AS1842856 is an inhibitor of GSK3B. (A) GSEA of genes exclusively regulated by either Foxo1-KO or AS1842856 were subjected to GSEA for the “c2” curated database as described in Figure 2D. The result for the gene set “WANG_RESPONSE_TO_GSK3_INHIBITOR_SB216763_UP” in both conditions is shown. (B) Structural similarity of AS1842856 to quinolizidinone carboxylic acid derivates that inhibit GSK3B. To predict the pharmacological activities of AS1842856, we used the SwissTargetPrediction tool (http://www.swisstargetprediction.ch/). The prediction was made by the high structural similarity of AS1842856 to quinolizidinone carboxylic acid derivatives. The 2 most similar compounds are shown (BindingDB and PubCHEM). (C) Investigation of GSK3B-inhibitory activity was performed by in vitro kinase luminescent assay. The highly specific GSK3B inhibitor CHIR-99021 was used as a positive control (n = 2). (D) An unbiased kinome screen was performed. Kinases inhibited by AS1842856 >70% are shown as red leaves. AS1842856 was tested at concentration 100 nM and adenosine triphosphate Km for each kinase. (E-F) RS4;11 (E) and 018Z cells (F) were treated with indicated compounds or vehicle (DMSO) for 24 hours. The expression of CTNNB1 or TUBB (loading control) was determined by immunoblot. The most representative of 3 independent experiments is shown. (G-I) AS1842856 reversibly inhibits CTNNB1 degradation. 018Z cells were cont. incubated with 100 nM AS1842856. After 2-hour incubation with AS1842856, CHX was added at a concentration of 50 μg/mL. In 1 of the groups (2 hours), AS1842856 was washed out before the addition of CHX. (G) The most representative of 3 independent experiments is shown (n = 3). (H) The immunoblots were quantified by ImageJ 1.53k software (https://imagej.net/ij). The relative CTNNB1 expression was calculated as (CTNNB1E/TUBBE)/(CTNNB1C/TUBBC), in which E are the results of measurements in experimental and C in control (time point 0) groups. The data are shown as mean and scattered experimental points (n = 3). (I) The CTNNB1 half-life was calculated with the help of GraphPad Prism 10.4.1 software (nonlinear regression; absolute IC50). CHX, cycloheximide; cont., continuously; FDR, false discovery rate; NES, normalized enrichment score.

AS1842856 is an inhibitor of GSK3B. (A) GSEA of genes exclusively regulated by either Foxo1-KO or AS1842856 were subjected to GSEA for the “c2” curated database as described in Figure 2D. The result for the gene set “WANG_RESPONSE_TO_GSK3_INHIBITOR_SB216763_UP” in both conditions is shown. (B) Structural similarity of AS1842856 to quinolizidinone carboxylic acid derivates that inhibit GSK3B. To predict the pharmacological activities of AS1842856, we used the SwissTargetPrediction tool (http://www.swisstargetprediction.ch/). The prediction was made by the high structural similarity of AS1842856 to quinolizidinone carboxylic acid derivatives. The 2 most similar compounds are shown (BindingDB and PubCHEM). (C) Investigation of GSK3B-inhibitory activity was performed by in vitro kinase luminescent assay. The highly specific GSK3B inhibitor CHIR-99021 was used as a positive control (n = 2). (D) An unbiased kinome screen was performed. Kinases inhibited by AS1842856 >70% are shown as red leaves. AS1842856 was tested at concentration 100 nM and adenosine triphosphate Km for each kinase. (E-F) RS4;11 (E) and 018Z cells (F) were treated with indicated compounds or vehicle (DMSO) for 24 hours. The expression of CTNNB1 or TUBB (loading control) was determined by immunoblot. The most representative of 3 independent experiments is shown. (G-I) AS1842856 reversibly inhibits CTNNB1 degradation. 018Z cells were cont. incubated with 100 nM AS1842856. After 2-hour incubation with AS1842856, CHX was added at a concentration of 50 μg/mL. In 1 of the groups (2 hours), AS1842856 was washed out before the addition of CHX. (G) The most representative of 3 independent experiments is shown (n = 3). (H) The immunoblots were quantified by ImageJ 1.53k software (https://imagej.net/ij). The relative CTNNB1 expression was calculated as (CTNNB1E/TUBBE)/(CTNNB1C/TUBBC), in which E are the results of measurements in experimental and C in control (time point 0) groups. The data are shown as mean and scattered experimental points (n = 3). (I) The CTNNB1 half-life was calculated with the help of GraphPad Prism 10.4.1 software (nonlinear regression; absolute IC50). CHX, cycloheximide; cont., continuously; FDR, false discovery rate; NES, normalized enrichment score.

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