the miniGα i recruitment assay, no signal decreases were observed. To assess whether this observation was caused by the inability of the ligands to induce inverse agonism in the G protein pathway, or rather by a limitation inherent to the employed A 3 AR-miniGα i recruitment assay, a GloSensor cAMP assay was performed. The outcome of the latter also suggests inverse agonism by the presumed neutral antagonists in this latter assay. These findings emphasize the importance of prior characterization of ligands in the relevant test system. Moreover, it showed the suitability of the NanoBiT βarr2 recruitment and the GloSensor cAMP assays to capture inverse agonism at the A 3 AR, as opposed to the NanoBiT miniGα i recruitment assay., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Serotonergic psychedelics are described to have activation of the serotonin 2A receptor (5-HT 2A ) as their main pharmacological action. Despite their relevance, the molecular mechanisms underlying the psychedelic effects induced by certain 5-HT 2A agonists remain elusive. One of the proposed hypotheses is the occurrence of biased agonism, defined as the preferential activation of certain signaling pathways over others. This study comparatively monitored the efficiency of a diverse panel of 4-position-substituted (and N -benzyl-derived) phenylalkylamines to induce recruitment of β-arrestin2 (βarr2) or miniGα q to the 5-HT 2A , allowing us to assess structure-activity relationships and biased agonism. All test compounds exhibited agonist properties with a relatively large range of both EC 50 and E max values. Interestingly, the lipophilicity of the 2C-X phenethylamines was correlated with their efficacy in both assays but yielded a stronger correlation in the miniGα q - than in the βarr2-assay. Molecular docking suggested that accommodation of the 4-substituent of the 2C-X analogues in a hydrophobic pocket between transmembrane helices 4 and 5 of 5-HT 2A may contribute to this differential effect. Aside from previously used standard conditions (lysergic acid diethylamide (LSD) as a reference agonist and a 2 h activation profile to assess a compound's activity), serotonin was included as a second reference agonist, and the compounds' activities were also assessed using the first 30 min of the activation profile. Under all assessed circumstances, the qualitative structure-activity relationships remained unchanged. Furthermore, the use of two reference agonists allowed for the estimation of both "benchmark bias" (relative to LSD) and "physiology bias" (relative to serotonin).

New psychoactive substances (NPS) are introduced on the illicit drug market at a rapid pace. Their molecular targets are often inadequately elucidated, which contributes to the delayed characterization of their pharmacological effects. Inspired by earlier findings, this study set out to investigate the µ opioid receptor (MOR) activation potential of a large set of psychedelics, substances which typically activate the serotonin (5-HT 2A ) receptor as their target receptor. We observed that some substances carrying the N-benzyl phenethylamine (NBOMe) structure activated MOR, as confirmed by both the NanoBiT® βarr2 recruitment assay and the G protein-based AequoScreen® Ca 2+ release assay. The use of two orthogonal systems proved beneficial as some aspecific, receptor independent effects were found for various analogs when using the Ca 2+ release assay. The specific 'off-target' effects at MOR could be blocked by the opioid antagonist naloxone, suggesting that these NBOMes occupy the same common opioid binding pocket as conventional opioids. This was corroborated by molecular docking, which revealed the plausibility of multiple interactions of 25I-NBOMe with MOR, similar to those observed for opioids. Additionally, structure-activity relationship findings seen in vitro were rationalized in silico for two 25I-NBOMe isomers. Overall, as MOR activity of these psychedelics was only noticed at high concentrations, we consider it unlikely that for the tested compounds there will be a relevant opioid toxicity in vivo at physiologically relevant concentrations. However, small modifications to the original NBOMe structure may result in a panel of more efficacious and potent MOR agonists, potentially exhibiting a dual MOR/5-HT 2A activation potential., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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-HT 2A ). 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-HT 2A and the serotonin 1A receptor (5-HT 1A ). The same compounds displayed affinity for 5-HT 2A and 5-HT 1A in mouse brain tissue in vitro and exhibited agonist efficacy in assays examining 5-HT 2A -mediated calcium mobilization and β-arrestin 2 recruitment. In mouse experiments, only the tertiary amines psilocin, psilocybin, and 4-acetoxy- N , N -dimethyltryptamine (psilacetin) induced head twitch responses (ED 50 0.11-0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT 2A antagonist pretreatment, supporting 5-HT 2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, the effects of which were blocked by 5-HT 1A 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., Competing Interests: The authors declare the following competing financial interest(s): A.R.C. has an ownership stake in CaaMTech, Inc., which owns patent applications covering new tryptamine compounds, their compositions, formulations, novel crystalline forms, methods of treatment, and methods for synthesis. No other authors report any competing financial interests related to the present work., (© 2022 American Chemical Society.)

