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

1. Introduction

The endocannabinoid system is comprised of two primary receptor subtypes, the cannabinoid receptor subtype 1 (CB₁) and subtype 2 (CB₂). While CB₁ is more abundantly expressed in the brain, CB₂ is expressed in the immune system and was initially thought to be a peripheral receptor [1,2]. CB₁ is a potential therapeutic target for indications including nausea [3], psychosis [4], anxiety [5], and pain [6]. Subsequent studies characterized CB₂ expression in the brain [7,8,9,10] and the CB₂ receptor has emerged as a therapeutic target for brain disorders ranging from anxiety [11] and depression [12], to addiction [13] and schizophrenia [14,15].

A number of CB₂ receptor agonists have been developed and tested in clinical trials for a variety of indications including pain and Alzheimer’s disease and several compounds have been shown to be safe for human administration. GW-842,166X is a CB₂ selective agonist developed by GlaxoSmithKline [16,17] and was evaluated in Phase I trials for the indications of pain and inflammation (NCT00511524) and Phase II trials for dental pain (NCT00444769). The trials were either withdrawn or completed with no results posted. MDA7 (NTRX-07) is a CB₂-preferring agonist developed by Naiguib et al. and NeuroTherapia [18,19,20]; it was progressed into Phase I trials for the indications of neuropathic pain (NCT04375436) and Alzheimer’s disease (NCT06194552) and, according to press releases, this compound is progressing into Phase II studies for Alzheimer’s disease.

As an alternative to direct CB₂ activation, the use of allosteric modulators may provide an improved approach by targeting neurotransmitter-dependent activity [21]. The spatial and temporal control made possible with allosteric potentiators, along with improved subtype-selectivity, suggests that efficacy may be improved with fewer adverse side effects [22,23]. The first published small molecule positive allosteric modulator (PAM) of CB₂, EC21a, has been shown to have diverse functional activity [24,25]. While reported initially as a potentiator of CB₂, it has subsequently shown inverse agonist activity in a variety of independent assays; thus, a pure PAM has yet to be identified [26,27]. To discover novel compounds with CB₂ PAM activity, we screened a collection of approximately 1000 FDA-approved drugs and identified mycophenolate mofetil as a putative PAM of the CB₂ receptor. Additional profiling, however, revealed that this compound was actually an agonist of CB₂. Mycophenolate mofetil is a widely used immunosuppressive agent [28,29,30]; this compound demonstrated CB₂ activity in a variety of assays, including G Protein Inwardly Rectifying Potassium (GIRK) channel-mediated thallium flux, phosphoinositide (PI) hydrolysis, cAMP inhibition, and β-arrestin recruitment. It was also selective for CB₂ versus CB₁. In contrast to the parent molecule, the active form of the drug that is responsible for immunosuppression, mycophenolic acid [28,29,30], was inactive at the CB₂ receptor. Radioligand binding studies indicate that mycophenolate mofetil does not interact with the binding site occupied by [³H]CP55,940 in a competitive manner. During the course of our studies, we also profiled the known CB₂ agonists, GW-842,166X and MDA7. In receptor-activated GIRK assays, our studies indicated comparable agonist activity between GW-842,166X and mycophenolate mofetil but MDA7 exhibited inverse agonist activity at CB₂; however, all three compounds demonstrated agonist activity in multiple signaling pathways using cAMP inhibition and β-arrestin assays. With respect to radioligand binding, GW-842,166X exhibited comparable binding activity to mycophenolate mofetil while MDA7 displayed a different binding profile. The FDA-approved immunosuppressant mycophenolate mofetil represents a novel CB₂ agonist with unique properties compared to known CB₂ agonists. While subtle, these differences may provide insight into the mechanism of action of CB₂ modulators and prove to be significant when evaluating novel compounds as possible therapeutics for brain disorders.

