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.

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.

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.

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.

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.

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.

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.

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.

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 .

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.

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.

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.

In Vitro Characterization of 6-Methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011) at the Type 1 Cannabinoid Receptor: Allosteric Agonist or Allosteric Modulator?

Orthosteric activation of CB1 is known to cause a plethora of adverse side effects in vivo. Allosteric modulation is an exciting therapeutic approach and is hoped to offer improved therapeutic potential and a reduced on-target side effect profile compared to orthosteric agonists. This study aimed to systematically characterize the in vitro activity of the positive allosteric modulator ZCZ011, explicitly considering its effects on receptor regulation. HEK293 cells expressing hCB1 receptors were used to characterize ZCZ011 alone and in combination with orthosteric agonists. Real-time BRET approaches were employed for G protein dissociation, cAMP signaling, and ß-arrestin translocation. Characterization also included ERK1/2 phosphorylation (PerkinElmer AlphaLISA) and receptor internalization. ZCZ011 is an allosteric agonist of CB1 in all pathways tested, with a similar signaling profile to that of the partial orthosteric agonist Δ9-tetrahydrocannabinol. ZCZ011 also showed limited positive allosteric modulation in increasing the potency and efficacy of THC-induced ERK1/2 phosphorylation, ß-arrestin translocation, and receptor internalization. However, no positive allosteric modulation was observed for ZCZ011 in combination with either CP55940 or AMB-FUBINACA, in G protein dissociation, nor cAMP inhibition. Our study suggests that ZCZ011 is an allosteric agonist, with effects that are often difficult to differentiate from those of orthosteric agonists. Together with its pronounced agonist activity, the limited extent of ZCZ011 positive allosteric modulation suggests that further investigation into the differences between allosteric and orthosteric agonism is required, especially in receptor regulation end points.

Synthesis and pharmacological evaluation of newly detected synthetic cannabinoid receptor agonists AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA, MDMB-4F-BUTINACA and their analogs.

Synthetic cannabinoid receptor agonists (SCRAs) continue to make up a significant portion new psychoactive substances (NPS) detected and seized worldwide. Due to their often potent activation of central cannabinoid receptors in vivo, use of SCRAs can result in severe intoxication, in addition to other adverse health effects. Recent detections of AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA and MDMB-4F-BUTINACA mark a continuation in the appearance of SCRAs bearing novel tail substituents. The proactive characterization campaign described here has facilitated the detection of several new SCRAs in toxicological case work. Here we detail the synthesis, characterization, and pharmacological evaluation of recently detected SCRAs, as well as a systematic library of 32 compounds bearing head, tail, and core group combinations likely to appear in future. In vitro radioligand binding assays revealed most compounds showed moderate to high affinity at both CB1 (pK i = < 5 to 8.89 ± 0.09 M) and CB2 (pK i = 5.49 ± 0.03 to 9.92 ± 0.09 M) receptors. In vitro functional evaluation using a fluorescence-based membrane potential assay showed that most compounds were sub-micromolar to sub-nanomolar agonists at CB1 (pEC50 = < 5 to 9.48 ± 0.14 M) and CB2 (pEC50 = 5.92 ± 0.16 to 8.64 ± 0.15 M) receptors. An in silico receptor-ligand docking approach was utilized to rationalize binding trends for CB2 with respect to the tail substituent, and indicated that rigidity in this region (i.e., 4-cyanobutyl) was detrimental to affinity.

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.

Putative Synthetic Cannabinoids MEPIRAPIM, 5F-BEPIRAPIM (NNL-2), and Their Analogues Are T-Type Calcium Channel (CaV3) Inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are a large and growing class of new psychoactive substances (NPSs). Two recently identified compounds, MEPIRAPIM and 5F-BEPIRAPIM (NNL-2), have not been confirmed as agonists of either cannabinoid receptor subtype but share structural similarities with both SCRAs and a class of T-type calcium channel (CaV3) inhibitors under development as new treatments for epilepsy and pain. In this study, MEPIRAPIM and 5F-BEPIRAPIM and 10 systematic analogues were synthesized, analytically characterized, and pharmacologically evaluated using in vitro cannabinoid receptor and CaV3 assays. Several compounds showed micromolar affinities for CB1 and/or CB2, with several functioning as low potency agonists of CB1 and CB2 in a membrane potential assay. 5F-BEPIRAPIM and four other derivatives were identified as potential CaV3 inhibitors through a functional calcium flux assay (>70% inhibition), which was further confirmed using whole-cell patch-clamp electrophysiology. Additionally, MEPIRAPIM and 5F-BEPIRAPIM were evaluated in vivo using a cannabimimetic mouse model. Despite detections of MEPIRAPIM and 5F-BEPIRAPIM in the NPS market, only the highest MEPIRAPIM dose (30 mg/kg) elicited a mild hypothermic response in mice, with no hypothermia observed for 5F-BEPIRAPIM, suggesting minimal central CB1 receptor activity.

