Rational design of cannabinoid type-1 receptor allosteric modulators: Org27569 and PSNCBAM-1 hybrids.

Allosteric modulation offers an alternate approach to target the cannabinoid type-1 receptor (CB1) for therapeutic benefits. Examination of the two widely studied prototypic CB1 negative allosteric modulators (NAMs) Org27569 and PSNCBAM-1 revealed structural resemblance and similar structure-activity relationships (SARs). In silico docking and dynamics simulation studies using the crystal structure of CB1 co-bound with CP55,940 and Org27569 suggested that Org27569 and PSNCBAM-1 occupied the same binding pocket and several common interactions were present in both series with the CB1 receptor. A new scaffold was therefore designed by merging the key structural features from the two series and the hybrids retained these binding features in the in silico docking studies. In addition, one such hybrid displayed similar functions to Org27569 in dynamic simulations by preserving a key R2143.50-D3386.30 salt bridge and maintaining an antagonist-like Helix3-Helix6 interhelical distance. Based on these results, a series of hybrids were synthesized and assessed in calcium mobilization, [35S]GTPγS binding and cAMP assays. Several compounds displayed comparable potencies to Org27569 and PSNCBAM-1 in these assays. This work offers new insight of the SAR requirement at the allosteric site of the CB1 receptor and provides a new scaffold that can be optimized for the development of future CB1 allosteric modulators.

Inconsistencies in histone acetylation patterns among different HD model systems and HD post-mortem brains.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in exon 1 of the huntingtin gene. Emerging evidence shows that additional epigenetic factors can modify disease phenotypes. Harnessing the ability of the epigenome to modify the disease for therapeutic purposes is therefore of interest. Epigenome modifiers, such as histone deacetylase inhibitors (HDACi), have improved pathology in a range of HD models. Yet in clinical trials, HDACi have failed to alleviate HD symptoms in patients. This study investigated potential reasons for the lack of translation of the therapeutic benefits of HDACi from lab to clinic. We analysed histone acetylation patterns of immuno-positive nuclei from brain sections and tissue microarrays from post-mortem human control and HD cases alongside several well-established HD models (OVT73 transgenic HD sheep, YAC128 mice, and an in vitro cell model expressing 97Q mutant huntingtin). Significant increases in histone H4 acetylation were observed in post-mortem HD cases, OVT73 transgenic HD sheep and in vitro models; these changes were absent in YAC128 mice. In addition, nuclear labelling for acetyl-histone H4 levels were inversely proportional to mutant huntingtin aggregate load in HD human cortex. Our data raise concerns regarding the utility of HDACi for the treatment of HD when regions of pathology exhibit already elevated histone acetylation patterns and emphasize the importance of searching for alternative epigenetic targets in future therapeutic strategies aiming to rescue HD phenotypes.

Divalent cannabinoid-1 receptor ligands: A linker attachment point survey of SR141716A for development of high-affinity CB1R molecular probes.

The cannabinoid-1 receptor (CB1R) inverse agonist SR141716A has proven useful for study of the endocannabinoid system, including development of divalent CB1R ligands possessing a second functional motif attached via a linker unit. These have predominantly employed the C3 position of the central pyrazole ring for linker attachment. Despite this precedent, a novel series of C3-linked CB1R-D2R divalent ligands exhibited extremely high affinity at the D2R, but only poor affinity for the CB1R. A systematic linker attachment point survey of the SR141716A pharmacophore was therefore undertaken, establishing the C5 position as the optimal site for linker conjugation. This linker attachment survey enabled the identification of a novel divalent ligand as a lead compound to inform ongoing development of high-affinity CB1R molecular probes.

Biased Agonism and Biased Allosteric Modulation at the CB1 Cannabinoid Receptor.

CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol], and HU-210 [11-hydroxy-Δ(8)-THC-dimethylheptyl] were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 [5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [(3)H]SR141716A [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and pregnenolone at CB1Rs. Pregnenolone but not lipoxin A4 displaced [(3)H]SR141716A, but there was no functional interaction between either of these ligands and cannabinoid agonists. This study demonstrates an approach to validating and quantifying ligand-biased signaling and allosteric modulation at CB1Rs, revealing ligand-biased "fingerprints" that may ultimately allow the development of improved CB1R-targeted therapies.