2. Results

2.1. Identification of Novel Modulators of the CB₂ Receptor

To identify new compounds capable of potentiating CB₂ responses to the endogenous agonist 2-AG, we used a thallium flux assay measuring the ability of ligands to enhance 2-AG-induced responses in cells expressing rCB₂ and GIRK channels to screen a collection of FDA-approved drugs. Compounds were added to cells loaded with thallium sensitive dye and allowed to incubate for 2.3 minutes, whereupon a submaximal concentration (EC₂₀) of 2-AG was added to the cells and the signal was monitored for an additional 2.7 minutes. The ~1000 compound collection was initially screened in single point at a 10 µM concentration of compound. Test compounds that potentiated the cellular response to 2-AG by a minimum of 3 standard deviations above the average EC₂₀ agonist response were selected as potential PAM hits. Sample kinetic traces of a hit from the screen, compared to EC₂₀ and EC_Max responses to 2-AG, are shown in Figure 1. 63 putative PAMs were identified; of these, 13 were confirmed as active with concentration-dependent activity at the rCB₂ receptor.

2.2. Mycophenolate Mofetil is a Potent and Selective Activator of rCB₂

One hit in particular, mycophenolate mofetil (Figure 2A, white circles), exhibited robust potency and efficacy at rCB₂ (pEC₅₀ = 6.64 ± 0.06, 228 nM; 74 ± 2 % Max) and was selected for additional studies. Mycophenolate mofetil is an immunosuppressant used to prevent organ transplant rejection and to treat autoimmune disorders such as lupus [28,29,30]. To determine if mycophenolate mofetil exerts its activity in the thallium flux assay through rCB₂, we tested it for activity in HEK cells expressing GIRK channels but not rCB₂. Submaximal concentrations of acetylcholine were used as the agonist, targeting the endogenous M₄ muscarinic receptor expressed in HEK/GIRK cells [31]. Mycophenolate mofetil was found to be inactive under these conditions (Figure 2A, red circles), suggesting that its activity is dependent on the presence of the rCB₂ receptor. It was also critical to determine if mycophenolate mofetil was selective for the rCB₂ versus rCB₁ receptors, as activation of rCB₁ receptors can have negative side effects including anxiety, paranoia, and psychosis [32-34]. To determine subtype selectivity, mycophenolate mofetil was tested for activity in cells co-expressing rCB₁ and GIRK channels using thallium flux. The compound did not enhance the agonist-induced response; however, mycophenolate mofetil exhibited a small inhibition of the rCB₁ response to 2-AG at high concentrations. Results are shown in Figure 2A (blue circles, pEC₅₀ < 5, -5.5 ± 1.1 % Max). Mycophenolate mofetil was also tested for activity at the human CB₂ receptor and found to be active (pEC₅₀ = 5.70 ± 0.11; 36 ± 4 % Max, data[32–34 not shown).

2.3. Mycophenolic Acid Is Inactive at CB Receptors

Mycophenolic acid is the active form of mycophenolate mofetil when used as an immunosuppressive drug or for the treatment of autoimmune disorders [35]. The morpholino ethyl ester of mycophenolic acid is a prodrug form which is rapidly metabolized to the active acid in the liver. Mycophenolic acid is a potent inhibitor of inosine-5′-monophosphate dehydrogenase [36]. To test the activity of this active metabolite, the acid was evaluated for activity at rCB₂, rCB₁, and parental HEK/GIRK cells using the thallium flux assay. It was found to be inactive when tested in each of these cell lines (Figure 2B), indicating mycophenolate mofetil, as opposed to the acid, is the active form of the molecule in rCB₂ cells.

2.4. Mycophenolate Mofetil Has Similar Activity in the Presence and Absence of 2-AG in Thallium Flux Assays

The initial high throughput screen and secondary assays were performed in the presence of a submaximal (EC₂₀) concentration of 2-AG to screen for PAMs, compounds that enhance the response of the receptor to an agonist. In the assay design used here, it is possible that compounds may have activity on their own in the absence of 2-AG and would instead be classified as agonists. To distinguish between these possibilities, mycophenolate mofetil was tested for activity in the presence and absence of 2-AG in cells expressing rCB₂/GIRK or rCB₁/GIRK using thallium flux. Increasing concentrations of mycophenolate mofetil enhanced the response of rCB₂/GIRK cells in a concentration-dependent manner with similar potency and efficacy values in the absence of 2-AG (Figure 3, red circles, pEC₅₀ = 6.81 ± 0.12, 154 nM; 71 ± 5 % Max). Comparing this activity to the PAM assay performed in Figure 2A shows that the only difference in activity between the two experiments is the elevated baseline observed in the presence of agonist due to the EC₂₀ response of 2-AG. These results suggest that mycophenolate mofetil acts as an agonist as opposed to a PAM in rCB₂/GIRK cells using thallium flux as a measure of activity. Mycophenolate mofetil decreased the response of rCB₁/GIRK cells both in the presence (Figure 2A, blue circles) and absence (Figure 3, black circles, pEC₅₀ < 5, -15 ± 3 % Max) of 2-AG to a similar value, again differentiated only by the EC₂₀ response of 2-AG. In rCB₁/GIRK cells, mycophenolate mofetil decreases the signal below baseline when added alone, although weakly, which may suggest inverse agonist activity. The decrease in signal in the presence of 30 µM mycophenolate mofetil compared to baseline is significant when added alone (p=0.002) and in the presence of 2-AG (p = 0.0003).

