Development of a membrane-based Gi-CASE biosensor assay for profiling compounds at cannabinoid receptors

Abstract

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB₁R) and 2 (CB₂R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB₁R and CB₂R are Gα_i protein-coupled receptors but detecting Gα_i protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [³⁵S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gα_i protein activity assays. Methods: Here, we describe the development of a membrane-based Gα_i signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB₁R or CB₂R, and components of the Gα_i-CASE biosensor. This BRET-based system allows direct detection of Gα_i signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the βγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10 μM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC₅₀) and efficacy (E_max) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gα_i protein activation assay is applicable to other Gα_i-coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation.

Keywords: BRET—bioluminescence resonance energy transfer; G-protein signaling; GPCR signalling assay; Gαi signaling pathway; Gαi signalling assay; cannabinoid receptors (CBRs); inverse agonism.

FIGURE 1

The Gi-CASE biosensor detects G protein activity based on changes in BRET signal. (A) Gi-CASE assay signal detection. The Gi-CASE biosensor plasmid genetically incorporates a NanoLuciferase^® donor on the Gα subunit and a Venus fluorescent acceptor fluorophore on the N-terminus of the Gγ subunit. Agonist-induced G protein activation results in dissociation of the Gα and Gβγ subunits causing a reduction in the BRET signal. Inverse agonist activity causes a reduction in Gα and Gβγ dissociation and promotes reassociation resulting in an increase in the relative BRET signal. (B) Simulated Gi-CASE data, shown as a change in (∆) BRET ratio for an inverse agonist (green) and agonist (red) of equal potency.

FIGURE 2

Gi-CASE activation/inhibition time courses and concentration-response curves in CB₁R and CB₂R-expressing HEK293TR cells, upon stimulation with reference compounds. Time courses at 37°C of (A) CB₁R- or (B) CB₂R-mediated Gi-CASE activation/inhibition following stimulation with HU-210, HU-308 and rimonabant or SR-144,528 at concentrations around the EC₅₀. (C) CB₁R- and (D) CB₂R-mediated Gi-CASE concentration-response curves following stimulation with HU-210, HU-308 and rimonabant or SR-144,528. Data are presented as mean ± S.E.M. from three or more experiments.

FIGURE 3

Gi-CASE activation/inhibition time courses and concentration-response curves in CB₁R- and CB₂R-expressing HEK293TR membranes, upon stimulation with reference compounds. Time courses at 28°C of (A) CB₁R- or (B) CB₂R-mediated Gi-CASE activation/inhibition following stimulation with HU-210, HU-308 and rimonabant or SR-144,528 at concentrations around the EC₅₀. (C) CB₁R- and (D) CB₂R-mediated Gi-CASE concentration-response curves following stimulation with HU-210, HU-308 and rimonabant or SR-144,528. Data are presented as mean ± S.E.M. from three or more experiments.

FIGURE 4

Gi-CASE activation/inhibition time courses and concentration-response curves in CB₁R- and CB₂R-expressing HEK293TR membranes, upon stimulation with reference compounds. Time courses at 37°C of (A) CB₁R- or (B) CB₂R-mediated Gi-CASE activation/inhibition following stimulation with HU-210, HU-308 and rimonabant or SR-144,528 at concentrations around the EC₅₀. (C) CB₁R- and (D) CB₂R-mediated Gi-CASE concentration-response curves following stimulation with HU-210, HU-308 and rimonabant or SR-144,528. Data are presented as mean ± S.E.M. from three or more experiments.

FIGURE 5

Gi-CASE activation/inhibition concentration-response curves in CB₁R- and CB₂R-expressing HEK293TR membranes, upon stimulation with cannabinoid agonists, inverse agonists and neutral compounds. Gi-CASE mediated concentration-response curves obtained in membranes expressing (A) CB₁R and (B) CB₂R. Data are presented as mean ± S.E.M. from three or more experiments.

FIGURE 6

A comparison of CB₁R and CB₂R activation/inhibition potency and efficacy estimates for test compounds obtained at 28°C and 37°C in the Gi-CASE membrane assay. Compound pEC₅₀ value comparison in (A) CB₁R- and (B) CB₂R-expressing Gi-CASE membranes at 28°C and 37°C. Compound intrinsic activity (efficacy) measure comparison expressed as fractional change in the basal BRET ratio in (C) CB₁R- and (D) CB₂R-expressing Gi-CASE membranes at 28°C and 37°C. The dashed line indicates the unity line for perfect correlation. Data are presented as mean ± S.E.M. from three or more experiments.

FIGURE 7

A direct comparison of CB₁R and CB₂R activation/inhibition potency and efficacy estimates for test compounds obtained at 28°C in the Gi-CASE membrane assay. Compound pEC₅₀ values obtained in (A) CB₁R- and (B) CB₂R-expressing Gi-CASE membranes. Compound intrinsic activity measures (efficacy) expressed as fractional change in the basal BRET ratio at (C) CB₁R and (D) CB₂R. The dashed line in A and B is the lower limit of potency detection with values below this level not determined (ND). Data are presented as mean ± S.E.M. from three or more experiments.

FIGURE 8

Inverse agonist-induced shifts in CB₁R- and CB₂R-expressing HEK293TR membranes. HU-210 Gi-CASE concentration-response curves obtained in HEK293TR (A) CB₁R membranes at 28°C, in the absence and presence of the reference CB₁R inverse agonist rimonabant and (B) CB₂R membranes at 28°C, in the absence and presence of the reference CB₁R inverse agonist SR-144,528 (C) CB₁R membranes at 37°C, in the absence and presence of rimonabant and (D) CB₂R membranes at 37°C, in the absence and presence of SR-144,528. Gi-CASE response data are presented as mean ± S.E.M. from three separate experiments performed in singlet.

FIGURE 9

Gi-CASE activation time courses and concentration-response curves in CB₂R-expressing HEK293TR membranes, upon stimulation with the reference agonist HU-308. Changes in Gi-CASE biosensor activation was detected by BRET in HEK293TR membranes. (A) Consecutive addition of increasing concentrations of HU-308 into the same assay wells. Data are displayed as mean $\pm$ S.E.M. from three experiments performed in triplicate. (B) Concentration-response curve of HU-308 generated from (A); 10 min after each consecutive injection of increasing HU-308 concentrations (triplicate determinations), or 60 min after stimulation of individual wells with increasing concentrations HU-308 (singlet determinations). Data are displayed as mean $\pm$ S.E.M. from three independent experiments.

FIGURE 10

Relationships between Gi-CASE and literature compound pEC₅₀ values. Correlation plots of Gi-CASE assay in intact cell and membrane-based assay compound pEC₅₀ values at (A) CB₁R and (B) CB₂R. Comparison of membrane-assay format derived (C) CB₁R and (D) CB₂R Gi-CASE compound pEC₅₀ values, obtained at 28°C, and literature derived pEC₅₀ values obtained in the commonly used GTPγS binding assay format (the source of the GTPγS binding data is provided in the Supplementary File). All Gi-CASE pEC₅₀ values were taken from experiments shown in Figures 2, 4, 5. All data used in these plots are detailed in Table 1, 2. Data are presented as mean ± S.E.M. from three or more experiments. The dashed line indicates the unity line for perfect correlation.