GPCR kinase subtype requirements for arrestin-2 and -3 translocation to the cannabinoid CB1 receptor and the consequences on G protein signalling.

Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of 'phosphorylation barcode,' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB1 receptor (CB1) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB1, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB1 must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.

A quantitative pharmacology model for cannabinoid CB1 receptor mediated by Gi/Gs protein competition.

BACKGROUND AND PURPOSE: Orthosteric agonism of the CB1 receptor normally associates with Gi signalling resulting in a net inhibition of cAMP production. Empirical evidence shows CB1 causes a net cAMP stimulation through Gs coupling under two conditions: co-stimulation with the D2 receptor and high-level CB1 expression. Two hypotheses have been proposed to account for these paradoxical effects, (1) Gi is consumed by coupling to D2 or extra CB1 and excess CB1 binds to Gs and (2), the formation of dimers CB1 -CB1 or CB1 -D2 switches Gi/Gs preference. This study explored the mechanisms of Gi/Gs preference based on a mathematical model of the CB1 receptor. EXPERIMENTAL APPROACH: The model was based on Hypothesis 1 and known mechanisms. The model was calibrated to align with multiple types of data (cAMP, Gi dissociation and internalisation). The key step of Hypothesis 1 was examined by simulation from the model. An experiment was proposed to distinguish Hypothesis 1 and 2. KEY RESULTS: The model successfully descripted multiple types of data under Hypothesis 1. Simulations from the model indicated that precoupling of G protein with receptors is necessary for this hypothesis. The model designed experiments to distinguish Hypothesis 1 and 2 by increasing Gi & Gs in parallel with CB1 overexpression. The two hypotheses result in distinct cAMP responses. CONCLUSION AND IMPLICATIONS: A mathematical model of CB1 -regulated Gi/Gs pathways was developed. It indicated Hypothesis 1 is feasible and G protein precoupling is a key step causing cAMP signalling switch. The model-designed experiments provided guides for future experimentation.

Determination of the Cannabinoid CB1 Receptor's Positive Allosteric Modulator Binding Site through Mutagenesis Studies.

Positive allosteric modulators (PAMs) of the cannabinoid CB1 receptor (CB1) offer potential therapeutic advantages in the treatment of neuropathic pain and addiction by avoiding the adverse effects associated with orthosteric CB1 activation. Here, molecular modeling and mutagenesis were used to identify residues central to PAM activity at CB1. Six putative allosteric binding sites were identified in silico, including novel sites previously associated with cholesterol binding, and key residues within each site were mutated to alanine. The recently determined ZCZ011 binding site was found to be essential for allosteric agonism, as GAT228, GAT229 and ZCZ011 all increased wild-type G protein dissociation in the absence of an orthosteric ligand; activity that was abolished in mutants F191A3.27 and I169A2.56. PAM activity was demonstrated for ZCZ011 in the presence of the orthosteric ligand CP55940, which was only abolished in I169A2.56. In contrast, the PAM activity of GAT229 was reduced for mutants R220A3.56, L404A8.50, F191A3.27 and I169A2.56. This indicates that allosteric modulation may represent the net effect of binding at multiple sites, and that allosteric agonism is likely to be mediated via the ZCZ011 site. This study underlines the need for detailed understanding of ligand receptor interactions in the search for pure CB1 allosteric modulators.