Since the recent clinical approval of siRNA-based drugs and COVID-19 mRNA vaccines, the potential of RNA therapeutics for patient healthcare has become widely accepted. Lipid nanoparticles (LNPs) are currently the most advanced nanocarriers for RNA packaging and delivery. Nevertheless, the intracellular delivery efficiency of state-of-the-art LNPs remains relatively low and safety and immunogenicity concerns with synthetic lipid components persist, altogether rationalizing the exploration of alternative LNP compositions. In addition, there is an interest in exploiting LNP technology for simultaneous encapsulation of small molecule drugs and RNA in a single nanocarrier. Here, we describe how well-known tricyclic cationic amphiphilic drugs (CADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes. We demonstrate that selected CADs, such as tricyclic antidepressants and antihistamines, self-assemble with the widely-used helper lipid DOPE to form cationic lipid vesicles for subsequent mRNA complexation and delivery, without the need for prior lipophilic derivatization. Selected CADosomes enabled efficient mRNA delivery in various in vitro cell models, including easy-to-transfect cancer cells (e.g. human cervical carcinoma HeLa cell line) as well as hard-to-transfect primary cells (e.g. primary bovine corneal epithelial cells), outperforming commercially available cationic liposomes and state-of-the-art LNPs. In addition, using the antidepressant nortriptyline as a model compound, we show that CADs can maintain their pharmacological activity upon CADosome incorporation. Furthermore, in vivo proof-of-concept was obtained, demonstrating CADosome-mediated mRNA delivery in the corneal epithelial cells of rabbit eyes, which could pave the way for future applications in ophthalmology. Based on our results, the co-formulation of CADs, helper lipids and mRNA into lipid-based nanocarriers is proposed as a versatile and straightforward approach for the rational development of drug combination therapies., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2022. Published by Elsevier B.V.)

The serotonin 2A receptor (5-HT 2A R) is the mediator of the psychedelic effects of serotonergic psychedelics, which have shown promising results in clinical studies for several neuropsychiatric indications. The 5-HT 2A R is able to signal through the Gα q and β-arrestin effector proteins, but it is currently not known how the different signaling pathways contribute to the therapeutic effects mediated by serotonergic psychedelics. In the present work, we have evaluated the subtype-selective 5-HT 2A R agonist 25CN-NBOH and a series of close analogues for biased signaling at this receptor. These ligands were designed to evaluate the role of interactions with Ser159 3×36 . The lack of interaction between this hydroxyl moiety and Ser159 3×36 resulted in detrimental effects on potency and efficacy in both βarr2 and miniGα q recruitment assays. Remarkably, Gα q -mediated signaling was considerably more affected. This led to the development of the first efficacious βarr2-biased 5-HT 2A R agonists 4a-b and 6e-f , βarr2 preferring, relative to lysergic acid diethylamide (LSD).

Serotonergic psychedelics are substances that induce alterations in mood, perception, and thought, and have the activation of serotonin (5-HT) 2A receptors (5-HT 2A Rs) as a main pharmacological mechanism. Besides their appearance on the (illicit) drug market, e.g. as new psychoactive substances, their potential therapeutic application is increasingly explored. This group of substances demonstrates a broad structural variety, leading to insufficiently described structure-activity relationships, hence illustrating the need for better functional characterization. This review therefore elaborates on the in vitro molecular techniques that have been used the most abundantly for the characterization of (psychedelic) 5-HT 2A R agonists. More specifically, this review covers assays to monitor the canonical G protein signaling pathway (e.g. measuring G protein recruitment/activation, inositol phosphate accumulation, or Ca 2+ mobilization), assays to monitor non-canonical G protein signaling (such as arachidonic acid release), assays to monitor β-arrestin recruitment or signaling, and assays to monitor receptor conformational changes. In particular, focus lies on the mechanism behind the techniques, and the specific advantages and challenges that are associated with these. Additionally, several variables are discussed that one should consider when attempting to compare functional outcomes from different studies, both linked to the specific assay mechanism and linked to its specific execution, as these may heavily impact the assay outcome., (© 2022 International Society for Neurochemistry.)

