41 research outputs found
Activity-based detection of new psychoactive substances as an alternative screening approach in forensic toxicology
No full textOff-target activity of NBOMes and NBOMe analogs at the mu opioid receptor
No full textNew 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 mu opioid receptor (MOR) activation potential of a large set of psychedelics, substances which typically activate the serotonin (5-HT2A) 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 (R) beta arr2 recruitment assay and the G protein-based AequoScreen (R) Ca2+ release assay. The use of two orthogonal systems proved beneficial as some aspecific, receptor independent effects were found for various analogs when using the Ca2+ 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-HT2A activation potential
In vitro structure-activity relationship determination of 30 psychedelic new psychoactive substances by means of beta-arrestin 2 recruitment to the serotonin 2A receptor
No full textSerotonergic psychedelics, substances exerting their efects primarily through the serotonin 2A receptor (5-HT2AR), 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 diferent 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-HT2AR, we here report on the setup of a stable HEK 293 T cell-based bioassay. Following verifcation 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 diferent subclasses of phenylalkylamine psychedelics, among which 12 phenethylamine derivatives (2C-X), 7 phenylisopropylamines (DOx) and 11 N-benzylderivatives (25X-NB). The resulting potency and efcacy values provide insights into the structure–activity relationship of the tested compounds, overall confrm fndings observed with other reported in vitro assays, and even show a signifcant correlation with estimated common doses. This approach, in which a large series of psychedelic NPS belonging to diferent 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
In vitro functional characterization of a panel of non-fentanyl opioid new psychoactive substances
No full textThe landscape of new psychoactive substances (NPS) is constantly evolving, with new compounds entering the illicit drug market at a continuous pace. Of these, opioid NPS form a threat given their high potency and prevalence. Whereas previously, the use of fentanyl and fentanyl derivatives was the main point of attention, legislations have reacted accordingly, whichmay have been a driving force towards the (ab)use of alternative µ-opioid receptor (MOR) agonists. In contrast to fentanyl (analogues), details on these novel non-fentanyl opioid NPS are scarce. We investigated the biological activity of a panel of 11 ‘alternative’, newly emerging MOR agonists (2-methyl-AP-237, AP-237, bromadol, brorphine, butorphanol, isotonitazene, mitragynine, 7-OH-mitragynine, MT-45, piperidylthiambutene, and tianeptine) using two closely related in vitro MOR activation bio-assays, monitoring either G protein (mini-Gi), or β-arrestin2 (βarr2) recruitment. Activity profles were obtained for all tested compounds, with values for potency (EC50) ranging from 1.89 nM (bromadol) to>3 µM (AP-237 and tianeptine).
Bromadol, brorphine, isotonitazene, piperidylthiambutene, and tianeptine had the highest efcacy (Emax) values, exceeding that of the reference compound hydromorphone≥1.3-fold (βarr2 assay) and>2.6-fold (mini-Gi assay). Information on the recruitment of two distinct signaling molecules additionally enabled evaluation of biased agonism; none of the evaluated opioids being signifcantly biased. Taken together, this study is the frst to systematically investigate the in vitro biological activity of a diverse panel of emerging non-fentanyl opioid NPS at MOR. Given the known danger of (fatal) intoxications with many opioid NPS, it is important to continuously monitor and characterize newly emerging compounds
The next generation of synthetic cannabinoids: Detection, activity, and potential toxicity of pent‐4en and but‐3en analogues including MDMB‐4en‐PINACA
No full textA new class of synthetic cannabinoids has emerged as new psychoactive substances (NPS). Similar in structure to JWH-022, these substances contain alkene modifications to the tail region of the synthetic cannabinoid core structure, and nomenclature denotes these new analogues as pent-4en or but-3en species. Internationally, two analogues from this new series recently emerged: MDMB-4en-PINACA and MMB-4en-PICA. Previously, data regarding activity and potential toxicity were not available.In vitroassessment of cannabinoid receptor 1 (CB1) activation via the beta-arrestin 2 recruitment was studied for three (3) pent-4en analogues, one (1) but-3en analogue, and one (1) principal metabolite. MDMB-4en-PINACA (2.47 nM, 239%), MDMB-4en-PICA (11.5 nM, 302%), and MDMB-3en-BINACA (14.3 nM, 286%) were highly potent and efficacious (comparison: JWH-018, 25.3 nM, 100%), while the potencies of MMB-4en-PICA and MDMB-4en-PINACA 3,3-dimethylbutanoic acid were markedly lower. Modifications to core and tail structural features (i.e., indole vs. indazole) led to relatively small differences in potency, while changes among the head region led to larger differences. Sample-mining and data-mining conducted on toxicology samples led to the identification of MDMB-4en-PINACA in 25 forensic toxicology cases, including postmortem and impaired driving investigations, with case details and limited histories described herein. Moderate geographical distribution of MDMB-4en-PINACA was noted in the United States with emergence in the Northeast, Midwest, South, and West regions. Results from toxicology testing paired with case history show the potential for MDMB-4en-PINACA to cause or contribute to impairment or death. Forensic scientists, public health and public safety officials, law enforcement, clinicians, medical examiners, and coroners should consider involvement of emergent synthetic cannabinoids in their work and that new analogues containing an alkene tail can retain similar or increased potency and toxicity
