Fig. 5: High-throughput screening applications.
a The high resolution and low background of the KM206 cannabinoid biosensor strain makes it well suited for high-throughput screening for GPCR ligands. The HTS screening workflow consists of (1) robotically dispensing the biosensor strain into 384-well plates, followed by (2) dispensing the library into these plates, (3) incubating for 3 h, (4) adding the developer solution (lysis buffer with luciferin) to the cells, and (5) measuring luciferase activity with a plate reader. b An agonist screen of the library led to the discovery of 2 CB2 agonists with markedly higher luciferase signal than the background. c A Dose–response curve (black) for AGO1 revealed an EC50 of 200 nM. While control curve (gray) shows no noticeable non-CB2 specific effect of AGO1 on the control strain KM207. d In the case of AGO2, a dose–response curve showed an EC50 2 of μM and no effect on control strain. These results clearly validate the hits from the screening experiments. e An antagonist screen of the library was performed by adding the library on top of cell supplemented with 2 nM CP55940. Here, compounds that resulted in lower luciferase activity than that of CP55940 alone were defined as hits (inhibitors). Their potency was calculated as [relative residual activity]−1. This revealed several potential antagonists (ANT1-7). f A dose–response curve for ANT2 revealed an IC50 of 29 μM. The control curve (gray) shows no non-CB2 specific effect of ANT2 on the control strain KM207. g For ANT5, a dose–response curve showed an IC50 of 710 nM) and no effect on the control strain. For (c), (d), (f), and (g), data presented as mean + /− standard deviation. n = 3 biologically independent samples. Source data are provided in the Source Data file.
