Biochemical characterization, regulation, and inhibition of human transcription kinases CDK12 and CDK13 and human cell cycle-related kinase CDK14

The transcription process is the first step in the molecular information flow from genome to proteome. In eukaryotes, the RNA polymerase II (RNAPII) transcribes genes that code for proteins by synthesizing pre-mature messenger RNAs (mRNAs). The Cyclin-dependent kinases CDK12 and CDK13 in association with Cyclin K are transcription regulating kinases by modulating the function of RNAPII. Both CDK/Cyclin pairs regulate transcriptional elongation as well as processes occurring co-transcriptionally through phosphorylation of the C-terminal domain (CTD) of RNAPII. They selectively affect the expression of genes involved in DNA damage response (DDR) and mRNA processing, respectively. CDK12 and CDK13 are involved in numerous types of cancer, representing potential targets for novel cancer treatments. Small molecule inhibitors that selectively target CDK12 and CDK13 in an ATP-competitive manner are used to investigate the consequences of their inhibition in healthy cells and cancer cells. In this thesis, using a protein structure-based approach, dinaciclib, a small molecule inhibitor of CDKs 1, 2 and 5, was identified as a potent inhibitor of CDK12 and CDK13. In comparison to flavopiridol, a known transcription-regulating CDK inhibitor, inhibition of CDK12 by dinaciclib was significantly more potent than by flavopiridol. By performing mutagenesis experiments of residues in the C-terminal extension helix of CDK12, we show that the histidine residue of the DCHEL motif causes a sterical hindrance that may reduce the inhibitory activity of flavopiridol. We also conclude that dinaciclib may readily accommodate its pyridine ring at the histidine binding site of the kinase leading to highly potent inhibition of CDK12 and CDK13. This might be achieved by a displacement mode of binding rather than by performing a base stacking interaction with the histidine imidazole ring. In a further approach, non-covalent reversible and covalent irreversible ATP-competitive small molecule inhibitors were characterized at different ATP ...