Cannabidiol is a negative allosteric modulator of the cannabinoid CB1 receptor

Abstract

Background and purpose: Cannabidiol has been reported to act as an antagonist at cannabinoid CB1 receptors. We hypothesized that cannabidiol would inhibit cannabinoid agonist activity through negative allosteric modulation of CB1 receptors.

Experimental approach: Internalization of CB1 receptors, arrestin2 recruitment, and PLCβ3 and ERK1/2 phosphorylation, were quantified in HEK 293A cells heterologously expressing CB1 receptors and in the STHdh(Q7/Q7) cell model of striatal neurons endogenously expressing CB1 receptors. Cells were treated with 2-arachidonylglycerol or Δ(9)-tetrahydrocannabinol alone and in combination with different concentrations of cannabidiol.

Key results: Cannabidiol reduced the efficacy and potency of 2-arachidonylglycerol and Δ(9)-tetrahydrocannabinol on PLCβ3- and ERK1/2-dependent signalling in cells heterologously (HEK 293A) or endogenously (STHdh(Q7/Q7)) expressing CB1 receptors. By reducing arrestin2 recruitment to CB1 receptors, cannabidiol treatment prevented internalization of these receptors. The allosteric activity of cannabidiol depended upon polar residues being present at positions 98 and 107 in the extracellular amino terminus of the CB1 receptor.

Conclusions and implications: Cannabidiol behaved as a non-competitive negative allosteric modulator of CB1 receptors. Allosteric modulation, in conjunction with effects not mediated by CB1 receptors, may explain the in vivo effects of cannabidiol. Allosteric modulators of CB1 receptors have the potential to treat CNS and peripheral disorders while avoiding the adverse effects associated with orthosteric agonism or antagonism of these receptors.

Figure 1

CBD reduced the rate and maximal BRET_Eff between CB₁ receptors and arrestin2 and also the internalization of these receptors in THC‐treated and 2‐AG‐treated STHdh ^Q7/Q7 cells. (A and B) STHdh ^Q7/Q7 cells were treated with THC (A) or 2‐AG (B) ± CBD for 10 min, and the fraction of CB₁ receptors at the plasma membrane was quantified using On‐cell and In‐cell Western analyses. Data were fit to a nonlinear regression model with variable slope. (C–E) STHdh ^Q7/Q7 cells were transfected with arrestin2‐Rluc‐containing and CB₁‐GFP²‐containing plasmids, and BRET² was measured every 10 s for 4 min (240 s) and again at 10 min (600 s) after treatment with THC (C) or 2‐AG (D) ± O‐2050 or CBD. Data were fit to a nonlinear regression model with variable slope. (E) The rate of arrestin2 recruitment to CB₁ receptors was measured as the change in BRET_Eff s⁻¹ during the first 4 min. (F–H) STHdh ^Q7/Q7 cells were treated with THC (F) or 2‐AG (G) ± CBD for 60 min, and the fraction of CB₁ receptors at the plasma membrane was quantified using On‐cell and In‐cell Western analyses. Data were fit to a nonlinear regression model with variable slope. (H) The rate of CB₁ receptor internalization was measured as the change in the fraction On‐cell CB₁/total CB₁ min⁻¹ prior to plateau. †P < 0.01 compared with 2‐AG or THC alone, *P < 0.01 compared with 0 CBD within orthosteric ligand treatment, ^P < 0.01 compared with 0.01 μM CBD (log[CBD] M = −8) within orthosteric ligand treatment; two‐way ANOVA with Bonferroni's post hoc test. N = 6.

Figure 2

CBD was a NAM of arrestin2 recruitment to CB₁ receptors following THC and 2‐AG treatment. HEK 293A (A–E) and STHdh ^Q7/Q7 (F–J) cells were transfected with arrestin2‐Rluc‐containing and CB₁‐GFP²‐containing plasmids, and BRET² was measured 30 min after treatment with 2‐AG or THC ± O‐2050 or CBD. CRCs were fit using Gaddum/Schild EC₅₀ shift (A, B, F and G) and operational model of allosterism (C, D, H and I) nonlinear regression models. (E and J) Schild regressions were plotted as the logarithm of 2‐AG or THC dose against the logarithm of the dose–response at EC₅₀ – 1. N = 6.

Figure 3

CBD was a NAM of CB₁ receptor‐dependent PLCβ3 phosphorylation following THC and 2‐AG treatment. HEK 293A cells expressing CB₁‐GFP² (A–E) and STHdh ^Q7/Q7 cells (F–J) were treated with 2‐AG or THC ± O‐2050 or CBD, and total and phosphorylated PLCβ3 levels were determined using In‐cell western. CRCs were fit using Gaddum/Schild EC₅₀ shift (A, B, F and G) and operational model of allosterism (C, D, H and I) nonlinear regression models. E and J Schild regressions were plotted as the logarithm of 2‐AG or THC dose against the logarithm of the dose–response at EC₅₀ – 1. N = 6.

Figure 4

CBD was a NAM of CB₁ receptor‐dependent ERK1/2 phosphorylation following 2‐AG treatment. HEK 293A cells expressing CB₁‐GFP² (A–C) and STHdh ^Q7/Q7 cells (D–F) were treated with 2‐AG ± O‐2050 or CBD, and total and phosphorylated ERK1/2 levels were determined using In‐cell western. CRCs were fit using Gaddum/Schild EC₅₀ shift (A and D) and operational model of allosterism (B and E) nonlinear regression models. (C and F) Schild regressions were plotted as the logarithm of 2‐AG or THC dose against the logarithm of the dose–response at EC₅₀ – 1. N = 6.

Figure 5

CBD was a NAM of AM251‐dependent inverse agonism and O‐2050 antagonism. STHdh ^Q7/Q7 cells were treated with AM251 ± CBD (A) or 2‐AG ± O‐2050, CBD or O‐2050 and CBD (B), and total and phosphorylated ERK1/2 levels were determined using In‐cell western. CRCs were fit using the operational model of allosterism (A) or nonlinear regression with variable slope (four parameters) (B) models. N = 6.

Figure 6

Cys⁹⁸ and Cys¹⁰⁷ coordinate the NAM activity of CBD at CB₁ receptors. (A and B) STHdh ^Q7/Q7 cells were transfected with arrestin2‐Rluc‐ and CB₁ ^C98A‐GFP²‐, and CB₁ ^C98S‐GFP²‐, CB₁ ^C107A‐GFP²‐ and CB₁ ^C107S‐GFP²‐containing plasmids, and BRET² was measured 30 min after treatment with THC (A) or 2‐AG (B) ± CBD. CRCs were fit using nonlinear regression with variable slope (four parameters) N = 4. (C) Diagram of the membrane‐proximal region of CB₁ receptors summarizing data presented in this figure (adapted from Fay and Farrens, 2013). Our observations and previous studies suggest that Cys⁹⁸ and Cys¹⁰⁷ contribute to CB₁ receptor allosterism, while the orthosteric site is near the second extracellular loop (orange box). In this diagram, green represents extracellular surface of CB₁ receptors. Black circles represent residues unique to the N‐terminus of CB_1A receptors Grey circles represent residues unique to the N‐terminus of CB_1B receptors. Yellow circles represent Cys. Purple circles represent N‐glycosylated residues. Residues mutated in this study are marked in bold. Non‐bold numbers indicate amino acid number relative to N‐terminus.