There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex–associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway–regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.
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This work was supported by Cancer Research UK (grant numbers C309/A11566, C368/A6743 and A368/A7990). We acknowledge Cancer Research UK funding to the Cancer Research UK Centre at The Institute of Cancer Research and The Royal Marsden National Health Service (NHS) funding to the National Institute for Health Research (NIHR) Biomedical Research Centre at the same institutions. We thank A. Mirza, M. Richards and M. Liu for their assistance with NMR, mass spectrometry and HPLC. We thank S. Gaus (Merck Serono) for excellent technical assistance. We thank the team of Proteros Biostructures GmbH, Martinsried, Germany for the Reporter Displacement Assay and in particular E.V. Schneider and A. Lammens for the X-ray co-crystal structure of CCT251545 with CDK8–cyclin C. We thank G.J. Feng and B. Lloyd Lewis for assistance with organoid growth experiments and F. Rudge for development of the esiRNA protocol (BBSRC grant number BB/G016887/1). We thank N. Evans for editorial assistance.
T.D., P.A.C., W.C., D.W., O.A.P., M.O.R., A.M., R.S.S., M.S., E.F., R.T.P., A.H.B., M.V., G.B., S.G., F. Raynaud, P.W., S.A.E. and J.B. are current or former employees of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. C.E., P.C., F. Rohdich, D.M., K. Schiemann, K. Schneider, D.S., R.S., O.P., A.B. and D.W. are current or former employees of Merck Serono, which has a commercial interest in the development of WNT pathway inhibitors.
Supplementary Results, Supplementary Tables 1–12 and Supplementary Figures 1–18. (PDF 22997 kb)
Compound synthetic methods. (PDF 635 kb)
Identification of significantly enriched transcription factor binding sites. (XLSX 30 kb)
Identification of significantly enriched pathways. (XLSX 25 kb)
Analysis of gene expression regulated by transcription factors with links to CDK8 and/or CDK19. (XLSX 110 kb)
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Dale, T., Clarke, P., Esdar, C. et al. A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease. Nat Chem Biol 11, 973–980 (2015). https://doi.org/10.1038/nchembio.1952
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