Classical psychedelics are a group of hallucinogens which trigger non-ordinary states of consciousness through activation of the 5-HT 2A receptor (5-HT 2A R) in the brain. However, the exact mechanism of how 5-HT 2A R agonism alters perception remains elusive. When studying receptor signaling, tools which work at the same spatiotemporal resolution as the receptor are exceptionally useful. To create such a tool, we designed a set of photoswitchable ligands based on the classical psychedelic N,N-dimethyltryptamine (DMT). By incorporation of the DMT-indole ring into the photoswitchable system, we obtained red-shifted ligands which can be operated by visible light. Among these azo-DMTs, compound 2 h ("Photo-DMT") stands out as its cis isomer exhibits DMT like activity while the trans isomer acts as weak partial agonist. Such a cis-on "efficacy switch" substantially expands the pharmacological toolbox to investigate the complex mechanisms of 5-HT 2A R signaling., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The P2Y 2 receptor (P2Y 2 R) is a G protein-coupled receptor that is activated by extracellular ATP and UTP, to a similar extent. This allows it to play roles in the cell's response to the (increased) release of these nucleotides, e.g., in response to stress situations, including mechanical stress and oxygen deprivation. However, despite its involvement in important (patho)physiological processes, the intracellular signaling induced by the P2Y 2 R remains incompletely described. Therefore, this study implemented a NanoBiT ® functional complementation assay to shed more light on the recruitment of β-arrestins (βarr1 and βarr2) upon receptor activation. More specifically, upon determination of the optimal configuration in this assay system, the effect of different (receptor) residues/regions on βarr recruitment to the receptor in response to ATP or UTP was estimated. To this end, the linker was shortened, the C-terminal tail was truncated, and phosphorylatable residues in the third intracellular loop of the receptor were mutated, in either singly or multiply adapted constructs. The results showed that none of the introduced adaptations entirely abolished the recruitment of either βarr, although EC 50 values differed and time-luminescence profiles appeared to be qualitatively altered. The results hint at the C-terminal tail modulating the interaction with βarr, while not being indispensable.

Following our study of 4'-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A 3 AR affinity, we incorporated dopamine-related N 6 substituents in the full agonist 5'-methylamide series. N 6 -(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed K i (nM) 0.563 at hA 3 AR (∼20,000-fold selective) and 1.54 at mA 3 AR. 2-Alkyl ethers maintained A 3 affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and β-arrestin 2 (βarr2)-dependent pathways indicate these are full agonists but not biased. Through use of computational modeling, we hypothesized that phenyl OH/OMe groups interact with polar residues, particularly Gln261, on the mA 3 AR extracellular loops as the basis for the affinity enhancement. Although the pharmacokinetics indicated facile clearance of parent O-methyl catechol nucleosides 21 and 31, prolonged mA 3 AR activation in vivo was observed in a hypothermia model, suggested potential formation of active metabolites through demethylation. Selected analogues induced mouse hypothermia following i.p. injection, indicative of peripheral A 3 AR agonism in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Masson SAS.)

Serotonergic psychedelics, substances exerting their pharmacological action through activation of the serotonin 2A receptor (5-HT 2A R), have continuously comprised a substantial fraction of the over 1000 reported New Psychoactive Substances (NPS) so far. Within this category, N -benzyl derived phenethylamines, such as NBOMes and NBFs, have shown to be of particular relevance. As these substances remain incompletely characterized, this study aimed at synthesizing positional isomers of 25H-NBF, with two methoxy groups placed on different positions of the phenyl group of the phenethylamine moiety. These isomers were then functionally characterized in an in vitro bioassay monitoring the recruitment of β-arrestin 2 to the 5-HT 2A R through luminescent readout via the NanoBiT technology. The obtained results provide insight into the optimal substitution pattern of the phenyl group of the phenethylamine moiety of N -benzyl derived substances, a feature so far mostly explored in the phenethylamines underived at the N -position. In the employed bioassay, the most potent substances were 24H-NBF (EC 50 value of 158 nM), 26H-NBF (397 nM), and 25H-NBF (448 nM), with 23H-NBF, 35H-NBF, and 34H-NBF yielding μM EC 50 values. A similar ranking was obtained for the compounds' efficacy: taking as a reference LSD (lysergic acid diethylamide), 24H-, 26H-, and 25H-NBF had an efficacy of 106-107%, followed by 23H-NBF (96.1%), 34H-NBF (75.2%), and 35H-NBF (58.9%). The stronger activity of 24H-, 25H-, and 26H-NBF emphasizes the important role of the methoxy group at position 2 of the phenethylamine moiety for the in vitro functionality of NBF substances.