Pharmacological evaluation of new generation OXIZID synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the largest classes of new psychoactive substances, and are increasingly associated with severe adverse effects and death compared to the phytocannabinoid Δ9-tetrahydrocannabinol (THC). In the attempt to circumvent the rapid emergence of novel SCRAs, several nations have implemented 'generic' legislations, or 'class-wide' bans based on common structural scaffolds. However, this has only encouraged the incorporation of new chemical entities, including distinct core and linker structures, for which there is a dearth of pharmacological data. The current study evaluated five emergent OXIZID SCRAs for affinity and functional activity at the cannabinoid CB1 receptor (CB1) in HEK 293 cells, as well as pharmacological equivalence with THC in drug discrimination in mice. All OXIZID compounds behaved as agonists in Gαi protein activation and ß-arrestin 2 translocation assays, possessing low micromolar affinity at CB1. All ligands also substituted for THC in drug discrimination, where potencies broadly correlated with in vitro activity, with the methylcyclohexane analogue BZO-CHMOXIZID being the most potent. Notably, MDA-19 (BZO-HEXOXIZID) exhibited partial efficacy in vitro, generating an activity profile most similar to that of THC, and partial substitution in vivo. Overall, the examined OXIZIDs were comparatively less potent and efficacious than previous generations of SCRAs. Further toxicological data will elucidate whether the moderate cannabimimetic activity for this series of SCRAs will translate to severe adverse health effects as seen with previous generations of SCRAs.

Insight into the mechanism of action of ORG27569 at the cannabinoid type one receptor utilising a unified mathematical model.

Allosteric modulation of CB1 is therapeutically advantageous compared to orthosteric activation as it potentially offers reduced on-target adverse effects. ORG27569 is an allosteric modulator that increases orthosteric agonist binding to CB1 but decreases functional signalling. ORG27569 is characterised by a delay in disinhibition of agonist-induced cAMP inhibition (lag); however, the mechanism behind this kinetic lag is yet to be identified. We aimed to utilise a mathematical model to predict data and design in vitro experiments to elucidate mechanisms behind the unique signalling profile of ORG27569. The established kinetic ternary complex model includes the existence of a transitional state of CB1 bound to ORG27569 and CP55940 and was used to simulate kinetic cAMP data using NONMEM 7.4 and Matlab R2020b. These data were compared with empirical cAMP BRET data in HEK293 cells stably expressing hCB1. The pharmacometric model suggested that the kinetic lag in cAMP disinhibition by ORG27569 is caused by signal amplification in the cAMP assay and can be reduced by decreasing receptor number. This was confirmed experimentally, as reducing receptor number through agonist-induced internalisation resulted in a decreased kinetic lag by ORG27569. ORG27569 was found to have a similar interaction with CP55940 and the high efficacy agonist WIN55,212-2, and was suggested to have lower affinity for CB1 bound by the partial agonist THC compared to CP55940. Allosteric modulators have unique signalling profiles that are often difficult to interrogate exclusively in vitro. We have used a combined mathematical and in vitro approach to prove that ORG27569 causes a delay in disinhibition of agonist-induced cAMP inhibition due to large receptor reserve in this pathway. We also used the pharmacometric model to investigate the common phenomenon of probe dependence, to propose that ORG27569 binds with higher affinity to CB1 bound by high efficacy orthosteric agonists.

Investigating selectivity and bias for G protein subtypes and ß-arrestins by synthetic cannabinoid receptor agonists at the cannabinoid CB1 receptor.

The cannabinoid CB1 receptor (CB1) is a G protein-coupled receptor (GPCR) with widespread expression in the central nervous system. This canonically G⍺i/o-coupled receptor mediates the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoid receptor agonists (SCRAs). Recreational use of SCRAs is associated with serious adverse health effects, making pharmacological research into these compounds a priority. Several studies have hypothesised that signalling bias may explain the different toxicological profiles between SCRAs and THC. Previous studies have focused on bias between G protein activation measured by cyclic adenosine monophosphate (cAMP) inhibition and ß-arrestin translocation. In contrast, the current study characterises bias between G⍺ subtypes of the G⍺i/o family and ß-arrestins; this method facilitates a more accurate assessment of ligand bias by assessing signals that have not undergone major amplification. We have characterised G protein dissociation and translocation of ß-arrestin 1 and 2 using real-time BRET reporters. The responses produced by each SCRA across the G protein subtypes tested were consistent with the responses produced by the reference ligand AMB-FUBINACA. Ligand bias was probed by applying the operational analysis to determine biases within the G⍺i/o family, and between G protein subtypes and ß-arrestins. Overall, these results confirm SCRAs to be balanced, high-efficacy ligands compared to the low efficacy ligand THC, with only one SCRA, 4CN-MPP-BUT7IACA, demonstrating statistically significant bias in one pathway comparison (towards ß-arrestin 1 when compared with G⍺oA/oB). This suggests that the adverse effects caused by SCRAs are due to high potency and efficacy at CB1, rather than biased agonism.