2.5. The Antagonist/Inverse Agonist AM630 Shifts the Mycophenolate Mofetil Concentration-Response Curve (CRC) to the Right Without Significantly Decreasing the Maximal Response

To further characterize the activity of mycophenolate mofetil, we investigated the effect of orthosteric antagonist/inverse agonist AM630 [37,38,39,40] on the concentration-response of rCB₂ cells to mycophenolate mofetil. If AM630 and mycophenolate mofetil bind to the same site on the receptor, increasing concentrations of AM630 would be expected to shift the CRC to the right with no decrease in maximal response, a pattern indicative of a competitive interaction. As seen in Figure 4, AM630 had little effect on the response to mycophenolate mofetil at or below a concentration of 1 µM. At 3 and 10 µM, AM630 significantly shifted the CRC to the right compared to vehicle (pEC₅₀ vehicle = 6.47 ± 0.16; pEC₅₀ 3 µM AM630 = 5.68 ± 0.09 (p = 0.01), pEC₅₀ 10 µM AM630 = 5.64 ± 0.16 (p = 0.02). There was no significant change in maximum signal compared to vehicle with either 3 or 10 µM AM630. While these results are not conclusive on their own, they suggest mycophenolate mofetil may have a competitive relationship with AM630.

2.6. Mycophenolate Mofetil Is Active in Phosphoinositide Hydrolysis Assays and Demonstrates Similar Activity in the Presence and Absence of 2-AG

While mycophenolate mofetil demonstrated potent agonist activity using thallium flux assays, it is important to confirm activity employing an assay that utilizes an alternate signaling pathway as CB receptors exhibit assay-dependent pharmacology [23,26,27,41,42,43,44]. In a complementary assay, we used phosphoinositide (PI) hydrolysis in the presence of the chimeric G protein, G_qi9, to assess activity. As seen in Figure 5, increasing concentrations of mycophenolate mofetil significantly increased PI hydrolysis in a concentration-dependent manner. Similar results were found in the presence (white circles, pIC₅₀ = 6.15 ± 0.09, 700 nM; 633 ± 122 Max IP1) and absence (red circles, pIC₅₀ = 6.28± 0.11, 521 nM; 438 ± 32 Max IP1) of 2-AG. These data are consistent with results from thallium flux assays, demonstrating mycophenolate mofetil acts as a CB₂ agonist in two distinct signaling pathways.

2.7. The CB₂ Receptor Agonist GW-842,166X Exhibits a Similar Functional Activity Profile Compared to Mycophenolate Mofetil

GW-842,166X is a CB₂ receptor agonist that demonstrates analgesic and anti-inflammatory activity in animal models [16,45,46] and was in clinical trials for inflammation and pain [1,47]. It is structurally distinct from mycophenolate mofetil and was selected as a comparator for these studies (see Table 1 for structure). When tested for activity in rCB₂/GIRK cells using thallium flux, GW-842,166X exhibited potent activity in the presence (white circles, pEC₅₀ = 7.13 ± 0.08, 74 nM; 82 ± 4 % Max) and absence (red circles, pEC₅₀ = 7.26 ± 0.05, 56 nM; 89 ± 5 % Max) of 2-AG as seen in Figure 6A. GW-842,166X was inactive at rCB₁ in both the presence (blue circles) and absence (black circles) of 2-AG. Overall, GW-842,166X exhibits a similar functional activity profile in thallium flux assays as that seen with mycophenolate mofetil.

