An Update on Cannabinoid Biosensors Research
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Over 200 million people worldwide consume marijuana (Cannabis sativa L.), making it the most commonly used drug after alcohol. With its legalization in Canada and in 22 states in the United States, cannabis has become more accessible for recreational use. Consumption of strains of cannabis containing high amounts of Δ9-THC, the major psychotropic agent in the plant, is associated with cognitive deficiencies and psychomotor impairment. For this reason, there is a growing concern that cannabis legalization will increase the incidence of automobile and work-related accidents.
Thus, it is important to develop technologies that can be utilized on-site to rapidly determine Δ9-THC concentrations in body fluids and exhaled breath. In addition, the clinical use of cannabinoids such as Δ9-THC and CBD will require methods for monitoring blood levels of these compounds. Finally, with advances in the heterologous production of cannabinoids in bacteria and yeast, sensors will be useful in scaling and optimizing cannabinoid output.
Traditionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the most common and accurate method for measuring cannabinoids. However, the large instrumentation of LC-MS/MS is not practical for on-site testing and LC-MS/MS measurements require long analysis times. These limitations in LC-MS/MS have spurred the development of portable, miniaturized cannabinoid sensing devices. Colorimetric assays using the diazonium compound Fast Blue BB (FBBB) have been applied for cannabinoid detection by drug-enforcement agencies. FBBB reacts with cannabinoids such Δ9-THC, CBD and cannabinol (CBN), resulting in the production of brightly colored diazo compounds (Figure 1).
Figure 1: Binding of Δ9-THC to FBBB results in the formation of a red diazo compound. Image credit: Kenneth B. Walsh.
However, FBBB detection has low cannabinoid specificity without prior separation of the individual compounds using chromatography. Semiconductor devices such as those composed of single-walled carbon nanotubes have successfully been employed to measure cannabinoids with a detection limit of 0.7 nanograms per milliliter (ng/ml) of Δ9-THC in liquid phase.
Recently, a research team led by Benoît Lessard and Adam Shuhendler used phythalocyanine-based organic thin-film transistors (OTFTs) in combination with FBBB to detect Δ9-THC. The researchers designed OTFT devices with a thin film of FBBB layered on top of different types of semiconducting materials. Binding of Δ9-THC to the FBBB-coated OTFT decreased the charge transport mobility and threshold voltage of the transistor. Importantly, the device could detect both Δ9-THC and CBD in liquefied and vaporized preparations.
Cannabinoid biosensors in yeast
G protein-coupled receptors (GPCRs) represent the largest group of transmembrane receptors found in nature with approximately 1000 genes identified that encode for these proteins. In addition to mediating the effects of a large number of drugs and neurotransmitters, GPCRs initiate cellular responses to external stimuli including light, odor and pheromones. Cannabinoids, including Δ9-THC and synthetic cannabinoids such as JWH-018 and CP55,940 (Figure 2), bring about their pharmacological actions primarily through cannabinoid CB1 and CB2 receptors. Like other GPCRs, cannabinoid receptors consist of seven transmembrane domains and an intracellular domain that interacts with heterotrimeric G proteins at the cytoplasmic side of the membrane. G proteins, such as the G inhibitory (Gi) protein, consist of the Gα, Gβ and Gγ subunits. Binding of the cannabinoids to the CB1 and CB2 receptors in various cell types stimulates the Gi protein reducing intracellular levels of cAMP and opening plasma membrane K+ channels. In addition, Gi activates the mitogen-activated protein kinase (MAPK) cascade that includes the extracellular regulated protein kinase (ERK) and Jun N-terminal kinase (JNK).
Figure 2. A cannabinoid biosensor was created in yeast cells by expressing the human CB2 receptor, modifying the G protein (Gpa1/Gαi) to increase its sensitivity and adding the GFP reporter gene. Fluorescent signals were measured in the presence of Δ9-THC and the synthetic cannabinoids CP55,940 and JWH-018. The figure is a modification of Figure 1 in Nature. Image credit: Miettinen K, Leelahakorn N, Almeida A, Zhao Y, Hansen LR, Nikolajsen IE, Andersen JB, Givskov M, Staerk D, Bak S, Kampranis SC.