Positive allosteric modulation of the cannabinoid CB1 receptor potentiates endocannabinoid signalling and changes ERK1/2 phosphorylation kinetics.

BACKGROUND AND PURPOSE: Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation. EXPERIMENTAL APPROACH: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and ß-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA. KEY RESULTS: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed. CONCLUSION AND IMPLICATIONS: Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including ß-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.

Clinical responses to selumetinib (AZD6244; ARRY-142886)-based combination therapy stratified by gene mutations in patients with metastatic melanoma.

BACKGROUND: The high prevalence of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) and neuroblastoma v-ras oncogene homolog (NRAS) mutations in melanoma provides a strong rationale to test the clinical efficacy of mitogen-activated protein kinase kinase (MEK) inhibition in this disease. The authors hypothesized that the presence of BRAF or NRAS mutations would correlate with clinical benefit among patients who received treatment with combination regimens that included the MEK inhibitor selumetinib. METHODS: BRAF and NRAS mutation status was determined retrospectively in available tissue specimens from patients with melanoma who were enrolled in a phase 1 trial of selumetinib in combination with 1 of 4 drugs (dacarbazine, docetaxel, temsirolimus, or erlotinib). The clinical response rate and the time to progression (TTP) were assessed as a function of BRAF and NRAS mutation status. RESULTS: Among 18 patients analyzed, 9 patients (50%) harbored a BRAF mutation (8 had a valine-to-glutamic acid substitution at residue 600 [V600E]; 1 had an arginine nonsense mutation at residue 603 [R603]), 4 patients (22%) harbored an NRAS mutation (2 had a glutamine-to-arginine substitution at residue 61 [Q61R], 1 had a glutamine-to-lysine substitution at residue 61 [Q61K], and 1 had a glycine-to-lysine substitution at residue 12 [G12S]), and 5 patient (28%) had the wild type of both genes. These mutations were mutually exclusive. Among the 9 patients who had BRAF mutations, 5 patients (56%) achieved a partial response, and 4 patients (44%) achieved stable disease for at least 6 weeks. No patient with the wild-type BRAF gene achieved a clinical response (P = .01 vs patients with BRAF mutations). The presence of an NRAS mutation did not correlate with the clinical response rate. The presence of a BRAF mutation was correlated significantly with the TTP in a multivariate model (hazard ratio, 0.22; P = .02 vs wild-type BRAF). CONCLUSIONS: Higher response rates and longer TTP were observed with selumetinib-containing regimens in patients who had tumors that harbored a BRAF mutation compared with patients who had wild-type BRAF.

Cannabinoid 1 (CB1 ) receptor arrestin subtype-selectivity and phosphorylation dependence.