2.8. In Contrast to Mycophenolate Mofetil and GW-842,166X, the CB₂ Receptor Agonist MDA7 Exhibits Inverse Agonist Activity at rCB₂ in Thallium Flux Assays

MDA7 is a CB₂ receptor modulator structurally distinct from both mycophenolate mofetil and GW-842,166X and was described as exhibiting agonist activity at CB₂ in GTPγS assays [18,19,20]. Studies reported MDA7 as active in models of inflammation and neuropathic pain [20,48,49,50] and the compound was shown to induce neuroprotective effects in rodent models [51,52,53]. Currently, MDA7 is being evaluated as a novel therapeutic for the treatment of Alzheimer's disease [1,47]. To compare to the other agonists described above, we tested MDA7 for activity in both rCB₂/GIRK and rCB₁/GIRK cells using thallium flux. In contrast to both mycophenolate and GW-842,166X, MDA7 exhibited inhibitory activity in the presence (white circles, pEC₅₀ < 5, -65 ± 15 Max) and absence (red circles, pEC₅₀ < 5, -64 ± 13 Max) of 2-AG stimulation of rCB₂ as seen in Figure 6B. In contrast, MDA7 activated rCB₁ in the presence (blue circles, pEC₅₀ = 6.40 ± 0.02, 395 nM; 48 ± 2 % Max) and absence (black circles, pEC₅₀ = 6.32 ± 0.03, 477 nM; 50 ± 2 % Max) of 2-AG (Figure 6B). The detection of opposing CB₂ activity in assays that target differential signaling pathways is difficult to predict but is not unanticipated, as it has been observed with other CB receptor modulators [26,27,54,55,56,57,58,59].

2.9. Mycophenolate Mofetil and Other CB₂ Modulators Differentially Bind to the Site Occupied by [³H]CP55,940

Results from functional assays including thallium flux and PI hydrolysis indicate that mycophenolate mofetil acts as an agonist of rCB₂ due to its ability to activate the receptor when added alone to cells expressing rCB₂; however, these assays do not provide information about the binding interaction between the receptor and mycophenolate mofetil. Agonists that bind to the receptor at the site occupied by the endogenous ligand, 2-AG in this case, are considered orthosteric agonists. It is also possible for an agonist to bind and activate a receptor via a site other than the orthosteric site; these ligands are classified as allosteric agonists. To characterize the nature of the agonist activity of mycophenolate mofetil, we tested its ability to bind to the receptor in equilibrium binding assays using membranes harvested from cells expressing rCB₂ using the radioligand [³H]CP55,940 [40,60]. CP55,940 has an affinity for both CB₁ and CB₂ receptors in the low nM range and is reported to bind to the site occupied by 2-AG [61]. The CB₂ antagonist/inverse agonist AM630 fully inhibited radioligand binding with a pK_i value of 7.61 ± 0.29, results consistent with those described in the literature [40]. Increasing concentrations of mycophenolate mofetil had little effect on the equilibrium binding of [³H]CP55,940 until reaching 1 µM and did not fully displace the binding when tested up to 30 µM (Figure 7, Table 1). Similar results were observed for the agonist GW-842,166X. The incomplete binding and discrepancy between the functional potency and binding affinity of these ligands suggest an overlapping or alternate binding site as opposed to a competitive interaction. In contrast, the CB₂ modulator MDA7 fully inhibited radioligand binding with a pK_i value of 6.60 ± 0.05. These binding results on their own suggest that MDA7 occupies the same site as [³H]CP55,940; however, the inverse agonist functional results suggest additional assays are needed to characterize the interaction.