Defining Steric Requirements at CB1 and CB2 Cannabinoid Receptors Using Synthetic Cannabinoid Receptor Agonists 5F-AB-PINACA, 5F-ADB-PINACA, PX-1, PX-2, NNL-1, and Their Analogues.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS). They commonly comprise N-alkylated indole, indazole, or 7-azaindole scaffolds with amide-linked pendant amino acid groups. To explore the contribution of the amino acid side chain to the cannabinoid pharmacology of SCRA NPS, a systematic library of side chain-modified SCRAs was prepared based on the recent detections of amino acid derivatives 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), and 20 (NNL-1). In vitro binding affinities and functional activities at cannabinoid type 1 and 2 receptors (CB1 and CB2, respectively) were determined for all the library members using radioligand competition experiments and a fluorescence-based membrane potential assay. Binding affinities and functional activities varied widely across compounds (Ki = 0.32 to >10 000 nM, EC50 = 0.24-1259 nM), with several clear structure-activity relationships (SARs) emerging. Affinity and potency at CB1 changed as a function of the heterocyclic core (indazole > indole > 7-azaindole) and the pendant amino acid side chain (tert-butyl > iso-propyl > iso-butyl > benzyl > ethyl > methyl > hydrogen). Ensemble docking at CB1 revealed a clear steric basis for observed SAR trends. Interestingly, although 15 (PX-1) and 19 (PX-2) have been detected in recreational drug markets, they failed to induce centrally CB1-mediated effects (e.g., hypothermia) in mice using radiobiotelemetry. Together, these data provide insights regarding structural contributions to the cannabimimetic profiles of 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), 20 (NNL-1), and other SCRA NPS.

Structure-activity relationships of valine, tert-leucine, and phenylalanine amino acid-derived synthetic cannabinoid receptor agonists related to ADB-BUTINACA, APP-BUTINACA, and ADB-P7AICA.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the most prevalent classes of new psychoactive substances (NPS) worldwide, and examples are generally poorly characterised at the time of first detection. We have synthesised a systematic library of amino acid-derived indole-, indazole-, and 7-azaindole-3-carboxamides related to recently detected drugs ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA, and characterised these ligands for in vitro binding and agonist activity at cannabinoid receptor subtypes 1 and 2 (CB1 and CB2), and in vivo cannabimimetic activity. All compounds showed high affinity for CB1 (K i 0.299-538 nM) and most at CB2 (K i = 0.912-2190 nM), and most functioned as high efficacy agonists of CB1 and CB2 in a fluorescence-based membrane potential assay and a ßarr2 recruitment assay (NanoBiT®), with some compounds being partial agonists in the NanoBiT® assay. Key structure-activity relationships (SARs) were identified for CB1/CB2 binding and CB1/CB2 functional activities; (1) for a given core, affinities and potencies for tert-leucinamides (ADB-) > valinamides (AB-) ≫ phenylalaninamides (APP-); (2) for a given amino acid side-chain, affinities and potencies for indazoles > indoles ≫ 7-azaindoles. Radiobiotelemetric evaluation of ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA in mice demonstrated that ADB-BUTINACA and ADB-P7AICA were cannabimimetic at 0.1 mg kg-1 and 10 mg kg-1 doses, respectively, as measured by pronounced decreases in core body temperature. APP-BUTINACA failed to elicit any hypothermic response up to the maximally tested 10 mg kg-1 dose, yielding an in vivo potency ranking of ADB-BUTINACA > ADB-P7AICA > APP-BUTINACA.

Exploring determinants of agonist efficacy at the CB1 cannabinoid receptor: Analogues of the synthetic cannabinoid receptor agonist EG-018.

Neutral antagonists of GPCRs remain relatively rare-indeed, a large majority of GPCR antagonists are actually inverse agonists. The synthetic cannabinoid receptor agonist (SCRA) EG-018 was recently reported as a low efficacy cannabinoid receptor agonist. Here we report a comparative characterization of EG-018 and 13 analogues along with extant putative neutral antagonists of CB1 . In HEK cells stably expressing human CB1 , assays for inhibition of cAMP were performed by real-time BRET biosensor (CAMYEL), G protein cycling was quantified by [35 S]GTPγS binding, and stimulation of pERK was characterized by AlphaLISA (PerkinElmer). Signaling outcomes for the EG-018 analogues were highly variable, ranging from moderate efficacy agonism with high potency, to marginal agonism at lower potency. As predicted by differing pathway sensitivities to differences in ligand efficacy, most EG-018-based compounds were completely inactive in pERK alone. The lowest efficacy analogue in cAMP assays, 157, had utility in antagonism assay paradigms. Developing neutral antagonists of the CB1 receptor has been a long-standing research goal, and such compounds would have utility both as research tools and in therapeutics. Although these results emphasize again the importance of system factors in determining signaling outcomes, some compounds characterized in this study appear among the lowest efficacy agonists described to date and therefore suggest that development of neutral antagonists is an achievable goal for CB1 .

Neuroprotective activity of new Δ3-N-acylethanolamines in a focal ischemia stroke model.

N-acylethanolamines (NAE, also called ethanolamides) are significant lipid signaling molecules with anti-inflammatory, pain-relieving, cell-protective, and anticancer properties. Here, we present the use of a hitherto unreported group of Δ3-NAE and also some Δ4- and Δ5-NAE, in in vitro and in vivo assays to gain a better understanding of their structure-bioactivity relationships. We have developed an efficient synthetic method to rapidly produce novel unlabeled and 13 C-labeled Δ3-NAE (NAE-18:5n-3, NAE-18:4n-6) and Δ4-NAE (NAE-22:5n-6). The new NAE with shorter carbon backbone structures confers greater neuroprotection than their longer carbon backbone counterparts, including anandamide (Δ5-NAE-20:4n-6) in a focal ischemia mouse model of stroke. This study highlights structure-dependent protective effects of new NAE following focal ischemia, in which some of the new NAE, administered intranasally, lead to significantly reduced infarct volume and improved recovery of limb use. The relative affinity of the new NAE toward cannabinoid receptors was assessed against anandamide, NAE-22:6n-3 and NAE-20:5n-3, which are known cannabinoid receptor ligands with high-binding constants. Among the newly synthesized NAE, Δ4-NAE-22:5n-6 shows the greatest relative affinity to cannabinoid receptors hCB1 and hCB2 , and inhibition of cyclic adenosine monophosphate activity through hCB2 compared to anandamide.