BACKGROUND AND PURPOSE: Arrestin or G protein bias may be desirable for novel cannabinoid therapeutics. Arrestin-2 and arrestin-3 translocation to CB1 receptor have been suggested to mediate different functions that may be exploited with biased ligands. Here, the requirement of a recently described phosphorylation motif 'pxxp' (where 'p' denotes phosphorylatable serine or threonine and 'x' denotes any other amino acid) within the CB1 receptor C-terminus for interaction with different arrestin subtypes was examined. EXPERIMENTAL APPROACH: Site-directed mutagenesis was conducted to generate nine different phosphorylation-impaired CB1 receptor C-terminal mutants. Bioluminescence resonance energy transfer (BRET) was employed to measure arrestin-2/3 translocation and G protein dissociation of a high efficacy agonist for each mutant. Immunocytochemistry was used to quantify receptor expression. KEY RESULTS: The effects of each mutation were shared for arrestin-2 and arrestin-3 translocation to CB1 receptor pxxp motifs are partially required for arrestin-2/3 translocation, but translocation was not completely inhibited until all phosphorylation sites were mutated. The rate of arrestin translocation was reduced with simultaneous mutation of S425 and S429. Desensitisation of G protein dissociation was inhibited in different mutants proportional to the extent of their respective loss of arrestin translocation. CONCLUSIONS AND IMPLICATIONS: These data do not support the existence of an 'essential' pxxp motif for arrestin translocation to CB1 receptor. These data also identify that arrestin-2 and arrestin-3 have equivalent phosphorylation requirements within the CB1 receptor C-terminus, suggesting arrestin subtype-selective biased ligands may not be viable and that different regions of the C-terminus contribute differently to arrestin translocation.

A kinetic model for positive allosteric modulator (PAM)-antagonists for the type 1 cannabinoid (CB1 ) receptor.

BACKGROUND AND PURPOSE: The cannabinoid (CB1 ) receptor is among the most abundant G protein-coupled receptors in brain. Allosteric ligands bind to a different site on receptors than the orthosteric ligand can have effects that are unique to the allosteric ligand and modulate orthosteric ligand activity. We propose a unified mathematical model describing the interaction effects of the allosteric ligand Org27569 and the orthosteric agonist CP55940 on CB1 receptor. EXPERIMENTAL APPROACH: A ternary complex model was constructed, which incorporated kinetic properties to describe the time course of effects of Org27569 and CP55940 reported in the literature: (i) enhanced receptor binding of CP55940, (ii) reduced internalisation and (iii), time-dependent modulation of cAMP. Underlying mechanisms of time-dependent modulation by Org27569 were evaluated by simulation. KEY RESULTS: A hypothetical transitional state of CP55940-CB1 -Org27569, which can internalise but cannot inhibit cAMP, was shown to be necessary and was sufficient to describe the allosteric modulation by Org27569, prior to receptors adopting an inactive conformation. The model indicated that the formation of this transitional CP55940-CB1 -Org27569 state and final inactive CP55940-CB1 -Org27569 state contributes to the enhanced CP55940 binding. The inactive CP55940-CB1 -Org27569 cannot internalise or inhibit cAMP, leading to reduced internalisation and cessation of cAMP inhibition. CONCLUSIONS AND IMPLICATIONS: In conclusion, a kinetic mathematical model for CB1 receptor allosteric modulation was developed. However, a standard ternary complex model was not sufficient to capture the data and a hypothetical transitional state was required to describe the allosteric modulation properties of Org27569.

Evaluating signaling bias for synthetic cannabinoid receptor agonists at the cannabinoid CB2 receptor.

The rapid structural evolution and emergence of novel synthetic cannabinoid receptor agonists (SCRAs) in the recreational market remains a key public health concern. Despite representing one of the largest classes of new psychoactive substances, pharmacological data on new SCRAs is limited, particularly at the cannabinoid CB2 receptor (CB2 ). Hence, the current study aimed to characterize the molecular pharmacology of a structurally diverse panel of SCRAs at CB2 , including 4-cyano MPP-BUT7AICA, 4F-MDMB-BUTINACA, AMB-FUBINACA, JWH-018, MDMB-4en-PINACA, and XLR-11. The activity of SCRAs was assessed in a battery of in vitro assays in CB2 -expressing HEK 293 cells: G protein activation (Gαi3 and GαoB ), phosphorylation of ERK1/2, and ß-arrestin 1/2 translocation. The activity profiles of the ligands were further evaluated using the operational analysis to identify ligand bias. All SCRAs activated the CB2 signaling pathways in a concentration-dependent manner, although with varying potencies and efficacies. Despite the detection of numerous instances of statistically significant bias, compound activities generally appeared only subtly distinct in comparison with the reference ligand, CP55940. In contrast, the phytocannabinoid THC exhibited an activity profile distinct from the SCRAs; most notably in the translocation of ß-arrestins. These findings demonstrate that CB2 is able to accommodate a structurally diverse array of SCRAs to generate canonical agonist activity. Further research is required to elucidate whether the activation of CB2 contributes to the toxicity of these compounds.

