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Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay.

Jing Li1, Hao Ji2, Donald C Porter3

  • 1Department of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC 29208, USA. jl13@email.sc.edu.

Cells
|October 9, 2019
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Summary

Researchers developed a novel cell-based assay to measure CDK8/19 kinase inhibition. This assay utilizes CRISPR-generated knockout cells and an NFκB reporter to specifically quantify CDK8/19 inhibitor activity, confirming their role in bone anabolic effects.

Keywords:
CDK inhibitorsCDK19CDK8NFκBcell-based assaysthienopyridines

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Drug Discovery

Background:

  • Cell-based assays for Cyclin-Dependent Kinase 8/19 (CDK8/19) inhibition are challenging due to the lack of unique cellular functions.
  • CDK8/19 are implicated in potentiating transcription factors like NFκB, but specific assays for their inhibition are needed.

Purpose of the Study:

  • To develop a robust and quantitative cell-based assay for CDK8/19 inhibition.
  • To validate the assay's ability to specifically measure CDK8/19 activity and its role in biological processes.

Main Methods:

  • Generation of 293 cell lines with CRISPR-Cas9 knockout of CDK8 and CDK19 (double knockout, dKO).
  • Development of a reporter assay using NFκB-dependent luciferase expression in wild-type (WT) and dKO cells.
  • Testing of selective and non-selective CDK inhibitors, including thienopyridines with known bone anabolic activity.

Main Results:

  • CDK8/19 dKO cells abrogated the effect of selective CDK8/19 inhibitors on NFκB-driven transcription.
  • Selective CDK8/19 inhibitors reduced TNFα-induced luciferase expression in WT cells but not dKO cells.
  • Thienopyridine compounds showed a strong correlation between their inhibitory activity in the reporter assay and their bone anabolic effects, mediated by CDK8/19.

Conclusions:

  • The developed reporter assay is a validated and quantitative method for assessing CDK8/19 inhibition.
  • CDK8/19 kinases play a crucial role in NFκB-mediated transcription and are the targets for the bone anabolic activity of thienopyridines.