Functional evaluation of carboxy metabolites of synthetic cannabinoid receptor agonists featuring scaffolds based on L‐valine or L‐tert ‐leucine
No full textIndole- and indazole-based synthetic cannabinoid receptor agonists (SCRAs), featuring valine or tert-leucine substituents, are commonly abused new psychoactive substances (NPS). A major metabolic pathway for these SCRAs is hydrolysis of the terminal amide or methylester functionalities. Although these hydrolysis products were already detected as main ingredients in some "legal highs," these metabolites are often poorly characterized. Here, we report a systematic investigation of the activity of 7 common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L-valine or L-tert-leucine in direct comparison to their parent compounds. An activity-based cannabinoid receptor 1 (CB1) bio-assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293T cell system. The recruitment of beta-arrestin2 to the activated CB1 (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96-well plate-reader. The major hydrolysis metabolites of 5F-AB-PINACA, ADB-CHMICA, ADB-CHMINACA, ADB-FUBICA, and their methyl- and ethylester derivatives showed no detectable CB1 activation at concentrations up to 1 mu M. On the other hand, metabolites of 5F-ADB-PINACA, AB-CHMINACA, and ADB-FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC50 values >100 nM). Activity-based characterization of SCRAs and their metabolites at CB1 may not only allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications, but may also allow application of the concept of "activity equivalents" present in biological fluids or, alternatively, in confiscated materials
Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system
No full textSynthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for "unknown" compounds. Therefore, an alternative screening method, not directly based on the structure but on the activity of the SC, may offer a solution for this problem. We generated stable CB1 and CB2 receptor activation assays based on functional complementation of a split NanoLuc luciferase and used these to test an expanded set of recent SCs (UR-144, XLR-11, and their thermal degradation products; AB-CHMINACA and ADB-CHMINACA) and their major phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at the cannabinoid receptors. These active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to the toxicity profile. Utility of the generated stable cell systems as a first -line screening tool for SCs in urine was also demonstrated using a relatively large set of authentic urine samples. Our data indicate that the stable CB reporter assays detect CB receptor, activation by extracts of urine in which SCs (or their metabolites) are present at low- or subnanomolar (ng/mL) level. Hence, the developed assays do not only allow activity profiling of SCs and their metabolites, it may also serve as a screening tool, complementing targeted and untargeted analytical assays and preceding analytical (mass spectrometry based) confirmation
Activity-based reporter assays for the screening of abused substances in biological matrices
No full textThe (ab)use of designer drugs and steroid hormones has gained popularity due to the lower chance of getting caught, as routine drug or doping tests may miss these (novel) compounds. Current analytical approaches mostly make use of targeted, structure-based techniques, such as immunoassays or mass spectrometry (MS)-based methods. However, these approaches have limitations, including a lack of cross-reactivity and the need for prior knowledge of molecular identity. This has initiated considerable interest in the so-called "untargeted" screening strategies to detect these compounds. The use of "untargeted" MS-based screening methods (e.g. gas chromatography MS and especially high-resolution MS) has gained considerable interest to detect and identify novel compounds. However, due to their expensive and time-consuming character, very sophisticated analytical methods are not ideal as a first-line screening method and are not routinely implemented in most laboratories. Given the above, it is clear that there lies potential in novel "untargeted" screening approaches, which are less expensive, more high-throughput-amenable and more routinely applicable. Activity-based assays, capable of monitoring the biological activity of an abused substance in a biological matrix, have been proposed as an alternative. These biological assays do not require knowledge about a compound's structure and could be used as a first-line screening tool to identify potentially positive samples. In this review, we focus on activity-based reporter bioassays for the detection of steroids and drugs of abuse in biological matrices. As for drugs of abuse, only bioassays for detecting cannabinoid or opioid activity in biological matrices are available, only (synthetic) cannabinoid receptor agonists and opioids are discussed
Setup of a serotonin 2A receptor (5-HT2AR) bioassay : demonstration of its applicability to functionally characterize hallucinogenic new psychoactive substances and an explanation why 5-HT2AR bioassays are not suited for universal activity-based screening of biofluids for new psychoactive substances
No full textClassic 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 (S-HT2AR), a G protein-coupled receptor, as their main pharmacological mechanism. Here, we report on the development of a 5-HT2AR bioassay, which monitors beta-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 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
