Signalling profiles of a structurally diverse panel of synthetic cannabinoid receptor agonists

Abstract

Synthetic cannabinoid receptor agonists (SCRAs) represent the most rapidly proliferating class of “designer drugs” or “new psychoactive substances”. SCRAs offer unregulated alternatives to cannabis that evade routine drug tests, but their use is increasingly associated with severe toxicity and death worldwide. Little is currently known about SCRA molecular pharmacology, or the mechanisms underpinning their toxicity, although the effects are believed to be primarily mediated by the type 1 cannabinoid receptor (CB₁). In this study, we aimed to characterise the signalling profiles of a structurally diverse panel of novel SCRAs at CB₁. We compare SCRAs to traditional reference cannabinoids CP55,940, WIN55,212-2, and THC. The activity of the SCRAs was assessed in key receptor signalling and regulatory pathways, including cAMP production, translocation of β-arrestin 1 and 2, and receptor internalisation. The activity profiles of the ligands were also evaluated using operational analysis to identify ligand bias. Results revealed that SCRAs activities were relatively balanced in the pathways evaluated (compared to WIN55,212-2), although 5F-CUMYL-P7AICA and XLR-11 possessed partial efficacy in cAMP stimulation and β-arrestin translocation. Notably, the SCRAs showed distinct potency and efficacy profiles compared to THC. In particular, while the majority of SCRAs demonstrated robust β-arrestin translocation, cAMP stimulation, and internalisation, THC failed to elicit high efficacy responses in any of these assays. Further study is required to delineate if these pathways could contribute to SCRA toxicity in humans.

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 Gα_i/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.