2.10. Mycophenolate Mofetil, GW-842,166X, and MDA7 Behave as Agonists in cAMP and β-Arrestin Assays

Recent publications highlight the complex pharmacology of CB₂ modulators such as EC21a and the importance of profiling CB₂ modulators in assays that target different signaling pathways [26,27,43,55,59]. We have described results for thallium flux assays that measure CB receptor-mediated opening of GIRK channels and PI hydrolysis assays measuring activity via a chimeric G protein. We also evaluated the ability of mycophenolate mofetil and its comparators to modulate CB₂ through two additional pathways, cAMP inhibition and β-arrestin recruitment. When added in the presence (Figure 8A) or absence (Figure 8C) of an EC₂₀ concentration of the agonist CP55,940, mycophenolate mofetil, GW-842,166X, and MDA7 all enhanced the cAMP response to CP55,940 in cells expressing human CB₂ receptors. The potency values for each (Table 2) were right-shifted compared to those observed for the thallium flux assay (Table 1). Mycophenolate mofetil also exhibited lower efficacy in cAMP assays compared to thallium flux and versus GW-842,166X and MDA7. Similarly, when tested in a β-arrestin recruitment assay in the presence (Figure 8B) or absence (Figure 8D) of an EC₂₀ concentration of the agonist CP55,940, mycophenolate mofetil, GW-842,166X, and MDA7 also induced similar responses in the absence and presence of CP55,940 and potency values were generally right-shifted compared to thallium flux assays. MDA7 was notable in that its efficacy was lower than the other two ligands with and without 2-AG when β-arrestin recruitment was measured. These data confirm mycophenolate mofetil and comparator GW-842,166X demonstrate agonist activity across multiple assays and signaling pathways and also confirm the signal bias induced by MDA7.

3. Discussion

The endocannabinoid system, composed of CB₁ and CB₂ receptors, has been extensively studied as a possible therapeutic target for various neurological and inflammatory disorders. CB₁ is widely expressed across brain regions, while CB₂ is primarily expressed in the immune system and microglia [1,2]. Recent studies have shown CB₂ expression in hippocampal, striatal, and dopaminergic neurons, suggesting the potential for CB₂ modulation for the treatment of disorders including pain, addiction, and schizophrenia [1,13,14,15,47,62,63]. Reduced CB₂ receptor expression and activity is correlated with an increased risk of developing schizophrenia [64,65,66,67]. We have shown that the antipsychotic effects of muscarinic M₄ receptor or metabotropic glutamate receptor 1 (mGlu₁) PAMs in rodent behavioral models are dependent on CB₂ activation, which occurs via retrograde signaling of 2-AG [14,15]. For these reasons, it is possible that CB₂ activation or potentiation may represent a novel strategy for treatment. The use of a PAM as opposed to an agonist may provide advantages, as a PAM would be predicted to produce lower levels of receptor desensitization and greater receptor specificity [22,23]. We recently profiled the first published small molecule PAM, EC21a, showing that this ligand exhibits complex pharmacology [26]. To discover novel CB₂ PAMs with improved properties compared to EC21a, we conducted a screen of FDA-approved drugs, resulting in the discovery of immunosuppressant mycophenolate mofetil as a selective activator of CB₂.

The initial design of the assay was intended to screen for PAMs, but subsequent assays determined mycophenolate mofetil was active in the absence of agonist. Other CB₂ agonists have been or are currently being studied in preclinical and clinical trials including GW-842,166X for pain [16,45,46] and MDA7 for pain [49,50,68] and Alzheimer’s disease [51,52], but to date no CB₂ agonist drugs are approved for clinical use. As shown here, both mycophenolate mofetil and GW-842,166X present clear profiles as selective agonists of CB₂ in multiple assays assessing various signaling pathways including thallium flux, cAMP, and β-arrestin assays. MDA7 instead exhibits a mixed profile. In cAMP and β-arrestin assays, it acts as an agonist of the CB₂ receptor; however, in thallium flux assays MDA7 demonstrates inverse agonist activity, clearly blocking the basal signal well below baseline levels. It is also not selective for CB₂ in thallium flux assays as it also significantly activates the CB₁ receptor. It is possible that this mixed activity profile may present challenges when assessing for potential utility as a treatment for brain disorders.