Two recent publications in the journal Nature, one from the University of Copenhagen and the other from Boston University and the Imperial College London, report exploiting an endogenous GPCR/MAPK pathway present in yeast (Saccharomyces cerevisiae) to develop a cannabinoid biosensor (Figure 2). Yeast cells express a pheromone GPCR known as Ste2p and a heterotrimeric G protein consisting of a Gα subunit (Gpa1p) and a Gβγ complex (Ste4p-Ste18p). Upon binding of a pheromone ligand, Ste2p undergoes a conformational change causing a dissociation of the Ste4p-Ste18p complex which stimulates the MAPK signaling cascade (Ste11p/Ste7p/Fus3p). MAPK then phosphorylates the transcription factor Ste12p. The investigators in the Nature papers made several changes to the yeast cells in order to generate a cannabinoid biosensor (Figure 2). First, Ste2p was deleted and replaced with the human CB2 receptor. Secondly, a chimeric Gpa1p/Gαi protein was created by substituting five amino acids at the C-terminus of Gpa1p with those of the human Gα subunit from the Gi protein. Finally, cannabinoid signaling was detected by putting a reporter gene encoding the green fluorescent protein (GFP) under control of the yeast promoter FIG1. In some experiments other strains of yeast were constructed containing luminescence- and color-based reporters. Thus, binding of cannabinoids to the CB2 receptor in the yeast cells was easily quantified by measuring changes in the green fluorescent, luminescent, or color signal.
In the paper by Kampranis and colleagues, the yeast biosensor was first tested using CP55,940, Δ9-THC, and JWH-018. The half-maximal effective concentration (EC50) of the cannabinoids increasing the fluorescent output of the biosensor was 4.6 nanomolars (nM) (CP55,940), 1.2 µM (micromolars) (Δ9-THC), and 169 nM (JWH-018). These values are consistent with EC50s obtained in previous studies with mammalian cells. Next, the biosensor was used to screen 1,600 compounds, chosen from a compound library at the University of Copenhagen, for activity at the CB2 receptor. The yeast cells were grown in 384-well plates and the compounds added to the wells using a liquid dispenser. Both CB2 receptor agonists and antagonists were identified using the assay with some of the compounds displaying EC50s (for an agonist) or a half maximal inhibitory concentrations (IC50s) (for an antagonist) in the high namomolar to low micromolar concentration range. In addition to screening purified compounds, the biosensor was used for evaluating methanol extracts obtained from a collection of 56 medicinal plants. One HPLC fraction, obtained from the prairie coneflower Ratibida columnifera, caused a 27-fold increase in the cannabinoid signal. From this fraction the authors isolated the active component Dugesialactone (Figure 2). This chemical was not previously known to possess CB2 receptor activity.
Ellis and colleagues evaluated the ability of the cannabinoid biosensor to measure microbially produced Δ9-THC. For these experiments, tetrahydrocannabinolic acid (THCA) was extracted from yeast cells expressing THCA synthase, the enzyme that converts cannabigerolic acid (CBGA) to THCA. The samples were then incubated at 140o C to allow the decarboxylation of THCA to Δ9-THC. After establishing a protocol for measuring Δ9-THC with the biosensor, the investigators created a library of yeast strains containing mutations in THCA synthase. Of the 109 mutant strains tested using the biosensor, five of them displayed a higher Δ9-THC-induced GFP signal than the unmutated THCA synthase yeast, indicating increased production of THCA in these strains. Overall, the GFP signal from the different yeast strains correlated well with Δ9-THC amounts measured with LC-MS.
In conclusion, the yeast CB2 receptor biosensor provides a low-cost and real-time method for screening compound libraries for new cannabinoid agonists and antagonists and for identifying new phytocannabinoids in medicinal plants and herbs. In addition, the biosensor will allow the monitoring and optimization of yeast strains that yield high levels of THCA and other CB2 receptor ligands. Future improvements will be needed to make the biosensor more selective for Δ9-THC and to give it greater portability.