Cargando…
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 (...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830309/ https://www.ncbi.nlm.nih.gov/pubmed/31590445 http://dx.doi.org/10.3390/cells8101208 |
_version_ | 1783465753982271488 |
---|---|
author | Li, Jing Ji, Hao Porter, Donald C. Broude, Eugenia V. Roninson, Igor B. Chen, Mengqian |
author_facet | Li, Jing Ji, Hao Porter, Donald C. Broude, Eugenia V. Roninson, Igor B. Chen, Mengqian |
author_sort | Li, Jing |
collection | PubMed |
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(50) values in the WT reporter assay showed near-perfect correlation (R(2) = 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. |
format | Online Article Text |
id | pubmed-6830309 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-68303092019-11-20 Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay Li, Jing Ji, Hao Porter, Donald C. Broude, Eugenia V. Roninson, Igor B. Chen, Mengqian Cells Article 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(50) values in the WT reporter assay showed near-perfect correlation (R(2) = 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. MDPI 2019-10-06 /pmc/articles/PMC6830309/ /pubmed/31590445 http://dx.doi.org/10.3390/cells8101208 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Li, Jing Ji, Hao Porter, Donald C. Broude, Eugenia V. Roninson, Igor B. Chen, Mengqian Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay |
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 | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830309/ https://www.ncbi.nlm.nih.gov/pubmed/31590445 http://dx.doi.org/10.3390/cells8101208 |
work_keys_str_mv | AT lijing characterizingcdk819inhibitorsthroughanfkbdependentcellbasedassay AT jihao characterizingcdk819inhibitorsthroughanfkbdependentcellbasedassay AT porterdonaldc characterizingcdk819inhibitorsthroughanfkbdependentcellbasedassay AT broudeeugeniav characterizingcdk819inhibitorsthroughanfkbdependentcellbasedassay AT roninsonigorb characterizingcdk819inhibitorsthroughanfkbdependentcellbasedassay AT chenmengqian characterizingcdk819inhibitorsthroughanfkbdependentcellbasedassay |