Differential activation of G protein‐mediated signaling by synthetic cannabinoid receptor agonists

Abstract Synthetic cannabinoid receptor agonists (SCRAs) are new psychoactive substances associated with acute intoxication and even death. However, the molecular mechanisms through which SCRAs may exert their toxic effects remain unclear—including the potential differential activation of G protein subtypes by cannabinoid receptor type 1 (CB1), a major target of SCRA. We measured CB1‐mediated activation of Gαs and Gαi/o proteins by SCRAs by examining stimulation (pertussis toxin, PTX treated) as well as inhibition (non‐PTX treated) of forskolin (FSK)‐induced cyclic adenosine monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably expressing CB1. Real‐time measurements of stimulation and inhibition of cAMP levels were made using a BRET biosensor. We found that the maximum concentration of SCRAs tested (10 µmol L−1), increased cAMP levels 12%‐45% above that produced by FSK alone, while the phytocannabinoid THC did not significantly alter cAMP levels in PTX‐treated HEK‐CB1 cells. All SCRAs had greater potency to inhibit FSK‐induced cAMP levels than to stimulate cAMP levels. The rank order of potencies for SCRA stimulation of cAMP (Gαs) was PB‐22 > 5F‐MDMB‐PICA > JWH‐018 ≈ AB‐FUBINACA > XLR‐11. By contrast, the potency of SCRAs for inhibition of cAMP (Gαi/o) was 5F‐MDMB‐PICA > AB‐FUBINACA > PB‐22 > JWH‐018 > XLR‐11. The different rank order of potency and EMax of the SCRAs to stimulate Gαs‐like signaling compared to Gαi/o signaling suggests differences in G protein preference between SCRAs. Understanding the apparent differences among these drugs may contribute to unravelling their complex effects in humans.


| INTRODUC TI ON
The use of synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) is associated with significant morbidity and mortality compared to use of ∆ 9 -tetrahydrocannabinol (THC), the main psychoactive ingredient of cannabis. 1,2 SCRAs are linked to a wide range of toxic effects including seizures, agitation, hypertension, cardiotoxicity, kidney damage, and sometimes death. 3,4 There has been a rapid increase in the number of structurally diverse SCRAs since 2010, with little known about their pharmacology and toxicology at time of identification. 5 The constant evolution of SCRA structures occurs in response to legislative restriction and development of urine drug screens for existing compounds. 6 SCRAs are usually agonists at both cannabinoid type-1 and type-2 receptors (CB1 and CB2, respectively 10 ); with the psychoactive effects attributed to the activation of CB1. 11 We have previously described the in vitro quantitative measurement of SCRA efficacy at CB1, where all SCRAs tested showed between 20-and 300-fold greater agonist activity at CB1 compared to THC. 12 Cannabinoid receptor-mediated G protein signaling is predominantly through the Gα i/o protein family 13 ; however, under some circumstances, CB1 can also stimulate adenylyl cyclase (AC) through Gα s -proteins. [14][15][16] For example, blockade of the canonical CB1-Gα i pathway with pertussis toxin (PTX) or sequestration of CB1-Gα i protein in the primary striatal rat neurons on coexpression with D2 results in an augmentation of cyclic adenosine monophosphate (cAMP) levels by cannabinoids, suggesting that CB1 couples to Gα s . 14,15 A recent study characterized the relationship between CB1 receptor expression and signaling, and showed that at very high receptor expression levels, the effect of CB1 activation on cAMP signaling was stimulatory, a phenotype that was reversed by systematic pharmacological knockdown at the receptor level. 17 The idea that certain SCRAs may preferentially activate different CB1 Gα subtypes is not unprecedented [18][19][20] ; in a study by Costain et al 21  The mechanism(s) through which SCRAs exert different behavioral and physiological effects remains unclear, and which pathways modulated by CB1 activation mediate the specific pharmacological effects of SCRAs is also unknown. Similarly, the question of whether these pathways are activated in a quantitatively or qualitatively similar way by SCRAs and THC is only beginning to be addressed. 22 Finally, the question of whether SCRA activity at noncannabinoid receptors is also important for their pharmacological effects is very much open. [23][24][25] With more than 250 SCRAs identified in the NPS market, 9 elucidation of the differential molecular mechanisms by which these compounds can exert distinct pharmacology, including their signaling via CB1, is essential for understanding their adverse effects. This study examined whether SCRAs that are representative of structural classes confirmed in patients admitted to emergency departments with presumed SCRA toxicity stimulate Gα s -like cAMP signaling via CB1. We measured the SCRA-mediated stimulation as well as inhibition of FSK-induced cAMP accumulation in HEK cells stably expressing CB1. We have observed SCRA-specific CB1-dependent activation of the two signaling pathways, but THC only coupled to inhibition, not stimulation of cAMP. While AB-CHIMINACA, previously identified as having a unique profile among SCRAs for elevating cAMP, appeared to signal, in part, through non-CB1 mechanisms.

