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

Background And Purpose: Activation of CB 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.

GPCR kinase subtype requirements for arrestin-2 and -3 translocation to the cannabinoid CB 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.

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

Positive allosteric modulators (PAMs) of the cannabinoid CB1 receptor (CB) offer potential therapeutic advantages in the treatment of neuropathic pain and addiction by avoiding the adverse effects associated with orthosteric CB activation. Here, molecular modeling and mutagenesis were used to identify residues central to PAM activity at CB. 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.

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

The cannabinoid CB receptor (CB) is a G protein-coupled receptor (GPCR) with widespread expression in the central nervous system. This canonically G⍺-coupled receptor mediates the effects of Δ-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.

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

Background And Purpose: Orthosteric agonism of the CB receptor normally associates with Gi signalling resulting in a net inhibition of cAMP production. Empirical evidence shows CB causes a net cAMP stimulation through Gs coupling under two conditions: co-stimulation with the D receptor and high-level CB expression. Two hypotheses have been proposed to account for these paradoxical effects, (1) Gi is consumed by coupling to D or extra CB and excess CB binds to Gs and (2), the formation of dimers CB -CB or CB -D switches Gi/Gs preference.

Evaluating signaling bias for synthetic cannabinoid receptor agonists at the cannabinoid CB 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 CB receptor (CB ). Hence, the current study aimed to characterize the molecular pharmacology of a structurally diverse panel of SCRAs at CB , including 4-cyano MPP-BUT7AICA, 4F-MDMB-BUTINACA, AMB-FUBINACA, JWH-018, MDMB-4en-PINACA, and XLR-11.

Signaling-specific inhibition of the CB 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 (CB-SSi). AEF0117 selectively inhibits a subset of intracellular effects resulting from Δ-tetrahydrocannabinol (THC) binding without modifying behavior per se.

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

Background And Purpose: The cannabinoid (CB ) 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 CB receptor.

RAMP and MRAP accessory proteins have selective effects on expression and signalling of the CB, CB, GPR18 and GPR55 cannabinoid receptors.

Background And Purpose: Receptor activity-modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB and CB, and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies.

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.

Characterisation of AMB-FUBINACA metabolism and CB-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.

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

Orthosteric activation of CB is known to cause a plethora of adverse side effects . 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 activity of the positive allosteric modulator ZCZ011, explicitly considering its effects on receptor regulation.

Cannabinoid 1 (CB ) 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 CB 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 CB receptor C-terminus for interaction with different arrestin subtypes was examined.

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 CB .

Development of 3-(4-Chlorophenyl)-1-(phenethyl)urea Analogues as Allosteric Modulators of the Cannabinoid Type-1 Receptor: RTICBM-189 is Brain Penetrant and Attenuates Reinstatement of Cocaine-Seeking Behavior.

We have shown that CB receptor negative allosteric modulators (NAMs) attenuated the reinstatement of cocaine-seeking behaviors in rats. In an effort to further define the structure-activity relationships and assess the druglike properties of the 3-(4-chlorophenyl)-1-(phenethyl)urea-based CB NAMs that we recently reported, we introduced substituents of different electronic properties and sizes to the phenethyl group and evaluated their potency in CB calcium mobilization, cAMP, and GTPγS assays. We found that 3-position substitutions such as Cl, F, and Me afforded enhanced CB potency, whereas 4-position analogues were generally less potent.

Delineating the interactions between the cannabinoid CB receptor and its regulatory effectors; β-arrestins and GPCR kinases.

Background And Purpose: The cannabinoid CB receptor (CB ) is a promising therapeutic target for modulating inflammation. However, little is known surrounding the mechanisms underpinning CB desensitisation and regulation, particularly the role of GPCR kinases (GRKs). Here, we evaluated the role of six GRK isoforms in β-arrestin recruitment to CB .

Exploring group size for statistical analysis of real-time signalling experiments.

Background And Purpose: Classical pharmacological bioassays generally use observed effects from a concentration series, at a single equilibrium time point to construct a concentration-effect curve, representing one experiment. However, if the full kinetic profile of the effect data for each concentration was evaluated simultaneously, then the analysis would be more powerful. In this work, we explore if more precise parameters can be achieved by using the full kinetic method.

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 (CB) for therapeutic benefits. Examination of the two widely studied prototypic CB 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 CB 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 CB receptor.

Pharmacological selection of cannabinoid receptor effectors: Signalling, allosteric modulation and bias.

The type-1 cannabinoid receptor (CB) is a promising drug target for a wide range of diseases. However, many existing and novel candidate ligands for CB have shown only limited therapeutic potential. Indeed, no ligands are currently approved for the clinic except formulations of the phytocannabinoids Δ-THC and CBD and a small number of analogues.

Biased agonism at the cannabinoid receptors - Evidence from synthetic cannabinoid receptor agonists.

The type 1 and type 2 cannabinoid receptors are G protein-coupled receptors implicated in a variety of physiological processes and diseases. Synthetic cannabinoid receptor agonists (SCRAs) were originally developed to explore the therapeutic benefits of cannabinoid receptor activation, although more recently, these compounds have been diverted to the recreational drug market and are increasingly associated with incidences of toxicity. A prominent concept in contemporary pharmacology is functional selectivity or biased agonism, which describes the ability of ligands to elicit differential activation of signalling pathways through stabilisation of distinct receptor conformations.

A novel allosteric modulator of the cannabinoid CB receptor ameliorates hyperdopaminergia endophenotypes in rodent models.

The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling.

Evaluation of the profiles of CB cannabinoid receptor signalling bias using joint kinetic modelling.

Background And Purpose: Biased agonism describes the ability of ligands to differentially regulate multiple signalling pathways when coupled to a single receptor. Signalling is affected by rapid agonist-induced receptor internalisation. Hence, the conventional use of equilibrium models may not be optimal, because (i) receptor numbers vary with time and, in addition, (ii) some pathways may show non-monotonic profiles over time.

Terpenoids From Cannabis Do Not Mediate an Entourage Effect by Acting at Cannabinoid Receptors.

The entourage effect was a proposed explanation for biological observations that endocannabinoid ligand activities can be modified by other lipids released from cells at the same time. An increasing volume of anecdotal reports and interest in the plant have provoked research into the activity of minor chemical constituents of the plant-including volatile terpenoids such as myrcene, α- and β- pinene, β-caryophyllene, and limonene. However, to date, no clear interaction has been identified.

In vitro and in vivo pharmacological evaluation of the synthetic cannabinoid receptor agonist EG-018.

Synthetic cannabinoid receptor agonists (SCRAs) possess high abuse liability and complex toxicological profiles, making them serious threats to public health. EG-018 is a SCRA that has been detected in both illicit products and human samples, but it has received little attention to date. The current studies investigated EG-018 at human CB and CB receptors expressed in HEK293 cells in [H]CP55,940 competition binding, [S]GTPγS binding and forskolin-stimulated cAMP production.