Serotonergic psychedelics are defined as compounds having serotonin 2A receptor (5-HT 2A R) activation as an important pharmacological mechanism. These compounds include the phenylalkylamine class, containing substances with e.g. 2C-X structures (phenethylamines) or their N -methoxybenzyl analogues (NBOMes). Besides their abuse potential, psychedelics are increasingly recognized for having therapeutic benefits. However, many psychedelics remain incompletely characterized, even concerning their structure-activity relationships. Here, five positional isomers of 25H-NBOMe, with two methoxy groups on the different positions of the phenyl ring of the phenethylamine moiety, were subjected to split-nanoluciferase assays assessing the in vitro recruitment of cytosolic proteins to the 5-HT 2A R. Furthermore, molecular docking at the 5-HT 2A R allowed estimation of which residues interact with the specific isomers' methoxy groups. Although the optimal substitution pattern of N -unsubstituted phenylalkylamines has been extensively studied, this is the first comparative evaluation of the functional effects of the positioning of the methoxy groups in the phenethylamine moiety of NBOMes., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Psychedelic new psychoactive substances (NPS), compounds exerting their main pharmacological effects through the activation of the serotonin 2A receptor (5-HT 2A R), continuously comprise a substantial portion of the reported NPS. However, these substances and their exact mechanism of action, differentiating them from non-psychedelic 5-HT 2A R agonists, require further characterization. One potentially relevant phenomenon is the occurrence of biased agonism, in which (a) certain signaling pathway(s) is preferentially activated over the other(s). To this end, a new bioassay was developed, monitoring the recruitment of an engineered miniGα q protein to the activated 5-HT 2A R. The setup was designed to be analogous to that of a previously developed bioassay monitoring β-arrestin 2 recruitment through the NanoBiT system, enabling estimation of the potential preference of a substance to trigger recruitment of one protein over the other. This approach yielded several statistically significantly biased agonists within the group of phenylalkylamine psychedelics, more specifically the N-benzyl substituted 25H analogues 25H-NBF, 25H-NBMD, 25H-NBOH and 25H-NBOMe. All four compounds show a statistically significant preference towards the recruitment of β-arrestin 2 over miniGα q , as compared to the reference psychedelic substance LSD. We identified markedly different responses for Bromo-DragonFLY in the two bioassays, suggesting biased agonism, though the calculated bias factor equalled out to approximately 0. This demonstrates that the accurate assessment of biased agonism requires both the consideration of the observed trends in addition to the numerical value of the bias factor. A second panel of structural (I-substituted) analogues of the former group of phenylalkylamines showed a similar trend in the ranking order of the bias factors, resulting in one additional compound (25I-NBF) being statistically significantly biased., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Serotonergic psychedelics, substances exerting their effects primarily through the serotonin 2A receptor (5-HT 2A R), continue to comprise a substantial portion of reported new psychoactive substances (NPS). The exact mechanisms of action of psychedelics still remain to be elucidated further, and certain pathways remain largely unexplored on a molecular level for this group of compounds. A systematic comparison of substances belonging to different subclasses, monitoring the receptor-proximal β-arrestin 2 recruitment, is lacking. Based on a previously reported in vitro bioassay employing functional complementation of a split nanoluciferase to monitor β-arrestin 2 recruitment to the 5-HT 2A R, we here report on the setup of a stable HEK 293 T cell-based bioassay. Following verification of the performance of this new stable cell system as compared to a system based on transient transfection, the stable expression system was deemed suitable for the pharmacological characterization of psychedelic NPS. Subsequently, it was applied for the in vitro assessment of the structure-activity relationship of a set of 30 substances, representing different subclasses of phenylalkylamine psychedelics, among which 12 phenethylamine derivatives (2C-X), 7 phenylisopropylamines (DOx) and 11 N-benzylderivatives (25X-NB). The resulting potency and efficacy values provide insights into the structure-activity relationship of the tested compounds, overall confirm findings observed with other reported in vitro assays, and even show a significant correlation with estimated common doses. This approach, in which a large series of psychedelic NPS belonging to different subclasses is comparatively tested, using a same assay setup, monitoring a receptor-proximal event, not only gives pharmacological insights, but may also allow prioritization of legal actions related to the most potent -and potentially dangerous- compounds.