The binding data generated with [³H]CP55,940 indicate multiple binding modes/sites are present within the CB₂ receptor. 2-AG is reported to share the same orthosteric binding site as CP55,940 [61] and we confirmed that 2-AG fully displaced radioligand binding (pK_i = 5.46 ± 0.05). Additionally, the inverse agonist AM630 fully inhibited the equilibrium binding of the radioligand with a K_i value consistent with that reported in the literature (pK_i = 7.61 ± 0.29) [40]. Mycophenolate mofetil was only able to inhibit radioligand binding to a level equal to 20% of [³H]CP55,940 total binding at 30 µM. This level of binding indicates a potential interaction with the binding site occupied by the radioligand, but the disconnect between the binding data and the functional potency generated in the same cell line (rCB₂/GIRK) using thallium flux suggests the binding of mycophenolate mofetil is not completely competitive with [³H]CP55,940. It is possible the two ligands occupy the same site but have different binding contacts or that the ligands share an overlapping portion of the site. It will be important to fully characterize the binding mode of mycophenolate mofetil using mutagenesis and additional molecular pharmacology studies to more fully understand the binding and functional relationship and how it relates to future drug development. This is also the case for GW-842,166X, which exhibited a similar binding profile to that of mycophenolate mofetil with incomplete binding reaching 20% of [³H]CP55,940 total binding. The potency value of this compound in thallium flux assays (pEC₅₀ = 7.13) revealed a left-shifted pEC₅₀ compared to the K_i. MDA7, on the other hand, presents a different profile and fully inhibited the equilibrium binding of [³H]CP55,940 (pK_i = 6.60 ± 0.05). In the rCB₂/GIRK cell line measuring thallium flux, MDA7 displayed weak inverse agonist activity with a potency value significantly right-shifted in comparison (pEC₅₀ < 5.0). The complete inhibition in the binding assay suggests a competitive interaction with the [³H]CP55,940 binding site, but the weak blockade in the functional assay indicates a disconnect between the binding affinity and the functional potency in the thallium flux assay.

In this study, we discovered that mycophenolate mofetil is a selective agonist of CB₂ through screening of an FDA-approved library of drugs. Its agonist activity was confirmed in multiple assays assessing activity through different signaling pathways, including thallium flux, PI hydrolysis, cAMP inhibition, and β-arrestin recruitment. The activity profiles of the CB₂ clinical candidates GW-842,166X and MDA7 were assessed and found to be similar to mycophenolate mofetil in the case of GW-842,166X but divergent for MDA7. These findings suggest that modulators of CB₂ have subtle pharmacological complexities and understanding these differences may provide insight into developing better treatments for brain disorders such as schizophrenia, pain, and Alzheimer's disease.

4. Materials and Methods

4.1. Chemicals

2-Arachidonoylglycerol (2-AG) and 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630) were purchased from Cayman Chemical (Ann Arbor, MI). 2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol (CP55,940) was purchased from Tocris Bioscience (Minneapolis, MN). [³H]-CP55,940 was obtained from Perkin Elmer (Boston, MA). 2-(2,4-dichloroanilino)-N-(oxan-4-ylmethyl)-4-(trifluoromethyl)pyrimidine-5-carboxamide (GW-842,166X) and 1-[(3-benzyl-3-methyl-2,3-dihydro-1-benzofuran-6-yl)carbonyl]piperidine (MDA7/NTRX-07) were synthesized by the Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, USA as previously described [16,18,19,20].

4.2. Cell Culture

Human Embryonic Kidney (HEK) 293 cells stably expressing rCB₂ and the G protein G_qi9 were maintained in Dulbecco's modified Eagle media (DMEM) containing 10% FBS, 1x antibiotic/antimycotic, 20 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 1x non-essential amino acids, 700 µg/mL G418 sulfate, and 0.6 µg/mL puromycin. HEK cells stably expressing rCB₂ or rCB₁ and the G protein inwardly rectifying potassium channel (GIRK) were maintained in DMEM/F12 containing 10% FBS, 1x antibiotic/antimycotic, 20 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 1x non-essential amino acids, 700 µg/mL G418 sulfate, and 0.6 µg/mL puromycin. Cells were monitored by periodic PCR detection using the LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich, St Louis, MO) to eliminate potential mycoplasma infection. Reagents were obtained from Invitrogen (Waltham, MA) unless otherwise noted.

