Synthetic Cannabinoid Signaling Elucidated by a BRET Biosensor

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The first thing that comes to mind for most people when thinking about the term cannabinoid is the best-known representative of the substance group—tetrahydrocannabinol, or THC. THC is the principal mildly psychedelic constituent of cannabis.¹ Cannabinoid psychedelic effects are primarily mediated by the interaction of THC with the cannabinoid receptors 1 and 2 (CB1 and CB2). Since THC is naturally found in the cannabis plant, it is categorized in the subgroup of phytocannabinoids.

Besides phytocannabinoids, there are also synthetic cannabinoid receptor agonists (SCRAs) which can interact with the receptors of the endocannabinoid system (Figure 1). As it is known that cannabinoids can have an analgesic effect, SCRAs were initially developed over the past 40 years as therapeutic agents or for legitimate scientific research. Many of the sub-stances are not structurally related to classical cannabinoids derived from the cannabis plant and generally form a diverse group. Unfortunately, SCRAs represent nowadays the most rapidly growing class of new psychoactive substances associated with drug misuse.² SCRAs are highly potent and addictive and pose serious health risks with adverse effects such as severe vomiting, chest pain, increased heart rate, kidney damage, seizures and unconsciousness, and even death. The increased danger posed by SCRAs, compared with regular marijuana, can be explained in part by many of the synthetic cannabinoids being full agonists of CB1 and CB2 receptors, compared to THC, which is only a partial agonist. Additionally, SCRAs may also have other actions in the body, independent of the cannabinoid receptors.

What is known so far about SCRA signaling

Unfortunately, little is known about the detailed toxicology and pharmacology of SCRAs including molecular mechanisms, and only a few human studies have been published. So far, previous studies have shown that SCRAs are agonists of both CB1 and CB2 receptors. Psychoactive effects are thereby mainly attributed to CB1. These cannabinoid receptors belong to the family of G-protein-coupled receptors (GPCRs). GPCRs span the outer membrane of cells and are coupled to a trimeric G-protein complex including Gα, Gβ, and Gγ at the inner side of the membrane. Binding of a receptor agonist may lead to dissociation of the trimeric G-protein complex and the release of the subunits into the cytoplasm, leading to the initiation of many cellular responses. Gα can be found in four main subtypes, Gα_i, Gα_s, Gα_q, and Gα_12/13, whose activation partly result in contrary effects. While the activation of Gα_s stimulates the adenyl cyclase leading to an increase in cAMP, activated Gα_i in contrast leads to a decrease in cAMP generation (Figure 1). SCRAs are known to mainly signal through Gα_i. Two groups of researchers from Macquarie University and the University of Sydney in Australia were interested in studying whether some selected SCARs signal via the Gi or the Gs pathway.

Monitoring cannabinoid signaling with the CAYMEL BRET biosensor

The CAYMEL BRET biosensor (BRET stands for bioluminescence resonance energy transfer) is composed of the EPAC protein bound to both a luciferase (RLuc) and a yellow fluorescent protein (YFP), as shown in Figure 1. In absence of cAMP, the conformation of EPAC allows Rluc and YFP to stay in proximity and to generate a BRET signal upon addition of the RLuc substrate coelenterazine. If cAMP is present in the cell, it binds EPAC, leading to a conformational change which diminishes the BRET signal.

To study whether a selection of cannabinoids signal via Gα_i or Gα_s, researchers used human HEK293 cells stably expressing the CB1 receptor.³ They additionally transfected the HEK293 cells with the gene for the CAYMEL plasmid, to monitor the intracellular cAMP concentration upon the addition of RLuc substrate. To be able to differentiate between Gα_i and Gα_s signaling, cells were treated or not-treated with pertussis toxin (PTX), which is known to inactivate the Gα_i-associated pathway. Forskolin (FSK) was added in all attempts, to generally raise levels of cAMP, which also allows the inhibition of cAMP generation to be measured.

Signaling was evaluated for 12 SCRAs and confirmed in patients admitted to emergency departments. Besides these SCRAs, four reference compounds, including THC, were tested. Living cells were stimulated with the different cannabinoids (10 µm) and FSK (3 µm) with or without PTX inhibition in a real-time kinetic experiment. During the stimulation and the cAMP monitoring, cells were incubated at 37°C in BMG LABTECH´s PHERAstar^® microplate reader for 20 min. The BRET signal consists of two emission signals, one for the luminescent signal of the BRET donor Rluc at 475 nm, and one for the emission of the acceptor YFP at 525 nm, which is only excited in the absence of cAMP. With its unique simultaneous dual emission detection system, the PHERAstar enables both emission signals to be measured at the same time, using a beam splitter and a matched pair of photomultiplier tubes for the signal detection. With this feature, each sample has to be read only once, which substantially reduces plate read times, increases sample throughput, and reduces variability. Inverse BRET ratios (475/535) were used for the evaluation, to allow an increase in cAMP to be related to an increase in the BRET ratio. Initially, the CB1-mediated activation of Gα_s was measured with PTX-treated cells. All tested cannabinoids increased the cAMP level above that produced by FSK alone, as displayed exemplarily for three compounds in Figure 2A. The area under the total signal curve was used to directly compare the different compounds regarding their ability to stimulate the Gα_s pathway (Figure 2B). In contrast to the SCRAs, THC did not significantly alter levels over FSK.

Differentiation between Gα_i and Gα_s signaling

The same measurements were repeated without PTX-driven inactivation of Gα_i, to monitor possible inhibition of cAMP generation. Concentration response curves were then constructed for five structurally different agents to determine EC₅₀ and E_max values for activation of both the Gα_s and Gα_i signaling pathways. All the cannabinoids tested activated CB1 via both pathways. However, all compounds were much more potent for Gα_i as compared to Gα_s pathways. It can therefore be concluded that all SCRAs had greater potency to inhibit FSK-induced cAMP levels than to stimulate cAMP levels. Furthermore, SCRAs showed a different rank order of potency in stimulating Gα_s-like signaling compared with Gα_i signaling. This suggests different preferences for G proteins between different SCRAs.

Acute adverse effects of SCRAs in humans range from intoxication to even death. Elucidation of the differential molecular mechanisms, triggered by these synthetic cannabinoids, may help unravel their complex effects in vivo. Thanks to its very high sensitivity, the PHERAstar is a reliable partner in this investigation process. The possibility of reading two emission signals simultaneously contributes significantly to the speed advantage of the microplate reader. This speeds up the experiments and enables more experiments to be conducted at the same time.

References
1. Köguel, C.C., López-Pelayo, H., Balcells-Olivero, M.M. et al. Psychoactive constituents of cannabis and their clinical implications: a systematic review. Adicciones. 2018; 30:140-151, doi: 10.20882/adicciones.858
2. Brandt SD, King LA, Evans‐Brown M. The new drug phenomenon. Drug Test Anal. 2014;6:587‐597, doi: 10.1002/dta.1686
3. Sachdev S, Banister SD, Santiago M et al; Differential activation of G protein mediated signaling by synthetic cannabinoid receptor agonists, Pharmacol Res Perspect. 2020; 00:e00566. doi: 10.1002/prp2.566

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