Fig. 2

Fig. 2

A high-throughput behavioral screen identifies GABAergic compounds. Zebrafish were treated with various compounds and analyzed for anesthetic-related behaviors. a This scatter plot compares phenoscores of individual wells treated with DMSO or etomidate (6.25 μΜ) (Z-factor = 0.7, n = 944 wells). b This contour plot scores each well from the large-scale behavior-based chemical screen (11,679 compounds, 2336 DMSO controls) by its phenoscore (y-axis) and immobilization index (x-axis). Labels indicate regions with 125 hit compounds (green), 44 toxic compounds (red), and the remaining screening compounds and DMSO controls (blue and gray, respectively). c Structural clustering of the top 125 hit compounds (y-axis) forms 14 clusters using a Tanimoto similarity metric (x-axis). d Example structures of selected compounds in the indicated clusters. e This scatter plot shows a 80.7% reproducibility rate for 57 primary hit compounds. Each point represents the average phenoscore of n = 12 wells at the indicated concentrations (colorbar). The first column represents DMSO controls; the order of other compounds are listed in Supplementary Table 4. f Human GABA_AR activation (y-axis) was measured by FLIPR analysis. Of 47 hit compounds, 23 potentiated GABA_ARs. Compounds 7013338 and 5942595 potentiated GABA_ARs significantly greater than positive controls (red asterisk = 7013338, two red asterisks = 5942595, P < 0.0001, two-tailed t-test, n = 2–4 replicates as indicated). The hit threshold was defined as 2× the average DMSO control group. Picrotoxin, BGC 20-761, progesterone, and DMSO were used as negative controls (x-axis) while etomidate, tracazolate, propofol, diazepam, and thiopental were used as positive controls. Arrows indicate compounds that were predicted by SEA to bind GABA_ARs (red arrows) and compounds that bound to TSPO in vitro (green arrowheads)