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E-GEOD-67847 - Identification of a potent and selective chemical probe for exploring the role of Mediator complex-associated protein kinases CDK8 and CDK19 in human disease [beta-catenin organoids]

Status
Released on 26 October 2015, last updated on 8 December 2015
Organism
Mus musculus
Samples (42)
Array (1)
Protocols (7)
Description
There is an 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 WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Compound 1,2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site. A 3D culture model of murine intestinal-derived organoids expressing a transgenic doxycycline-inducible mutant beta-catenin expression following removal of doxycycline results in reduced WNT signalling and was compared to treatment for 24 hours with coumpound 2, 6 and 9. Compound 2 (n=3), compound 6 (n=4), compound 9 (n=3), doxycycline removal (n=4), doxycycline treatment (n=7). Samples were hybridized agianst mouse reference. Compounds 2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.
Experiment type
transcription profiling by array 
Contacts
Robert te Poele <robert.te-poele@icr.ac.uk>, A Lammens, Alexis de Haven Brandon, Andree Blaukat, Aurelie Mallinger, Christina Esdar, Daniel Schwarz, Dennis Waalboer, Dirk Wienke, Djordje Musil, E V Schneider, Elizabeth Fraser, Felix Rohdich, Florence Raynaud, Gary Box, Julian Blagg, Kai Schiemann, Ken Ewan, Klaus Schneider, Maria-Jesus Ortiz-Ruiz, Mark Stubbs, Melanie Valenti, Olajumoke Adeniji-Popoola, Oliver Poeschke, Paul A Clarke, Paul Czodrowski, Paul Workman, Rahul Samant, Richard Schneider, Robert H te Poele, Sharon Gowan, Suzanne A Eccles, Trevor Dale, Will Court
Citation
A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease. Dale T, Clarke PA, Esdar C, Waalboer D, Adeniji-Popoola O, Ortiz-Ruiz MJ, Mallinger A, Samant RS, Czodrowski P, Musil D, Schwarz D, Schneider K, Stubbs M, Ewan K, Fraser E, TePoele R, Court W, Box G, Valenti M, de Haven Brandon A, Gowan S, Rohdich F, Raynaud F, Schneider R, Poeschke O, Blaukat A, Workman P, Schiemann K, Eccles SA, Wienke D, Blagg J. , PMID:26502155
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