4.3. Thallium Flux Assay

Thallium flux assays were performed as previously described in Niswender et al., 2008 [31]. HEK/GIRK cells stably expressing rCB₁ or rCB₂ (15,000 cells/20 µL/well) were seeded in 384-well, poly-D-lysine coated assay plates (Corning BioCoat®) (Corning Inc., Corning, NY) and incubated overnight at 37 °C in the presence of 5% CO₂. The next day, medium was removed and replaced with 20 μL of 1.2 µM thallium-sensitive dye Thallos-AM (ION biosciences, San Marcos, TX), prepared as a DMSO stock solution mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127, and diluted in assay buffer (Hank's balanced salt solution, 20 mM HEPES, pH 7.4). Following 1 hour at room temperature, dye solution was removed and replaced with 20 µL assay buffer. For screening of the FDA-Approved Drug Collection, 10 mM DMSO stocks of library compounds were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA) and diluted in assay buffer to a 2x final concentration. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10-point concentration response curves in DMSO, transferred to daughter plates using the Echo or Bravo Automated Liquid Handling Platform (Agilent Technologies, Santa Clara, CA), and diluted in assay buffer to a 2x final concentration. Thallium flux was measured using the Hamamatsu FDSS/μCELL Kinetic Plate Imager (Bridgewater, NJ). After establishment of a fluorescence baseline (excitation, 480 ± 20 nm; emission, 540 ± 30 nm), 20 µL (2x) of test compound was added to the cells at 1 second and the response was measured. 140 seconds later, 10 µL (5x) of an EC₂₀ concentration of agonist (PAM mode) or vehicle (AGO mode) in thallium stimulus buffer (125 mM NaHCO₃, 1.8 mM CaSO₄, 1 mM MgSO₄, 5 mM glucose, 12 mM Tl₂SO₄, 10 mM HEPES, 0.5% BSA, pH 7.4) was added to the cells, and the response of the cells was measured for an additional 159 seconds (data were acquired for 300 seconds total at 0.5 Hz for 140 seconds and 1 Hz for 160 seconds). Raw kinetic data were analyzed in a multi-step process. 1) 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. 2) The slope value for each kinetic trace was calculated for the time window of 145-155 seconds, a window occurring directly after the second addition. 3) The average slope was calculated for wells containing vehicle and this value was subtracted from all wells. 4) Vehicle-subtracted slope was normalized to the relevant maximal agonist signal for each assay. For concentration response curves, normalized data were fit to a four-parameter logistic equation using GraphPad Prism (La Jolla, CA) or the Dotmatics software platform (Dotmatics, Bishop’s Stortford, UK).

$$y=bottom+{\displaystyle \frac{top-bottom}{1+{10}^{\left(LogEC50-A\right)Hillslope}}}$$

where A is the molar concentration of the compound; bottom and top denote the lower and upper plateaus of the concentration-response curve; Hillslope is the Hill coefficient that describes the steepness of the curve; and EC₅₀ is the molar concentration of compound required to generate a response halfway between the top and bottom. Data shown represent the mean ± standard error of the pEC₅₀ or maximal response. Experiments were performed in duplicate or triplicate and repeated a minimum of three separate times.

4.4. PI Hydrolysis Assay

One day prior to experimentation, selected HEK/G_qi9 monoclonal cells stably expressing rat CB₂ were plated onto poly-D-lysine coated clear-bottom 384-well plates (15,000 cells per well) in DMEM supplemented with 10% FBS and 20 mM HEPES. Ten minutes prior to the assay, cell culture medium was replaced with 20 µL 37 °C Hank's balanced salt solution (HBSS, with Ca²⁺, Mg²⁺, glucose). IP₁ stimulation was then initiated by adding 5 µL of 5x agonist dissolved in HBSS plus 40 mM Li⁺, and cells were incubated for an additional hour before aspiration and addition of Lysis Buffer. IP₁ levels were determined using the Cisbio HTRF IP-ONE assay kit per the manufacturer’s instructions, and fluorescence was measured using an Envision plate reader (PerkinElmer, Waltham, MA). Data were acquired as HTRF ratio (665/620) and expressed as nanomolar levels of IP₁.

4.5. Radioligand Binding Assays

Membranes were made from HEK/GIRK cells stably expressing rat CB₂. Radioligand competition binding assays were performed as previously described with minor modifications. In brief, compounds were serially diluted into assay buffer with 0.1% bovine serum albumin (BSA) and added to each well of a 96-well plate, along with 20 μg/well cell membrane and approximately 500 pM [³H]-CP55,940 (specific activity = 104 Ci/mmol, PerkinElmer, Waltham, MA). Following a 3-hour incubation period on shaker at room temperature, the membrane-bound ligand was separated from free ligand by filtration through glass fiber 96-well filter plates (Unifilter-96, GF/B; PerkinElmer, Waltham, MA). Forty microliters of scintillation fluid were added to each well, and the membrane-bound radioactivity was determined by scintillation counting (Microbeta2; Revvity, Waltham, MA). Nonspecific binding was determined using 10 μM of cold CP55,940.

