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

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.