MICROPLATE READERS
A
B
Figure 1: TR-FRET assay to study binding affinity of phytocannabinoids to CB1 receptor
respectively (figure 1B). The affinity for ∆9-THCA was considerably lower with a pKi of 5.8 ± 0.6. The Simultaneous Dual Emission
(SDE) feature on the PHERAstar FSX allows both emission signals (from donor and acceptor) to be measured simultaneously. In addition to its high sensitivity and reliability, where even the smallest changes in receptor- ligand interactions can be measured, SDE results in further time savings and more robust data.
SPEED AND ACCURACY SCRAs were originally developed for scientific research or as therapeutic agents. Unfortunately, they have emerged as drugs of abuse and are associated with serious health risks including death.[3] Little is known about the detailed toxicology and pharmacology of SCRAs.[4] However, details of their mode of action are needed to reduce the risks they pose and pave the way for their use as therapeutic agents.
A To study whether a selection of
cannabinoids signal via the Gαi- or Gαs- subunit of the CB1 receptor, cAMP, a downstream second messenger of the Gαs-stimulated cascade was analysed in the following study. Binding of a receptor agonist to CB1 (a G-protein coupled receptor) may lead to dissociation of the trimeric G-protein complex and the release of the subunits into the cytoplasm, initiating various signalling pathways and cellular responses. cAMP levels were quantified with a BRET-based CAYMEL biosensor. The sensor is composed of an EPAC protein, a Renilla luciferase (Rluc) and a yellow fluorescent protein (YFP). Upon binding of cAMP by EPAC, Rluc and YFP are spatially separated owing to a conformational change. This leads to a reduction of the BRET signal which is generated in the absence of cAMP owing to proximity of Rluc and YFP (figure 2A). The overall levels of cAMP generated confirm that cannabinoid receptor agonists have significantly
B
different efficacies in the activation of the Gαs pathway (figure 2B). Again, the high detection speed
enabled the study of several potential compounds in parallel in a kinetic approach. The PHERAstar FSX also offers compatibility with other automation solutions which make it an ideal choice for high-throughput applications in all reading modes. More information on cannabinoid
research is available in this blog post or you can email applications@
bmglabtech.com with questions.
REFERENCES: 1. DOI: 10.1093/jat/bkab107 2. DOI: 10.1007/s10681-015-1585-y 3. DOI: 10.1056/NEJMp1505328 4. DOI: 10.3389/fpsyt.2020.00464
For more information visit:
www.bmglabtech.com
Figure 2: Monitoring cannabinoid signalling with a BRET assay
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