4.6. cAMP Assays

Assays were performed by Eurofins Discovery (Freemont, CA). cAMP Hunter cell lines were expanded from freezer stocks according to standard procedures. Cells were seeded in a total volume of 20 μL into white walled, 384-well microplates and incubated at 37 °C for the appropriate time prior to testing. cAMP modulation was determined using the DiscoverX HitHunter cAMP XS+ assay. For agonist determination, cells were incubated with sample in the presence of EC₈₀ forskolin to induce response. Media was aspirated from cells and replaced with 10 μL HBSS/10mM HEPES. Intermediate dilution of sample stocks was performed to generate 4X sample in assay buffer and 5 μL of 4X sample was added to cells. 5 μL of 4X EC₈₀ forskolin diluted in HBSS/HEPES was added and incubated at 37 °C for 30 minutes. Final assay vehicle concentration was 1%. For allosteric determination, cells were pre-incubated with sample followed by agonist induction at the EC₂₀ concentration. Media was aspirated from cells and replaced with 10 μL HBSS/10mM HEPES. Intermediate dilution of sample stocks was performed to generate 4X sample in assay buffer. 5 μL of 4X compound was added to the cells and incubated at room temperature or 37 °C for 5 minutes. 5 μL of 4X EC₂₀ agonist was added to the cells and incubated at 37 °C for 30 minutes. EC₈₀ forskolin was included.

After appropriate compound incubation, assay signal was generated through incubation with 5μL cAMP XS+ Ab reagent followed by 20 μL cAMP XS+ ED/CL lysis cocktail for one hour at room temperature. 20 μL cAMP XS+ EA reagent for two hours at room temperature. Microplates were read following signal generation with a PerkinElmer EnvisionTM instrument for chemiluminescent signal detection.

Compound activity was analyzed using CBIS data analysis suite (ChemInnovation, CA). For agonist mode assays, percentage activity is calculated using the following formula: % Activity = 100% x (1 - (mean RLU of test sample - mean RLU of MAX control) / (mean RLU of vehicle control - mean RLU of MAX control)).

For Gi positive allosteric mode assays, percentage modulation was calculated using the following formula: % Modulation =100% x (1-(mean RLU of test sample - mean RLU of MAX control) / (mean RLU of EC₂₀ control - mean RLU of MAX control)).

4.7.β-. Arrestin Assays

Assays were performed by Eurofins Discovery (Freemont, CA). PathHunter cell lines were expanded from freezer stocks according to standard procedures. Cells were seeded in a total volume of 20 μL into white walled, 384-well microplates and incubated at 37 °C for the appropriate time prior to testing. For agonist determination, cells were incubated with sample to induce response. Intermediate dilution of sample stocks was performed to generate 5X sample in assay buffer. 5 μL of 5X sample was added to cells and incubated at 37 °C or room temperature for at least 120 minutes. Final assay vehicle concentration was 1%. For allosteric modulator determination, cells were pre-incubated with sample followed by agonist challenge at the EC₂₀. Intermediate dilution of sample stocks was performed to generate 5X sample in assay buffer. 5 μL of 5X sample was added to cells and incubated at 37 °C or room temperature for 15 minutes. Vehicle concentration was 1%. 5 μL of 6X EC₂₀ agonist in assay buffer was added to the cells and incubated at 37 °C or room temperature for at least 120 minutes.

Assay signal was generated through a single addition of 15 μL (50% v/v) of PathHunter Detection reagent cocktail, followed by a one-hour incubation at room temperature. Microplates were read following signal generation with a PerkinElmer EnvisionTM instrument for chemiluminescent signal detection.

Compound activity was analyzed using CBIS data analysis suite (ChemInnovation, CA). For agonist mode assays, percentage activity was calculated using the following formula: % Activity = 100% x (mean RLU of test sample — mean RLU of vehicle control) / (mean MAX control ligand — mean RLU of vehicle control).

For positive allosteric mode assays, percentage modulation was calculated using the following formula: % Modulation = 100% x ((mean RLU of test sample — mean RLU of EC₂₀ control) / (mean RLU of MAX control ligand — mean RLU of EC₂₀ control)).