The 'ABC' of split-nanoluciferase HIF heterodimerization bioassays: Applications, Benefits & Considerations.

Hypoxia-inducible factors (HIF) are interesting targets for multiple therapeutic indications. While HIF activation is desired for the treatment of anemia-related and ischemic diseases, HIF inhibition is of tremendous interest to anti-cancer drug development. Different signaling events within the HIF pathway are being targeted by drug discovery programs, with a special interest in HIF-selective (possibly also HIF1/2 isoform-selective) compounds. In this study, we applied recently developed cell-based split-nanoluciferase HIF heterodimerization assays to study the effects of compounds, targeting HIF activity by various mechanisms of action. This study shows that the application of similar or diverse assay protocols allows to detect various influences on HIF heterodimerization as a key signaling event in the oxygen sensing pathway: increased HIF heterodimerization (roxadustat, MG-132), decreased HIF heterodimerization (PX-478, ibuprofen) and direct (HIF isoform-selective) heterodimerization inhibiting effects (PT-2385). Changes in treatment time and in the assay protocol allowed to assess direct and indirect effects on HIFα-HIFß heterodimerization. In addition to the evaluation of applications of these new bioassays regarding pharmacological characterizations, benefits and considerations are discussed related to the use of cellular, luminescent-based bioassays. Briefly, benefits include the bidirectional nature of the biological readout, the upstream mechanism of detection, the differentiation between HIF1 and HIF2 effects and the simulation of various conditions. Specific and general considerations include cell-based, technical and disease/drug-related aspects (e.g., non-specific effects, color interference). In summary, the versatility of these bioassays offers benefits in widespread applications regarding drug discovery and pharmacological characterization of various therapeutics, applying either the same or optimized experimental protocols.

Interpreting mono- and poly-SCRA intoxications from an activity-based point of view: JWH-018 equivalents in serum as a comparative measure.

Synthetic cannabinoid receptor agonists (SCRAs) are a class of synthetic drugs that mimic and greatly surpass the effect of recreational cannabis. Acute SCRA intoxications are in general difficult to assess due to the large number of compounds involved, differing widely in both chemical structure and pharmacological properties. The rapid pace of emergence of unknown SCRAs hampers on one hand the timely availability of methods for identification and quantification to confirm and estimate the extent of the SCRA intoxication. On the other hand, lack of knowledge about the harm potential of emerging SCRAs hampers adequate interpretation of serum concentrations in intoxication cases. In the present study, a novel comparative measure for SCRA intoxications was evaluated, focusing on the cannabinoid activity (versus serum concentrations), which can be measured in serum extracts with an untargeted bioassay assessing ex vivo CB1 activity. Application of this principle to a series of SCRA intoxication cases (n = 48) allowed for the determination of activity equivalents, practically entailing a conversion from different SCRA serum concentrations to a JWH-018 equivalent. This allowed for the interpretation of both mono- (n = 34) and poly-SCRA (n = 14) intoxications, based on the intrinsic potential of the present serum levels to exert cannabinoid activity (cf. pharmacological/toxicological properties). A non-distinctive toxidrome was confirmed, showing no relation to CB1 activity. The JWH-018 equivalent was partly related to the poison severity score (PSS) and causality of the clinical intoxication elicited by the SCRA. Altogether, this equivalent concept allows to comparatively and timely interpret (poly-)SCRA intoxications based on CB1 activity.

Investigation of the intrinsic cannabinoid activity of hemp-derived and semisynthetic cannabinoids with ß-arrestin2 recruitment assays-and how this matters for the harm potential of seized drugs.

Cultivation of industrial low-Δ9-tetrahydrocannabinol (Δ9-THC) hemp has created an oversupply of cannabidiol (CBD)-rich products. The fact that phytocannabinoids, including CBD, can be used as precursors to synthetically produce a range of THC variants-potentially located in a legal loophole-has led to a diversification of cannabis recreational drug markets. 'Hemp-compliant', 'hemp-derived' and 'semisynthetic' cannabinoid products are emerging and being advertised as (legal) alternatives for Δ9-THC. This study included a large panel (n = 30) of THC isomers, homologs, and analogs that might be derived via semisynthetic procedures. As a proxy for the abuse potential of these compounds, we assessed their potential to activate the CB1 cannabinoid receptor with a ß-arrestin2 recruitment bioassay (picomolar-micromolar concentrations). Multiple THC homologs (tetrahydrocannabihexol, THCH; tetrahydrocannabiphorol, THCP; tetrahydrocannabinol-C8, THC-C8) and THC analogs (hexahydrocannabinol, HHC; hexahydrocannabiphorol, HHCP) were identified that showed higher potential for CB1 activation than Δ9-THC, based on either higher efficacy (Emax) or higher potency (EC50). Structure-activity relationships were assessed for Δ9-THC and Δ8-THC homologs encompassing elongated alkyl chains. Additionally, stereoisomer-specific differences in CB1 activity were established for various THC isomers (Δ7-THC, Δ10-THC) and analogs (HHC, HHCP). Evaluation of the relative abundance of 9(S)-HHC and 9(R)-HHC epimers in seized drug material revealed varying epimeric compositions between batches. Increased abundance of the less active 9(S)-HHC epimer empirically resulted in decreased potency, but sustained efficacy for the resulting diastereomeric mixture. In conclusion, monitoring of semisynthetic cannabinoids is encouraged as the dosing and the relative composition of stereoisomers can impact the harm potential of these drugs, relative to Δ9-THC products.

Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay.

Hypoxia-inducible factor (HIF) stabilizers are listed in the World Anti-Doping Agency's prohibited list as they can increase aerobic exercise capacity. The rapid pace of emergence of highly structurally diverse HIF stabilizers could pose a risk to conventional structure-based methods in doping control to detect new investigational drugs. Therefore, we developed a strategy that is capable of detecting the presence of any HIF stabilizer, irrespective of its structure, by detecting biological activity. Previously developed cell-based HIF1/2 assays were optimized to a stable format and evaluated for their screening potential toward HIF stabilizers. Improved pharmacological characterization was established by the stable cell-based formats, and broad specificity was demonstrated by pharmacologically characterizing a diverse set of HIF stabilizers (including enarodustat, IOX2, IOX4, MK-8617, JNJ-42041935). The methodological (in solvent) limit of detection of the optimal HIF1 stable bioassay toward detecting the reference compound roxadustat was 100 nM, increasing to 50-100 ng/mL (corresponding to 617-1233 nM in-well) in matching urine samples, owing to strong matrix effects. In a practical context, a urinary limit of detection of 1.15 µg/mL (95% detection rate) was determined, confirming the matrix-dependent detectability of roxadustat in urine. Pending optimization of a universal sample preparation strategy and/or a methodology to correct for the matrix effects, this untargeted approach may serve as a complementing method in antidoping control, as theoretically, it would be capable of detecting any unknown substance with HIF stabilizing activity.

Comprehensive Characterization of a Systematic Library of Alkyl and Alicyclic Synthetic Cannabinoids Related to CUMYL-PICA, CUMYL-BUTICA, CUMYL-CBMICA, and CUMYL-PINACA.

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 (ßarr2d366) ß-arrestin2 to the activated cannabinoid 1 receptor (CB1) or monitoring Gßγ-mediated membrane hyperpolarization. Altered compound characterization was observed when comparing derived potency (EC50) and efficacy (Emax) 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 CB1 activation approximating an n-pentyl (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 CB1 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.

Linking in vitro and ex vivo CB1 activity with serum concentrations and clinical features in 5F-MDMB-PICA users to better understand SCRAs and their metabolites.

Synthetic cannabinoid receptor agonists (SCRAs) pose a danger to public health. This study focused on individuals experiencing recreational drug toxicity who had used 5F-MDMB-PICA.Patient records were evaluated regarding vital signs, Glasgow Coma Scale (GCS) and clinical features. Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) confirmed and quantified the presence of 5F-MDMB-PICA (and/or metabolites) as the only SCRA present in the serum of 71 patients. Cannabinoid activity was evaluated by a cannabinoid receptor (CB1) bioassay, to assess the relationship between serum concentrations and ex vivo human CB1 activation potential. Furthermore, a link with the clinical presentation was appraised.5F-MDMB-PICA and five metabolites were pharmacologically profiled in vitro, revealing theoretically possible contributions of two active in vivo metabolites to overall cannabinoid activity. Serum concentrations of 5F-MDMB-PICA were correlated to the ex vivo cannabinoid activity, revealing a sigmoidal relationship. The latter could also be predicted based on pharmacological characterization of 5F-MDMB-PICA and its metabolites and an in-depth investigation of the bioassay outcome. Clinically, the GCS showed a significant trend (decrease) with increasing ex vivo cannabinoid activity.This is the first study to evaluate possible toxic effects of 5F-MDMB-PICA in a unique large patient cohort. It allows a better understanding of 5F-MDMB-PICA and metabolites in humans, suggesting a negligible contribution by 5F-MDMB-PICA metabolites to the overall cannabinoid activity in serum. Additionally, this work shows that in vitro pharmacological characterization allows close prediction of an individual's ex vivo CB1 activity, the latter showing a relationship with the level of consciousness.

Machine Learning to Assist in Large-Scale, Activity-Based Synthetic Cannabinoid Receptor Agonist Screening of Serum Samples.

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 intoxication, imposing serious health risks. Hence, continuous monitoring of these compounds is important, but 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.

Sensing an Oxygen Sensor: Development and Application of Activity-Based Assays Directly Monitoring HIF Heterodimerization.

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/HIFß 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/HIFß 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/HIFß-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 (Emax) 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.

In vitro activity profiling of Cumyl-PEGACLONE variants at the CB1 receptor: Fluorination versus isomer exploration.

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 ß-arrestin2 (ß-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 (ß-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.