| Assay for cAMP measurement
Intracellular cAMP levels were measured using pcDNA3L-His-CAMYEL plasmid, which encodes the cAMP sensor YFP-Epac-RLuc (CAMYEL) as outlined in Ref. [ 27,28 ] Cells were detached from the flask using trypsin/ EDTA (Sigma-Aldrich), and resuspended in DMEM supplemented with 10% FBS, 100 units mL −1 penicillin, and 100 µg mL −1 streptomycin. Cells were seeded in 10 cm dishes at a density of 7 000 000 such that they would be 60%-70% confluent the next day. On the following day, the cells were transiently transfected with 5 µg of pcDNA3L-His-

| Data analysis
Raw data are presented as inverse bioluminescence resonance energy transfer (BRET) ratio of emission at 475/535 nm, such that an increase in ratio corresponds with increase in cAMP production. Real-time (raw) cAMP time course data were then analyzed using area under curve analysis in GraphPad PRISM (Graph Pad Software Inc). Data were normalized to the change produced by FSK over 20 minutes (set as 100%) for each experiment. The percent change values were fit to three or four-parameter non-linear regression curves in PRISM to derive EC 50 and E max . In the three parameter fit the Hill slope was constrained to 1, in the four parameter fit it was free to vary. All final datasets passed the Shapiro-Wilk test for normality. Unless otherwise stated, the data represent mean ± SEM of at least five independent experiments, each conducted in duplicate.

F I G U R E 1
Concentration response curve for CP55940 and WIN55212-2. Treatment with CP55940 or WIN55212-2 produced a concentration-dependent inhibition of forskolin-mediated cAMP production in human embryonic kidney 293-cannabinoid receptor type 1. Curves were generated by area under the curve analysis for CP55940 or WIN55212-2 in the presence of 3 μmol L −1 forskolin. Data were normalized to forskolin (100%) and vehicle (0%), and plotted as mean ± SEM for at least five independent experiments performed in duplicate. cAMP, cyclic adenosine monophosphate  The differences between groups were tested using unpaired Student's t test, and one-way ANOVA as appropriate when comparing multiple groups (PRISM). Statistical significance is defined as P < .05. University. Chemical structure of SCRAs can be found elsewhere. 12 All the SCRAs were prepared in DMSO and stored in aliquots of 30 mmol L −1 in −30°C until needed.

| Real-time cAMP BRET measurement of the Gα s -mediated signaling of SCRAs
Using the CAMYEL assay, we measured the effect of seventeen cannabinoids (10 µmol L −1 each) on the FSK-stimulated cellular cAMP levels in HEK-CB1 cells following pretreatment with PTX.
All the SCRAs produced an increase in cAMP levels above that produced by FSK alone (100%). Examples of raw traces are shown for some SCRAs (Figure 2A), note that the stimulation of cAMP by SCRAs in the presence of FSK and PTX plateaued approximately after 12 minutes, and maintained at that level for the entire course of the assay (20 minutes). The effects of SCRAs tested ranged from 12% to 45% increase in signal relative to FSK alone.

| Differential SCRAs-induced stimulation and inhibition of cAMP signaling in HEK-CB1
To assess whether there was any evidence of preferential coupling to Gα i/o over Gα s among SCRAs, we assessed the pharmacological activity (EC 50 and E max ) of a selection of SCRAs belonging to different structural classes (JWH-018, PB-22, AB-FUBINACA, XLR-11, and 5F-MDMB-PICA), to stimulate and inhibit cAMP in HEK-CB1 cells.
AB-CHMINACA has previously been reported to stimulate Gα slike cAMP signaling pathway in a concentration-dependent manner in HEK-CB1 cells. 21 Following PTX treatment, AB-CHMINACA increased cAMP levels above that of FSK alone ( Figure 5A Figure 5B). To confirm that this response was at least in part non-CB1-mediated, AB-CHMINACA was tested in HEK 293 wild-type cells; in these cells, AB-CHMINACA (10 µmol L −1 ) also produced a small increase in FSK-stimulated cAMP accumulation ( Figure 5C, 29 ± 10%), suggesting that some of these stimulatory effects were occurring via mechanism(s) unrelated to CB1 receptor activity.

