Signalling profiles of a structurally diverse panel of synthetic cannabinoid receptor agonists
Graphical abstract
Introduction
A long history of recreational and medicinal use of cannabis (Cannabis sativa) prompted the investigation into, and the subsequent discovery of the endocannabinoid system. This included the study of the related Gαi/o protein-coupled receptors; type 1 cannabinoid receptor (CB1) [1], found abundantly throughout the central nervous system and type 2 cannabinoid receptor (CB2), located primarily in immune cells [2], [3]. Following the discovery and elucidation of the structure of the primary bioactive component of cannabis, Δ9-tetrahydrocannabinol (THC) [4], synthetic analogues of THC were developed for pharmacological interrogation of the endocannabinoid system for potential medicinal effects.
The THC-like in vivo effects of many synthetic cannabinoids has resulted in their diversion to recreational drugs in an attempt to create “legal” or unregulated alternatives to cannabis. In the 2000s, designer synthetic cannabinoids were included in herbal incense products branded “Spice” or “AK-47 24 Karat Gold”, which contained the compounds JWH-018 and JWH-073 [5], [6]. New generation synthetic cannabinoid receptor agonists (SCRAs) with no precedent in the scientific literature are constantly emerging, enabling producers to change the composition of compounds in order to circumvent laws [7]. SCRAs now represent the most rapidly proliferating class of New Psychoactive Substances (NPS), with over 260 reported to the United Nations Office on Drugs and Crime as of 2018 [8].
Synthetic cannabinoid use is a growing social and health issue. Cases of severe adverse effects and death linked to recreational use of SCRAs have been reported internationally, in contrast to the relatively low incidence of toxicity reported for cannabis [9], [10], [11]. However, molecular pharmacology data and potential mechanisms of toxicity for many of these compounds are currently limited. The apparently greater abuse liability and toxicity of SCRAs compared to cannabis, may be linked to their ability to potently and efficaciously activate cannabinoid receptors (Fig. 1). For example, when compared to traditional research agonists like CP55,940, THC induces potent CB1-mediated activation of ERK phosphorylation and inhibition of adenylate cyclase but with lower efficacy in these pathways [12], [13]. In contrast, indole or indazole derivatives detected in illicit synthetic preparations, such as 5F-CUMYL-PICA, AB-PINACA, AMB-FUBINACA and MDMB-FUBINACA display higher potency and efficacy at CB1-mediated inhibition of cAMP production [14], [15], [16]. This suggests the profile or pattern of responses produced by toxic SCRAs differs from THC, which may help explain their enhanced toxicity.
Interestingly, within the indole and indazole SCRA families, varying degrees of activity (and potentially toxicity) have been associated with specific structural features. For instance, the inclusion of an azaindole core (an indole analogue) in 5F-CUMYL-P7AICA conferred reduced CB1 affinity and functional activity compared to the indazole and indole cores of 5F-CUMYL PINACA and 5F-CUMYL PICA, respectively [17]. Similarly, terminal fluorination of the N-pentyl indole of XLR-11 was found to increase CB1 potency compared to the non-fluorinated analogue, UR-144 [18], [19]. Additionally, particular classes of compounds (indoles and indazole-3-carboxamides with amino acid derivative side-chains, like AMB-FUBINACA and XLR-11) seem to be more frequently encountered in cases of SCRA-related toxicity and death, in contrast to structurally-related compounds like 5F-CUMYL-PINACA and 5F-MDMB-PICA – despite all compounds being in the NPS marketplace and exhibiting equivalently high efficacy in in vitro signalling assays [9], [17], [19], [20]. While it is challenging to compare toxicities because the exact use and dose profiles of sufficient numbers of SCRAs are not known, this might suggest that small structural alterations between ligands may induce different active receptor conformations to differentially activate downstream signalling pathways (termed “biased agonism” or “functional selectivity”), which may have implications for toxicity in vivo [21]. Further insight into SCRA pharmacology may help identify such biased signalling profiles, and particular structural conformations or chemical features responsible for their effects.
This study has characterised the molecular pharmacology of a structurally-diverse panel of novel synthetic cannabinoids at CB1 (Fig. 2). The activity profiles of select compounds were compared in a battery of in vitro assays; cAMP production, β-arrestin translocation and receptor internalisation. To explore differences in the activity profiles of the synthetic cannabinoids, evaluation of functional selectivity was also performed to allow comparisons between the phytocannabinoid THC, and classical research cannabinoids, CP55,940 and WIN55,212-2.
Section snippets
Drugs
CP55,940 was purchased from Cayman Chemical Company (Ann Arbour, MI, USA); forskolin and WIN55,212-2 were obtained from Tocris Bioscience (Bristol, UK); (−)-trans-Δ9- tetrahydrocannabinol (THC) was purchased from THC Pharm GmbH (Frankfurt, Germany). Synthetic cannabinoids were provided by Dr Samuel Banister (University of Sydney, Australia), with the exception of 4-cyano CUMYL-BUTINACA (4CN-CUMYL-BUTINACA), AB-CHMINACA, AB-FUBINACA and XLR-11, which were obtained from Cayman Chemical Company
SCRAs display full efficacy in cAMP signalling
The canonical signalling pathway of CB1 involves coupling to Gαi proteins, resulting in inhibition of adenylate cyclase and consequent reduction in cAMP concentration [2], [31]. To determine CB1 agonist behaviour, we examined the ability of a select group of SCRAs to inhibit adenylate cyclase using a real-time BRET-CAMYEL assay in HEK 293 3HA-hCB1 cells. Congruent with previous studies [12], [26], we found WIN55,212-2, CP55,940, and THC generated concentration-dependent inhibition of
Discussion
The increasing emergence of novel SCRAs as recreational drugs raises concerns surrounding their pharmacological action and subsequent effects upon human consumption [38]. SCRA-related fatalities and hospitalisation are a growing global public health problem [39], with over 70 deaths linked to synthetic cannabinoid use in New Zealand in the past two years [40]. The current study has utilised several methods to explore the molecular pharmacology of synthetic cannabinoids and found that a diverse
Acknowledgements
MP and JJM are recipients of the University of Otago Doctoral Scholarship. DBF is supported by a Lottery Health Research Postdoctoral Fellowship. Research was supported by grants from the Maurice and Phyllis Paykel Trust and Health Research Council of New Zealand to MG.
Conflicts of interest
The authors declare no conflicts of interest.
Author contributions
MP designed and performed experiments, analysed data and wrote the paper. JJM and DBF performed experiments, assisted with data analysis, and revised drafts of the manuscript. JAJ provided the arrestin assay. SDB synthesised compounds used in experiments. NG contributed to experimental design and oversaw aspects of the project. MG oversaw all elements of the project, obtained the funding, and reviewed drafts of the paper. All authors reviewed and approved the final version of the paper.
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