Characterisation of AMB-FUBINACA metabolism and CB1-mediated activity of its acid metabolite.

PURPOSE: AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The metabolising enzymes associated with this biotransformation remain unknown. This study aimed to determine if AMB-FUBINACA metabolism could be reduced in the presence of carboxylesterase (CES) inhibitors and recreational drugs commonly consumed with it. The affinity and activity of the AMB-FUBINACA acid metabolite at the cannabinoid type-1 receptor (CB1) was investigated to determine the activity of the metabolite. METHODS: The effect of CES1 and CES2 inhibitors, and delta-9-tetrahydrocannabinol (Δ9-THC) on AMB-FUBINACA metabolism were determined using both human liver microsomes (HLM) and recombinant carboxylesterases. Radioligand binding and cAMP assays comparing AMB-FUBINACA and AMB-FUBINACA acid were carried out in HEK293 cells expressing human CB1. RESULTS: AMB-FUBINACA was rapidly metabolised by HLM in the presence and absence of NADPH. Additionally, CES1 and CES2 inhibitors both significantly reduced AMB-FUBINACA metabolism. Furthermore, digitonin (100 µM) significantly inhibited CES1-mediated metabolism of AMB-FUBINACA by ~ 56%, while the effects elicited by Δ9-THC were not statistically significant. AMB-FUBINACA acid produced only 26% radioligand displacement consistent with low affinity binding. In cAMP assays, the potency of AMB-FUBINACA was ~ 3000-fold greater at CB1 as compared to the acid metabolite. CONCLUSIONS: CES1A1 was identified as the main hepatic enzyme responsible for the metabolism of AMB-FUBINACA to its less potent carboxylic acid metabolite. This biotransformation was significantly inhibited by digitonin. Since other xenobiotics may also inhibit similar SCRA metabolic pathways, understanding these interactions may elucidate why some users experience high levels of harm following SCRA use.

The piperazine analogue para-fluorophenylpiperazine alters timing of the physiological effects of the synthetic cannabinoid receptor agonist AMB-FUBINACA, without changing its discriminative stimulus, signalling effects, or metabolism.

AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA), which has been associated with substantial abuse and health harm since 2016 in many countries including New Zealand. A characteristic of AMB-FUBINACA use in New Zealand has included the observation that forensic samples (from autopsies) and drugs seized by police have often been found to contain para-fluorophenylpiperazine (pFPP), a relatively little-characterised piperazine analogue that has been suggested to act through 5HT1a serotonin receptors. In the current study, we aimed to characterise the interactions of these two agents in rat physiological endpoints using plethysmography and telemetry, and to examine whether pFPP altered the subjective effects of AMB-FUBINACA in mice trained to differentiate a cannabinoid (THC) from vehicle. Though pFPP did not alter the ability of AMB-FUBINACA to substitute for THC, it did appear to abate some of the physiological effects of AMB-FUBINACA in rats by delaying the onset of AMB-FUBINACA-mediated hypothermia and shortening duration of bradycardia. In HEK cells stably expressing the CB1 cannabinoid receptor, 5HT1a, or both CB1 and 5HT1a, cAMP signalling was recorded using a BRET biosensor (CAMYEL) to assess possible direct receptor interactions. Although low potency pFPP agonism at 5HT1a was confirmed, little evidence for signalling interactions was detected in these assays: additive or synergistic effects on potency or efficacy were not detected between pFPP and AMB-FUBINACA-mediated cAMP inhibition. Experiments utilising higher potency, classical 5HT1a ligands (agonist 8OH-DPAT and antagonist WAY100635) also failed to reveal evidence for mutual CB1/5HT1a interactions or cross-antagonism. Finally, the ability of pFPP to alter the metabolism of AMB-FUBINACA in rat and human liver microsomes into its primary carboxylic acid metabolite via carboxylesterase-1 was assessed by HPLC; no inhibition was detected. Overall, the effects we have observed do not suggest that increased harm/toxicity would result from the combination of pFPP and AMB-FUBINACA.

