Gemma L. Baillie

Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro

A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB1 and CB2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB1 receptors expressed in mouse brain and to human CB2 receptors that have been transfected into CHO cells.

Pregnenolone Can Protect the Brain from Cannabis Intoxication

Counteracting Cannabis What is the role of steroid hormones in vulnerability to addiction? Working with rodents, Vallée et al. (p. 94) found that all major drugs of abuse (morphine, cocaine, alcohol, nicotine) increase neurosteroid levels, with the active ingredient in cannabis (THC) inducing a particularly large increase. THC and other drugs increased levels of pregnenolone, long thought to be an inactive precursor of downstream active steroids. Pregnenolone antagonized most of the known behavioral and somatic effects of THC. The universal precursor of steroid hormones acts as a negative allosteric modulator of cannabinoid receptors. Pregnenolone is considered the inactive precursor of all steroid hormones, and its potential functional effects have been largely uninvestigated. The administration of the main active principle of Cannabis sativa (marijuana), ∆9-tetrahydrocannabinol (THC), substantially increases the synthesis of pregnenolone in the brain via activation of the type-1 cannabinoid (CB1) receptor. Pregnenolone then, acting as a signaling-specific inhibitor of the CB1 receptor, reduces several effects of THC. This negative feedback mediated by pregnenolone reveals a previously unknown paracrine/autocrine loop protecting the brain from CB1 receptor overactivation that could open an unforeseen approach for the treatment of cannabis intoxication and addiction.

Evidence that the plant cannabinoid Δ9-tetrahydrocannabivarin is a cannabinoid CB1 and CB2 receptor antagonist

Δ9‐tetrahydrocannabivarin (THCV) displaced [3H]CP55940 from specific binding sites on mouse brain and CHO‐hCB2 cell membranes (Ki=75.4 and 62.8 nM, respectively). THCV (1 μM) also antagonized CP55940‐induced stimulation of [35S]GTPγS binding to these membranes (apparent KB=93.1 and 10.1 nM, respectively). In the mouse vas deferens, the ability of Δ9‐tetrahydrocannabinol (THC) to inhibit electrically evoked contractions was antagonized by THCV, its apparent KB‐value (96.7 nM) approximating the apparent KB‐values for its antagonism of CP55940‐ and R‐(+)‐WIN55212‐induced stimulation of [35S]GTPγS binding to mouse brain membranes. THCV also antagonized R‐(+)‐WIN55212, anandamide, methanandamide and CP55940 in the vas deferens, but with lower apparent KB‐values (1.5, 1.2, 4.6 and 10.3 nM, respectively). THCV (100 nM) did not oppose clonidine, capsaicin or (−)‐7‐hydroxy‐cannabidiol‐dimethylheptyl‐induced inhibition of electrically evoked contractions of the vas deferens. Contractile responses of the vas deferens to phenylephrine hydrochloride or β,γ‐methylene‐ATP were not reduced by 1 μM THCV or R‐(+)‐WIN55212, suggesting that THCV interacts with R‐(+)‐WIN55212 at prejunctional sites. At 32 μM, THCV did reduce contractile responses to phenylephrine hydrochloride and β,γ‐methylene‐ATP, and above 3 μM it inhibited electrically evoked contractions of the vas deferens in an SR141716A‐independent manner. In conclusion, THCV behaves as a competitive CB1 and CB2 receptor antagonist. In the vas deferens, it antagonized several cannabinoids more potently than THC and was also more potent against CP55940 and R‐(+)‐WIN55212 in this tissue than in brain membranes. The bases of these agonist‐ and tissue‐dependent effects remain to be established.

CB1 Receptor Allosteric Modulators Display Both Agonist and Signaling Pathway Specificity

We have previously identified allosteric modulators of the cannabinoid CB1 receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB1 receptor agonist [3H]CP55940 but producing a decrease in CB1 receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB1 receptor activation. We assessed the effect of these compounds on CB1 receptor agonist–induced [35S]GTPγS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and β-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB1 agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signaling as compared with WIN55212 and having little effect on [3H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding, simulation (Gαs-mediated), and inhibition (Gαi-mediated) of cAMP production and β-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic-binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB1 agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.

Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids

Background: The endogenous l-α-lysophosphatidylinositol activates GPR55. Results: Structural analogues of SR141716A act both as agonists alone and as inhibitors of l-α-lysophosphatidylinositol. Certain CB2 receptor agonists also modulate GPR55 activity. Conclusion: Certain cannabinoids can both activate GPR55 and attenuate l-α-lysophosphatidylinositol-mediated phosphorylated ERK1/2 activation. This has mechanistic implications for the antinociceptive effects of certain CB2 agonists. Significance: Cannabinoid ligands have complex interactions with the l-α-lysophosphatidylinositol/GPR55 signaling system. GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

MODULATION OF L-α-LYSOPHOSPHATIDYLINOSITOL /GPR55 MAP KINASE SIGNALLING BY CANNABINOIDS

Background: The endogenous l-α-lysophosphatidylinositol activates GPR55. Results: Structural analogues of SR141716A act both as agonists alone and as inhibitors of l-α-lysophosphatidylinositol. Certain CB2 receptor agonists also modulate GPR55 activity. Conclusion: Certain cannabinoids can both activate GPR55 and attenuate l-α-lysophosphatidylinositol-mediated phosphorylated ERK1/2 activation. This has mechanistic implications for the antinociceptive effects of certain CB2 agonists. Significance: Cannabinoid ligands have complex interactions with the l-α-lysophosphatidylinositol/GPR55 signaling system. GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

Allosteric Modulation of a Cannabinoid G Protein-coupled Receptor BINDING SITE ELUCIDATION AND RELATIONSHIP TO G PROTEIN SIGNALING

Background: Cannabinoid-1 (CB1) receptor allosteric modulator ORG27569 increases CP55,940 binding, yet antagonizes G protein signaling. Results: ORG27569 binding sterically blocks movement in CB1 extracellular loops and transmembrane helix 6 (TMH6). Conclusion: ORG27569 increases CP55,940 binding by promoting an intermediate receptor conformation where changes important for signaling are blocked. Significance: This information may lead to the rational design of new allosteric modulators. The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [3H](−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of ORG27569 at CB1. To this end, we used computation, synthesis, mutation, and functional studies to identify the ORG27569-binding site in the CB1 TMH3-6-7 region. This site is consistent with the results of K3.28192A, F3.36200A, W5.43279A, W6.48356A, and F3.25189A mutation studies, which revealed the ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28192 that is important for ORG27569 to act as an inverse agonist. At this allosteric site, ORG27569 promotes an intermediate conformation of the CB1 receptor, explaining ORG27569s ability to increase equilibrium binding of CP55,940. This site also explains ORG27569s ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63176. 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.

The pentafluorosulfanyl group in cannabinoid receptor ligands: synthesis and comparison with trifluoromethyl and tert-butyl analogues

An array of cannabinoid ligands, bearing meta- and para-substituted pentafluorosulfanyl (SF5) aniline groups in position 3 of the pyrazole ring, was efficiently synthesised and compared with the exact trifluoromethyl and tert-butyl analogues. In general, the SF5 substituted ligands showed higher lipophilicity (i.e. log P values) than the CF3 counterparts and lower lipophilicity than the tert-butyl ones. In terms of pharmacological activity, SF5 pyrazoles generally showed slightly higher or equivalent CB1 receptor affinity (Ki), always in the nanomolar range, and selectivity towards the CB2 relative to both CF3 and tert-butyl analogues. Functional β-arrestin recruitment assays were used to determine equilibrium dissociation constants (Kb) and showed that all of the tested SF5 and CF3 compounds are CB1 neutral antagonists. These results confirm the possibility of successfully using an aromatic SF5 group as a stable, synthetically accessible and effective bioisosteric analogue of the electron-withdrawing CF3 group, and possibly also of bulky aliphatic groups, for drug discovery and development applications.

Development of indole sulfonamides as cannabinoid receptor negative allosteric modulators

Existing CB1 negative allosteric modulators (NAMs) fall into a limited range of structural classes. In spite of the theoretical potential of CB1 NAMs, published in vivo studies have generally not been able to demonstrate the expected therapeutically-relevant CB1-mediated effects. Thus, a greater range of molecular tools are required to allow definitive elucidation of the effects of CB1 allosteric modulation. In this study, we show a novel series of indole sulfonamides. Compounds 5e and 6c (ABD1075) had potencies of 4 and 3nM respectively, and showed good oral exposure and CNS penetration, making them highly versatile tools for investigating the therapeutic potential of allosteric modulation of the cannabinoid system.

4-Cyano-5-(2-thiophenyl)-pyrazoles are high affinity CB1 receptor ligands

Pyrazoles bearing a 5-thiophenyl and a 4-cyano group were synthesised and tested for their affinity to the cannabinoid CB1 receptor showing in many cases single digit nanomolar Ki values and moderate to good selectivity towards the CB2 receptor. Some of these pyrazole ligands, such as 8g, displayed relatively low lipophilicity (experimental logP 90) suggesting that these compounds may behave as peripherally restricted CB1 ligands. Furthermore, 2-fluoroethyl carboxamides 8d, 8h and 8l are interesting candidates for further development into PET tracers.