Pharmacological characterization of the endocannabinoid sensor GRAB_eCB2.0

Abstract

IntroductionThe endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid receptors with distinct pharmacology, and differentially regulate pathophysiological processes. The genetically encoded sensor, GRABeCB2.0, detects real-time changes in eCB levels in cells in culture and preclinical model systems; however, its activation by eCB analogues produced by cells and by phyto-cannabinoids remains uncharacterized, a current limitation when interpreting changes in its response. This information could provide additional utility for the tool in in vivo pharmacology studies of phyto-cannabinoid action.MethodsGRABeCB2.0was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements.Results2-AG increased GRABeCB2.0fluorescent signal (EC50= 85 nM), and the cannabinoid 1 receptor (CB1R) antagonist, SR141617, decreased GRABeCB2.0signal (SR1, IC50= 3.3 nM), responses that mirror their known potencies at cannabinoid 1 receptors (CB1R). GRABeCB2.0fluorescent signal also increased in response to AEA (EC50= 815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (2-LGand2-OG, EC50s = 1.5 and 1.0 μM, respectively), Δ9-tetrahydrocannabinol (Δ9-THC) andΔ8-THC(EC50s = 1.6 and 2.0 μM, respectively), and the artificial CB1R agonist, CP55,940 (CP, EC50= 82 nM); however their potencies were less than what has been described at CB1R. Cannabidiol (CBD) did not affect basal GRABeCB2.0fluorescent signal and yet reduced the 2-AG stimulated GRABeCB2.0responses (IC50= 8.8 nM).Conclusions2-AG and SR1 modulate the GRABeCB2.0fluorescent signal with EC50s that mirror their potencies at CB1R whereas AEA, eCB analogues, THC and CP increase GRABeCB2.0fluorescent signal with EC50s significantly lower than their potencies at CB1R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRABeCB2.0retains the negative allosteric modulator (NAM) property of CBD at CB1R. This study describes the pharmacological profile of GRABeCB2.0to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB1R ligands.

Correspondence of fentanyl brain pharmacokinetics and behavior measured via engineering opioids biosensors and computational ethology

“…A general approach to detecting opioids using OpuBC mutants. A common strategy in generating optical biosensors involves merging a conformationally switching protein with circularly permuted GFP (cpGFP) so that ligand binding elicits an increase in fluorescence [35][36][37][38][39][40][41] . The µ-opioid receptor could be a natural choice for detecting opioid drugs; however, unlike several other GPCRs, the µ-opioid receptor merged with cpGFP has been only minimally responsive to its ligands 42 .…”

Functional principles of genetically encoded fluorescent biosensors for metabolism and their quantitative use

“…The effects of indirect modulation can be significant. In the case of GRAB eCD2.0 , 1 μM CBD was reported to reduce the sensor response to 1 μM 2-arachidonoylglycerol by 26% (Singh et al, 2023).…”

“…This is identified in competition assays in which the cognate ligand is titrated over a constant level of the interfering ligand, or vice versa. The interfering ligand can either compete for the same binding site, as in the case of NAD + sensors that are modulated by ATP and ADP (Zou et al, 2020) or lactate sensors that are modulated by pyruvate (Bekdash et al, 2021; San Martin et al, 2013); or it can modulate the sensor response by some other allosteric means, as was recently suggested for CBD modulation of an endocannabinoid sensor (Singh et al, 2023).…”

“…The effects of indirect modulation can be significant. In the case of GRAB eCD2.0 , 1 μM CBD was reported to reduce the sensor response to 1 μM 2‐arachidonoylglycerol by 26% (Singh et al, 2023). As another example, for the NAD + sensor FiNAD, neither ATP nor ADP is capable of generating a sensor response on their own.…”

P2X7 receptor‐dependent increase in endocannabinoid 2‐arachidonoyl glycerol production by neuronal cells in culture: Dynamics and mechanism

“…GRAB eCB2.0 and mut-GRAB eCB2.0 DNA were subcloned into an AM/CBA-WPRE-bGH plasmid as previously described (Singh et al, 2023) using an In-Fusion cloning system (Takara Bio Inc, Japan).…”