Abstract PR02: Identification of a potent and selective chemical probe for exploring the role of CDK8/19 in cancer biology
Authors: Clarke Paul A. , Dale Trevor , Esdar Christina , Waalboer Dennis , Adeniji-Popoola Olajumoke , Ortiz-Ruiz Maria-Jesus , Mallinger Aurelie , Ewan Ken , te Poele Robert , Gowan Sharon , Workman Paul , Schiemann Kai , Eccles Suzanne A. , Wienke Dirk , Blagg Julian 
Published: May 2016

The discovery of chemical probes by testing libraries of small molecules against cellular pathway screens has re-emerged as a hit discovery strategy. We previously reported a series of 3,4,5-trisubstituted pyridines identified from a high-throughput cell-based reporter assay of WNT pathway signalling. We were able to optimise this series and identified CCT251545 as a chemical tool that potently inhibits readouts of WNT signalling pathway activity with evidence for in vivo activity. A series of cell-based assays activating WNT signalling at distinct loci identified the TCF locale as the likely target. CCT251545 was not a general inhibitor of the transcription machinery and did not affect expression of TCFs. Regulation of beta-catenin/TCF transcription involves recruitment or loss of DNA binding proteins, histone modification and also interaction with additional protein networks. Given the potential complexity of these multiple networked interactions, we employed an unbiased chemical proteomics strategy to identify molecular targets of CCT251545. Knowledge of the structure-activity-relationships of the series allowed us to identify derivatives that retained cellular potency and were suitable for linker coupling to generate an affinity matrix. SILAC-based quantitative mass spectrometry identified target proteins captured by incubation of the immobilised compound with lysates from cells grown in media with different forms of isotopically-labeled amino acids. Competition experiments with unconjugated analogues allowed us to identify affinities of proteins bound to the immobilised probe. These experiments identified Mediator complex-associated protein kinases CDK8 and CDK19 as targets of the 3,4,5-trisubstituted pyridine series. We show that CCT251545, is a selective and potent ATP competitive chemical probe for these two kinases, with &gt;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-like CDK8/19 ligands, CCT251545 displays potent cell-based activity. We demonstrate that CCT251545 not only alters WNT-pathway regulated gene expression, but also other CDK8/19 targets including 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 show in vivo activity of CCT251545 in WNT-dependent tumours. The potential role for CDK8, and by implication CDK19, as an oncoprotein further highlights the need for an active and specific probe compound that can be used as a complementary reagent to RNAi tools. Here we demonstrate that the small molecule CCT251545 fulfils this requirement as a potent, selective, cell-active chemical probe that can be used with confidence to explore the consequences of CDK8/19 kinase function in cellular and in vivo animal models.

Citation Format: Paul A. Clarke, Trevor Dale, Christina Esdar, Dennis Waalboer, Olajumoke Adeniji-Popoola, Maria-Jesus Ortiz-Ruiz, Aurelie Mallinger, Ken Ewan, Robert te Poele, Sharon Gowan, Paul Workman, Kai Schiemann, Suzanne A. Eccles, Dirk Wienke, Julian Blagg. Identification of a potent and selective chemical probe for exploring the role of CDK8/19 in cancer biology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR02.