| D ISCUSS I ON
In this study, we set out to systematically characterize the ability of several SCRAs to activate Gα s and Gα i/o proteins by examining F I G U R E 3 Concentration response curves for SCRAs-induced stimulation and inhibition of cAMP signaling. Pooled concentration response relationship for five SCRAs (PB-22, 5F-MDMB-PICA, AB-FUBINACA, XLR-11, and JWH-018) for two signaling outputs of cannabinoid receptor type 1-stimulation and inhibition of cAMP levels following overnight treatment in the absence (−PTX, black), or presence (+PTX, red) of PTX. Data were normalized to forskolin (FSK, 100%) and vehicle (0%), and plotted as mean ± SEM for at least five independent experiments performed in duplicate. For some points, the error bars are shorter than the height of the symbol. BRET, bioluminescence resonance energy transfer; cAMP, cyclic adenosine monophosphate; PTX, pertussis toxin; SCRA, synthetic cannabinoid receptor agonist We further sought to investigate SCRA differential activation of distinctive G protein subsets-inhibition and stimulation of FSKmediated cAMP signaling. The relative ability of SCRAs to induce inhibition of cAMP production via Gα i/o is very similar to that observed in previous studies in assays of membrane potential and [ 35 S]GTPγS binding. 12,25,33,34 The similar E max observed for the SCRA-mediated activation of Gα i/o -CB1 signaling in this study probably reflects receptor reserve for inhibition of cAMP accumulation in these cells, wherein maximal responses are elicited at less than maximal receptor occupancy because the system maximum is already achieved. 12 SCRA-induced stimulation of cAMP showed significant differences in E max (Table 1), suggesting an absence of receptor reserve for most of the Gs-dependent signaling we observed for the SCRA in these conditions. This may (at least for the drugs with a lower E max ) reflect an accurate representation of intrinsic efficacy of the ligands at this pathway. 35 The observed dynamic range of E max for cannabinoids is consistent with CB1 having low coupling efficiency to both Gα s pathway and β-arrestin-2 (as observed previously 32 ), compared to  TA B L E 1 Comparison of pharmacological activity (EC 50 and E max ) of SCRAs-induced stimulation (G s (+PTX)) and inhibition (G i (−PTX)) of cAMP signaling in HEK-CB1 cells that of Gα i pathway. 17,36,37 Future studies could examine the structure of SCRA-bound CB1-Gα s complexes, which might assist in explaining the observed cAMP signaling profiles. This is particularly interesting given that the interaction of SCRA MDMB-FUBINACA with the "toggle twin switch" in the CB1 binding pocket coupled to Gα i was recently studied. 38 The rigid C-shape geometry of MDMB-FUBINACA along with the strong pi-pi interaction of its indazole ring with "toggle twin switch" residues, might help distinguish the high efficacy agonist activity of SCRA from partial agonists like THC lacking "toggle twin switch" interaction. 38  which might explain some of the toxicity experienced by recreational users of these drugs. [43][44][45][46][47][48][49] Our data shows that SCRA-induced cAMP increase was abolished after SR141716A treatment, supporting the hypothesis that SCRAs Gα s -like effects were mediated through CB1 receptor. All the SCRAs tested in this study exhibited greater potency at Gα i -than Gα s -like pathways, and the efficacies of these SCRAs have previously been measured in response to Gα i -mediated activation of GIRK channel in AtT20-CB1 cells. 12 The rank order of greater efficacy for the Gα s pathway compared to its Gα i -mediated activity profile in the membrane potential assay. 12 Evaluating the differences in G protein preference between SCRAs may be an important part of understanding the apparent differences in effect between these drugs in humans. However, the biological significance of SCRA-mediated differential coupling of CB1 to G i/o and G s is not well  understood. The G s signaling of CB1 arises in circumstances where G i/o is exhausted or sequestered, and has been measured after PTX treatment or when other G i -coupled receptors are concomitantly activated. The phenomenon was first observed in primary rat striatal neurons natively expressing CB1 and D2 receptors, 14,15 while a switch in G i -G s signaling due to high CB1 expression has subsequently been defined in recombinant systems. 17 The phenomenon of CB1-Gs coupling may be relevant in specific cancer conditions where upregulation in CB1 receptor was reported (eg colorectal cancer, human epithelia ovarian tumors, and prostate cancer). 17 Our study showed that SCRAs have significantly different

D I SCLOS U R E S
The authors declare that they have no conflict of interest related to this work.