Signaling-specific inhibition of the CB1 receptor for cannabis use disorder: phase 1 and phase 2a randomized trials.

Cannabis use disorder (CUD) is widespread, and there is no pharmacotherapy to facilitate its treatment. AEF0117, the first of a new pharmacological class, is a signaling-specific inhibitor of the cannabinoid receptor 1 (CB1-SSi). AEF0117 selectively inhibits a subset of intracellular effects resulting from Δ9-tetrahydrocannabinol (THC) binding without modifying behavior per se. In mice and non-human primates, AEF0117 decreased cannabinoid self-administration and THC-related behavioral impairment without producing significant adverse effects. In single-ascending-dose (0.2 mg, 0.6 mg, 2 mg and 6 mg; n = 40) and multiple-ascending-dose (0.6 mg, 2 mg and 6 mg; n = 24) phase 1 trials, healthy volunteers were randomized to ascending-dose cohorts (n = 8 per cohort; 6:2 AEF0117 to placebo randomization). In both studies, AEF0117 was safe and well tolerated (primary outcome measurements). In a double-blind, placebo-controlled, crossover phase 2a trial, volunteers with CUD were randomized to two ascending-dose cohorts (0.06 mg, n = 14; 1 mg, n = 15). AEF0117 significantly reduced cannabis' positive subjective effects (primary outcome measurement, assessed by visual analog scales) by 19% (0.06 mg) and 38% (1 mg) compared to placebo (P < 0.04). AEF0117 (1 mg) also reduced cannabis self-administration (P < 0.05). In volunteers with CUD, AEF0117 was well tolerated and did not precipitate cannabis withdrawal. These data suggest that AEF0117 is a safe and potentially efficacious treatment for CUD.ClinicalTrials.gov identifiers: NCT03325595 , NCT03443895 and NCT03717272 .

Cannabinoid receptor 2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway.

Cannabinoid receptor 2 (CB2) is a GPCR highly expressed on the surface of cells of the immune system, supporting its role in immunomodulation. This study has investigated the trafficking properties of this receptor when stably expressed by HEK-293 cells. As previously reported, cell surface CB2 rapidly internalized upon exposure to agonist. Direct evidence of CB2 recycling was observed upon competitive removal of the stimulating agonist by inverse agonist. CB2 also underwent slow constitutive internalization when agonist was absent and was up-regulated in the presence of inverse agonist. Co-expression of CB2 and dominant negative Rab5 resulted in a significantly reduced capacity for receptors to internalize with no effect on recycling of the internalized receptors. Conversely, co-expression with dominant negative Rab11 did not alter the ability of CB2 to internalize but did impair their ability to return to the cell surface. Co-expression of wild-type, dominant negative or constitutively active Rab4 with CB2 did not alter basal surface expression, extent of internalization, or extent of recycling. These results suggest that Rab5 is involved in CB2 endocytosis and that internalized receptors are recycled via a Rab11 associated pathway rather than the rapid Rab4 associated pathway. This report provides the first comprehensive description of CB2 internalization and recycling to date.

Covalent cannabinoid receptor ligands - structural insight and selectivity challenges.

X-ray crystallography and cryogenic electronic microscopy have provided significant advancement in the knowledge of GPCR structure and have allowed the rational design of GPCR ligands. The class A GPCRs cannabinoid receptor type 1 and type 2 are implicated in many pathophysiological processes and thus rational design of drug and tool compounds is of great interest. Recent structural insight into cannabinoid receptors has already led to a greater understanding of ligand binding sites and receptor residues that likely contribute to ligand selectivity. Herein, classes of heterocyclic covalent cannabinoid receptor ligands are reviewed in light of the recent advances in structural knowledge of cannabinoid receptors, with particular discussion regarding covalent ligand selectivity and rationale design.