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Figure 3. (A) Shown are the results of the PNT domain PCA with compound plates from the Prestwick (335, 338, 339), Biomol (342), Sigma (348, 355), and <t>Microsource</t> (368) libraries. The circles represent the luminescence reading of each well of a 96-well plate. The red bars represent the mean relative luminescence and standard deviation of that plate. Pink circles represent wells that were exposed to only DMSO. An initial screening hit is defined as being more than 2 standard deviations away from the mean (blue circles). (B) Compounds identified as hits in the initial high-throughput screen were retested for a concentration-dependent response in the PNT domain and leucine zipper PCAs. Compounds were tested in duplicate at 1, 3, 10, and 30 µM, and cells were visually examined to determine toxicity or compound precipitation. Those that were not cytotoxic and elicited a similar response in both PCA systems were likely inhibitors of split luciferase reconstitution, luciferase activity itself, or another variable. Compounds 1 and 2 represent examples of artifacts of the original, large-scale screen, whereas compounds 3–5 represent examples of concentration- dependent responses. Compound 1, (±)-pindobind; compound 2, gitoxigenin diacetate; compound 3, rosolic acid; compound 4, gambogic acid amide; compound 5, 3,4-dimethoxydalbergione.
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Figure 3. (A) Shown are the results of the PNT domain PCA with compound plates from the Prestwick (335, 338, 339), Biomol (342), Sigma (348, 355), and <t>Microsource</t> (368) libraries. The circles represent the luminescence reading of each well of a 96-well plate. The red bars represent the mean relative luminescence and standard deviation of that plate. Pink circles represent wells that were exposed to only DMSO. An initial screening hit is defined as being more than 2 standard deviations away from the mean (blue circles). (B) Compounds identified as hits in the initial high-throughput screen were retested for a concentration-dependent response in the PNT domain and leucine zipper PCAs. Compounds were tested in duplicate at 1, 3, 10, and 30 µM, and cells were visually examined to determine toxicity or compound precipitation. Those that were not cytotoxic and elicited a similar response in both PCA systems were likely inhibitors of split luciferase reconstitution, luciferase activity itself, or another variable. Compounds 1 and 2 represent examples of artifacts of the original, large-scale screen, whereas compounds 3–5 represent examples of concentration- dependent responses. Compound 1, (±)-pindobind; compound 2, gitoxigenin diacetate; compound 3, rosolic acid; compound 4, gambogic acid amide; compound 5, 3,4-dimethoxydalbergione.
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Figure 3. (A) Shown are the results of the PNT domain PCA with compound plates from the Prestwick (335, 338, 339), Biomol (342), Sigma (348, 355), and Microsource (368) libraries. The circles represent the luminescence reading of each well of a 96-well plate. The red bars represent the mean relative luminescence and standard deviation of that plate. Pink circles represent wells that were exposed to only DMSO. An initial screening hit is defined as being more than 2 standard deviations away from the mean (blue circles). (B) Compounds identified as hits in the initial high-throughput screen were retested for a concentration-dependent response in the PNT domain and leucine zipper PCAs. Compounds were tested in duplicate at 1, 3, 10, and 30 µM, and cells were visually examined to determine toxicity or compound precipitation. Those that were not cytotoxic and elicited a similar response in both PCA systems were likely inhibitors of split luciferase reconstitution, luciferase activity itself, or another variable. Compounds 1 and 2 represent examples of artifacts of the original, large-scale screen, whereas compounds 3–5 represent examples of concentration- dependent responses. Compound 1, (±)-pindobind; compound 2, gitoxigenin diacetate; compound 3, rosolic acid; compound 4, gambogic acid amide; compound 5, 3,4-dimethoxydalbergione.

Journal: SLAS discovery : advancing life sciences R & D

Article Title: A Multipronged Screening Approach Targeting Inhibition of ETV6 PNT Domain Polymerization.

doi: 10.1177/2472555220979599

Figure Lengend Snippet: Figure 3. (A) Shown are the results of the PNT domain PCA with compound plates from the Prestwick (335, 338, 339), Biomol (342), Sigma (348, 355), and Microsource (368) libraries. The circles represent the luminescence reading of each well of a 96-well plate. The red bars represent the mean relative luminescence and standard deviation of that plate. Pink circles represent wells that were exposed to only DMSO. An initial screening hit is defined as being more than 2 standard deviations away from the mean (blue circles). (B) Compounds identified as hits in the initial high-throughput screen were retested for a concentration-dependent response in the PNT domain and leucine zipper PCAs. Compounds were tested in duplicate at 1, 3, 10, and 30 µM, and cells were visually examined to determine toxicity or compound precipitation. Those that were not cytotoxic and elicited a similar response in both PCA systems were likely inhibitors of split luciferase reconstitution, luciferase activity itself, or another variable. Compounds 1 and 2 represent examples of artifacts of the original, large-scale screen, whereas compounds 3–5 represent examples of concentration- dependent responses. Compound 1, (±)-pindobind; compound 2, gitoxigenin diacetate; compound 3, rosolic acid; compound 4, gambogic acid amide; compound 5, 3,4-dimethoxydalbergione.

Article Snippet: Screening compounds for the cellular assays consisted of 16,000 compounds from the Maybridge Hitfinder collection, 10,000 compounds from the ChemBridge DIVERset collection, 1120 compounds from Prestwick Chemicals, 1280 compounds from the Sigma LOPAC library, 2000 compounds from the Microsource Spectrum collection, 2697 compounds from the Selleck L1700 Bioactive Compound library, and 500 compounds from Biomol.

Techniques: Standard Deviation, High Throughput Screening Assay, Concentration Assay, Luciferase, Activity Assay