Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest groups of new psychoactive substances monitored in Europe. SCRAs are known to typically exert higher cannabinoid activity than tetrahydrocannabinol from cannabis, thereby entailing a greater health risk. Both Cumyl‐PEGACLONE and 5F‐Cumyl‐PEGACLONE were not controlled by the national legislation upon their first detection in Germany in 2016 and 2017, respectively, and have been linked to several fatalities. In this study, the CB1 receptor activity of these compounds, together with two newly synthesized structural isomers (Cumyl‐PEGACLONE ethylbenzyl isomer and n‐propylphenyl isomer), was assessed using two different in vitro receptor‐proximal bioassays, monitoring the recruitment of either β‐arrestin2 (β‐arr2) or a modified G protein (mini‐Gαi) to the activated CB1 receptor. In terms of both potency and relative efficacy, Cumyl‐PEGACLONE and 5F‐Cumyl‐PEGACLONE were found to exert strong CB1 activation, with sub‐nanomolar EC50 values and efficacy values exceeding those of the reference agonist JWH‐018 threefold (β‐arr2 assay) or almost twofold (mini‐Gαi assay). The ethylbenzyl and n‐propylphenyl isomers exhibited a strongly reduced CB1 activity (EC50 values >100 nM; efficacy <40% relative to JWH‐018), which is hypothesized to originate from steric hindrance in the ligand‐binding pocket. None of the evaluated compounds exhibited significant biased agonism. In conclusion, the functional assays applied here allowed us to demonstrate that 5‐fluorination of Cumyl‐PEGACLONE is not linked to an intrinsically higher CB1 activation potential and that the ethylbenzyl and n‐propylphenyl isomers yield a strongly reduced CB1 activation.

Conventionally, hypoxia-inducible factor (HIF) activation by prolyl hydroxylase domain enzyme (PHD) inhibition is monitored by gene reporter assays. The principle relies on the monitoring of an upstream event (HIF stabilization) by the downstream transcriptional activity. Here, we developed a novel approach to directly sense HIF activation by monitoring the heterodimerization of the HIFα/HIFβ subunits, constituting the functional HIF transcription factor. Two live cell-based biosensor assay setups were designed, utilizing functional complementation of split-nanoluciferase as a tool to measure HIFα/HIFβ protein–protein interaction resulting from the stabilization of HIF1α or HIF2α. The assay setup in a 96-well format was optimized for a duration of 2 h, and a HEK293T transfection protocol was introduced for the optimal configuration of HIFα/HIFβ-fusion proteins. These new bioassays outperformed hypoxia response element-based gene reporter assay, the current state-of-the-art assay, in terms of sensitivity. Applicability was demonstrated using a panel of PHD inhibitors, including roxadustat, molidustat, daprodustat, desidustat, vadadustat, and FG-2216, for which concentration–response curves were generated, allowing for the derivation of potency (EC50) and efficacy (E max) data. The broad applicability of the biosensors was established via applying hypoxia mimetic CoCl2, iron chelator desferrioxamine, proteasome inhibitor MG-132, and 2-OG mimetic dimethyloxalylglycine on the assays, indicating concentration-dependent effects.

BACKGROUND Synthetic cannabinoid receptor agonists (SCRAs) are amongst the largest groups of new psychoactive substances (NPS). Their often-high activity at the CB1 cannabinoid receptor frequently results in intoxications, imposing serious health risks. Hence, continuous monitoring of these compounds is important, but is challenged by the rapid emergence of novel analogues that are missed by traditional targeted detection strategies. We addressed this need by performing an activity-based, universal screening on a large set (n = 968) of serum samples from patients presenting to the emergency department with acute recreational drug or NPS toxicity. METHODS We assessed the performance of an activity-based method in detecting newly circulating SCRAs compared with liquid chromatography coupled to high-resolution mass spectrometry. Additionally, we developed and evaluated machine learning models to reduce the screening workload by automating interpretation of the activity-based screening output. RESULTS Activity-based screening delivered outstanding performance, with a sensitivity of 94.6% and a specificity of 98.5%. Furthermore, the developed machine learning models allowed accurate distinction between positive and negative patient samples in an automatic manner, closely matching the manual scoring of samples. The performance of the model depended on the predefined threshold, e.g., at a threshold of 0.055, sensitivity and specificity were both 94.0%. CONCLUSION The activity-based bioassay is an ideal candidate for untargeted screening of novel SCRAs. The combination of this universal screening assay and a machine learning approach for automated sample scoring is a promising complement to conventional analytical methods in clinical practice.

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Over 200 synthetic cannabinoid receptor agonists (SCRAs) have been identified as new psychoactive substances. Effective monitoring and characterization of SCRAs are hindered by the rapid pace of structural evolution. Ahead of possible appearance on the illicit drug market, new SCRAs were synthesized to complete a systematic library of cumyl-indole-(e.g., CUMYL-CPrMICA, CUMYL-CPMICA) and cumyl-indazole-carboxamides (e.g., CUMYL-CPrMINACA, CUMYL-CPMINACA), encompassing butyl, pentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl tails. Comprehensive pharmacological characterization was performed with three assay formats, monitoring the recruitment of either wild-type or C-terminally truncated (βarr2d366) β-arrestin2 to the activated cannabinoid 1 receptor (CB 1 ) or monitoring G βγ -mediated membrane hyperpolarization. Altered compound characterization was observed when comparing derived potency (EC 50 ) and efficacy (E max ) values from both assays monitoring the same or a different signaling event, whereas ranges and ranking orders were similar. Structure−activity relationships (SAR) were assessed in threefold, resulting in the identification of the pendant tail as a critical pharmacophore, with the optimal chain length for CB 1 activation approximating an npentyl (e.g., cyclopentylmethyl or cyclohexylmethyl tail). The activity of the SCRAs encompassing cyclic tails decreased with decreasing number of carbons forming the cyclic moiety, with CUMYL-CPrMICA showing the least CB 1 activity in all assay formats. The SARs were rationalized via molecular docking, demonstrating the importance of the optimal steric contribution of the hydrophobic tail. While SAR conclusions remained largely unchanged, the differential compound characterization by both similar and different assay designs emphasizes the importance of detailing specific assay characteristics to allow adequate interpretation of potencies and efficacies.