Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay
Cell-based assays for CDK8/19 inhibition are not easily defined, since there are no known cellular functions unique to these kinases. To solve this problem, we generated derivatives of 293 cells with CRISPR knockout of one or both of CDK8 and CDK19. Double knockout (dKO) of CDK8 and CDK19 together (...
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| Format: | Article |
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MDPI AG
2019-10-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/8/10/1208 |
| _version_ | 1797714360163893248 |
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| author | Jing Li Hao Ji Donald C. Porter Eugenia V. Broude Igor B. Roninson Mengqian Chen |
| author_facet | Jing Li Hao Ji Donald C. Porter Eugenia V. Broude Igor B. Roninson Mengqian Chen |
| author_sort | Jing Li |
| collection | DOAJ |
| description | Cell-based assays for CDK8/19 inhibition are not easily defined, since there are no known cellular functions unique to these kinases. To solve this problem, we generated derivatives of 293 cells with CRISPR knockout of one or both of CDK8 and CDK19. Double knockout (dKO) of CDK8 and CDK19 together (but not individually) decreased the induction of transcription by NFκB (a CDK8/19-potentiated transcription factor) and abrogated the effect of CDK8/19 inhibitors on such induction. We generated wild type (WT) and dKO cell lines expressing luciferase from an NFκB-dependent promoter. Inhibitors selective for CDK8/19 over other CDKs decreased TNFα-induced luciferase expression in WT cells by ~80% with no effect on luciferase induction in dKO cells. In contrast, non-selective CDK inhibitors flavopiridol and dinaciclib and a CDK7/12/13 inhibitor THZ1 (but not CDK4/6 inhibitor palbociclib) suppressed luciferase induction in both WT and dKO cells, indicating a distinct role for other CDKs in the NFκB pathway. We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor. Thienopyridines inhibited luciferase induction in the WT but not dKO cells and their IC<sub>50</sub> values in the WT reporter assay showed near-perfect correlation (R<sup>2</sup> = 0.98) with their reported activities in a bone anabolic activity assay, confirming that the latter function is mediated by CDK8/19 and validating our assay as a robust and quantitative method for CDK8/19 inhibition. |
| first_indexed | 2024-03-12T07:50:45Z |
| format | Article |
| id | doaj.art-6209016a158c420290e1a7a6f1e25b80 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-12T07:50:45Z |
| publishDate | 2019-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-6209016a158c420290e1a7a6f1e25b802023-09-02T20:36:28ZengMDPI AGCells2073-44092019-10-01810120810.3390/cells8101208cells8101208Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based AssayJing Li0Hao Ji1Donald C. Porter2Eugenia V. Broude3Igor B. Roninson4Mengqian Chen5Department of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC 29208, USADepartment of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC 29208, USASenex Biotechnology, Inc., Columbia, SC 29208, USADepartment of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC 29208, USADepartment of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC 29208, USADepartment of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC 29208, USACell-based assays for CDK8/19 inhibition are not easily defined, since there are no known cellular functions unique to these kinases. To solve this problem, we generated derivatives of 293 cells with CRISPR knockout of one or both of CDK8 and CDK19. Double knockout (dKO) of CDK8 and CDK19 together (but not individually) decreased the induction of transcription by NFκB (a CDK8/19-potentiated transcription factor) and abrogated the effect of CDK8/19 inhibitors on such induction. We generated wild type (WT) and dKO cell lines expressing luciferase from an NFκB-dependent promoter. Inhibitors selective for CDK8/19 over other CDKs decreased TNFα-induced luciferase expression in WT cells by ~80% with no effect on luciferase induction in dKO cells. In contrast, non-selective CDK inhibitors flavopiridol and dinaciclib and a CDK7/12/13 inhibitor THZ1 (but not CDK4/6 inhibitor palbociclib) suppressed luciferase induction in both WT and dKO cells, indicating a distinct role for other CDKs in the NFκB pathway. We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor. Thienopyridines inhibited luciferase induction in the WT but not dKO cells and their IC<sub>50</sub> values in the WT reporter assay showed near-perfect correlation (R<sup>2</sup> = 0.98) with their reported activities in a bone anabolic activity assay, confirming that the latter function is mediated by CDK8/19 and validating our assay as a robust and quantitative method for CDK8/19 inhibition.https://www.mdpi.com/2073-4409/8/10/1208cdk8cdk19cdk inhibitorsnfκbthienopyridinescell-based assays |
| spellingShingle | Jing Li Hao Ji Donald C. Porter Eugenia V. Broude Igor B. Roninson Mengqian Chen Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay Cells cdk8 cdk19 cdk inhibitors nfκb thienopyridines cell-based assays |
| title | Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay |
| title_full | Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay |
| title_fullStr | Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay |
| title_full_unstemmed | Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay |
| title_short | Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay |
| title_sort | characterizing cdk8 19 inhibitors through a nfκb dependent cell based assay |
| topic | cdk8 cdk19 cdk inhibitors nfκb thienopyridines cell-based assays |
| url | https://www.mdpi.com/2073-4409/8/10/1208 |
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