It has been brought to the authors' attention that Fig. 1 of "In vitro structure-activity relationship determination of 30 psychedelic new psychoactive substances by means of β-arrestin 2 recruitment to the serotonin 2A receptor" contained a mistake in the structures for 2C-B-FLY and Bromo-DragonFLY. This has now been corrected. The authors apologize for any inconvenience caused.

The A 3 adenosine receptor (A 3 AR) is a G protein-coupled receptor that is involved in a wide variety of physiological and pathological processes, such as cancer. However, the use of compounds pharmacologically targeting this receptor remains limited in clinical practice, despite extensive efforts for compound synthesis. Moreover, the possible occurrence of biased agonism further complicates the interpretation of the functional characteristics of compounds. Hence the need for simple assays, which are comparable in terms of the used cell lines and read-out technique. We previously established a stable β-arrestin 2 (βarr2) bioassay, employing a simple, luminescent read-out via functional complementation of a split nanoluciferase enzyme. Here, we developed a complementary, new bioassay in which coupling of an engineered miniGα i protein to activated A 3 AR is monitored using a similar approach. Application of both bioassays for the concurrent determination of the potencies and efficacies of a set of 19 N 6 -substituted adenosine analogues not only allowed for the characterization of structure-activity relationships, but also for the quantification of biased agonism. Although a broad distribution in potency and efficacy values was obtained within the test panel, no significant bias was observed toward either the βarr2 or miniGα i pathway., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Classic or serotonergic hallucinogens comprise the third largest number of reported new psychoactive substances (NPS), according to the United Nations Office on Drugs and Crime. While being structurally very divergent, they share activation of the serotonin 2A receptor (5-HT2AR), a G protein-coupled receptor, as their main pharmacological mechanism. Here, we report on the development of a 5-HT2AR bioassay, which monitors β-arrestin2 recruitment to the receptor via a split-luciferase system, as a measure of receptor activation. Possible applications of the bioassay would lie in the characterization of serotonergic hallucinogens and the screening of these compounds in biofluids based on their serotonergic activity, rather than on their specific structures. The developed bioassay allowed the determination of the potency and efficacy of representatives of different classes of hallucinogens (LSD, 5-MeO-DALT, mescaline) and of a selected group of 2C hallucinogens and their corresponding NBOMes, with EC50 values from the subnanomolar (NBOMes) to micromolar (mescaline) range. When implementing the bioassay for the screening of plasma, a pronounced receptor activation was already observed in blank samples, which could be ascribed to endogenous serotonin, as suggested by annihilation of this activity by a 5-HT2AR antagonist or after incubation with MAO-A (monoamine oxidase-A). The presence and degradability by MAO-A of serotonin in plasma extracts were confirmed by LC-HRMS. Due to the possible metabolism of certain hallucinogens by MAO-A, which would cause a bias in the detectability of compounds in biofluids, the main application potential of this bioassay lies in the characterization of these scarcely characterized serotonergic hallucinogens.

Tailored developed nanoliposomes loaded with a siRNA against the transcription factor E2F1 (siE2F1), were produced and delivered to human colorectal adenocarcinoma cell lines and to intestinal human biopsies. siE2F1 loaded nanoliposomes were produced through a dedicated ultrasound assisted technique producing particles with about 40nm size (Small Unilamellar Vesicles, SUVs) and 100% siRNA encapsulation efficiency. Compared to other production methods, the one proposed here can easily produce particles in the nanometric scale by suitable ultrasonic duty cycle treatments. Furthermore, SUVs have a high degree of size homogeneity, a relevant feature for uniform delivery behaviour. siE2F1-loaded SUVs demonstrated a very low cytotoxicity in cells when compared to a commercial transfection agent. Moreover, SUVs loaded with siE2F1 were effective in the down regulation of the target in cultured colon carcinoma cells and in the consequent reduction of cell growth. Finally, a remarkable uptake and target silencing efficiencies were observed in cultured human biopsy of colonic mucosa. In conclusion, whereas further studies in more complex models are required, the siE2F1-SUVs generated have the potential to contribute to the development of novel effective inflammatory bowel diseases-associated colorectal cancer therapies for a future personalized medicine., (Copyright © 2017 Elsevier B.V. All rights reserved.)