Characterization of Novel and Known Activators of Cannabinoid Receptor Subtype 2 Reveals Mixed Pharmacology That Differentiates Mycophenolate Mofetil and GW-842,166X from MDA7

Abstract

CB₁ and CB₂ cannabinoid receptors are members of the GPCR superfamily that modulate the effects of endocannabinoids. CB₁ is the most abundant CB receptor in the central nervous system, while CB₂ is present both peripherally and in the brain. CB₂ plays a role in inflammation, as well as neurodegenerative and psychiatric disorders. To identify new ligands for CB₂, we screened a library of FDA-approved drugs for activity at the receptor using a thallium flux assay, resulting in the discovery of the immunosuppressant mycophenolate mofetil as a potent, selective activator of CB₂. Further characterization of the compound confirmed agonist activity in a variety of complementary assays, including PI hydrolysis, cAMP inhibition, and β-arrestin recruitment. Radioligand binding assays established a non-competitive interaction with the site occupied by [³H]CP55,940. CB₂ agonists GW-842,166X and MDA7 were also profiled, revealing that GW-842,166X exhibits a similar activity profile to mycophenolate mofetil, whereas MDA7 presents a distinct profile. These differences provide insight into the complex CB₂ pharmacology impacting preclinical and clinical studies, and ultimately, new treatment strategies for brain disorders.

Keywords: GW-842,166X; MDA7; allosteric modulator; brain disorders; cannabinoid receptor; mycophenolate mofetil.

Figure 1

Sample traces from screen of FDA collection exemplify a PAM hit. Test compounds or vehicle were added at t = 1 s, and the fluorescence ratio was monitored. 2-AG (EC₂₀ or EC_Max) was added at t = 141 s. Fluorescence readings for each time point in a well were divided by the fluorescence reading at the initial time point to account for differences in cell number, non-uniform illumination, and dye-loading. The slope value for each kinetic trace was calculated for the time window of 145–155 s. The average slope was calculated for wells containing vehicle and this value was subtracted from all wells. Vehicle-subtracted slopes were normalized to the relevant maximal agonist signal for each assay. Traces for a sub-maximal agonist response (EC₂₀, green) are compared to a maximal response (EC_Max, black) and PAM hit (red).

Figure 2

Mycophenolate mofetil is potent and selective for rCB₂, while mycophenolic acid is inactive at CB receptors and parental HEK/GIRK cells. Concentration–response curves in thallium flux assays for (A) mycophenolate mofetil and (B) mycophenolic acid in the presence of a sub-maximal (EC₂₀) concentration of agonist (2-AG or acetylcholine). Results are shown for rCB₂ (white), HEK/GIRK (no CB receptor, red), and rCB₁ (blue) cells. Data represent the mean ± SEM of at least three independent experiments run in duplicate or triplicate, except for mycophenolic acid at HEK/GIRK cells, which was run in two independent experiments. Data are plotted as a percentage of maximal 2-AG response for rCB₂ and rCB₁, or acetylcholine response for HEK/GIRK cells.

Figure 3

Mycophenolate mofetil is active at CB receptors in the absence of 2-AG. Concentration–response curves in thallium flux assays for mycophenolate mofetil in the absence of agonist. Results are shown for rCB₂ (red) and rCB₁ (black) cells. Data represent the mean ± SEM of at least three independent experiments run in duplicate or triplicate. Data are plotted as a percentage of maximal 2-AG response.

Figure 4

Increasing concentrations of AM630 shift the CRC of mycophenolate mofetil in cells expressing rCB₂. Concentration–response curves (CRCs) in thallium flux assays for mycophenolate mofetil in the presence of AM630. Data represent the mean ± SEM of at least three independent experiments run in duplicate or triplicate. Data are plotted as a percentage of maximal 2-AG response.

Figure 5

Mycophenolate mofetil exhibits similar activity in presence and absence of 2-AG in PI hydrolysis. Concentration–response curves in PI hydrolysis assays for mycophenolate mofetil in the presence (white) and absence (red) of a sub-maximal (EC₂₀) concentration of agonist in cells expressing rCB₂. Data represent the mean ± SEM of three independent experiments run in duplicate or triplicate. Data are plotted as IP1 concentration in nM.

Figure 6

The CB₂ agonists GW-842,166X and MDA7 exhibit distinctions in pharmacological activity in rCB₂ thallium flux assays. Concentration–response curves in thallium flux assays are shown for GW-842,166X (A) and MDA7 (B). Results are presented for rCB₂ in the presence (white) or absence (red) of a sub-maximal (EC₂₀) concentration of agonist. Results are presented for rCB₁ in the presence (blue) or absence (black) of a sub-maximal (EC₂₀) concentration of agonist. Data represent the mean ± SEM of three independent experiments run in duplicate or triplicate. Data are plotted as a percentage of the maximal 2-AG response.

Figure 7

CB receptor ligands exhibit varying effects on [³H]CP55,940 binding under equilibrium conditions. Competition binding concentration–response curves were obtained in the presence of 0.5 nM [³H]CP55,940 using membranes harvested from HEK/GIRK cells expressing rCB₂. Data represent the mean ± SEM of three independent experiments run in triplicate. Data are plotted as a percentage of specific [³H]CP55,940 binding. Non-specific binding was determined in the presence of 10 µM CP55,940.

Figure 8

Differential activity of CB₂ agonists in cAMP and β-arrestin assays. Concentration–response curves in (A) cAMP assays and (B) β-arrestin recruitment assays, in the presence or absence of an EC₂₀ concentration of agonist CP55,940, are shown for mycophenolate mofetil (red), GW-842,166X (blue), and MDA7 (black). Control concentration–response curves for CP55,940 are shown in white. Data represent at least one independent experiment run in duplicate. Data are plotted as a percentage of the maximal CP55,940 response.