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Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors., Competing Interests: PAC: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. M-JO-R: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. RT: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. OA-P: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. GB: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. WC: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. SC: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. SEB: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. CE: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. SG: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. ADHB: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. PH: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. SMH: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. WK: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. AM: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. FRa: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. TR: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. FRo: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. KS: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. SS: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. RS: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. MV: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. SW: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. JB: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. AB: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. SAE: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. SH: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. KU: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. PW: Current or former employee of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. DW: Current or former employee of Merck KGaA (Darmstadt, Germany), which has a commercial interest in the development of WNT pathway inhibitors. The other authors declare that no competing interests exist.

The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer.

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 STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

Purpose: To investigate pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R-roscovitine; seliciclib) in the HCT116 human colon carcinoma model., Experimental Design: The in vitro activity of the agents was determined in a human tumor panel using the sulforhodamine B assay. The concentration and time dependence was established in HCT116 cells. Molecular biomarkers, including RB phosphorylation and cyclin expression, were assessed by Western blotting. Pharmacokinetic properties were characterized in mice following analysis by liquid chromatography-tandem mass spectrometry. Based on these studies, a dosing regimen was developed for CYC202 that allowed therapeutic exposures in the HCT116 tumor xenograft., Results: The antitumor potency of the agents in vitro was in the order olomoucine (IC50, 56 micromol/L) < bohemine (IC50, 27 micromol/L) < CYC202 (IC50, 15 micromol/L), corresponding to their activities as cyclin-dependent kinase inhibitors. Antitumor activity increased with exposure time up to 16 hours. The agents caused inhibition of RB and RNA polymerase II phosphorylation and depletion of cyclins. They exhibited relatively rapid clearance following administration to mice. CYC202 displayed the slowest clearance from plasma and the highest tumor uptake, with oral bioavailability of 86%. Oral dosing of CYC202 gave active concentrations in the tumor, modulation of pharmacodynamic markers, and inhibition of tumor growth., Conclusions: CYC202 showed therapeutic activity on human cancer cell lines in vitro and on xenografts. Pharmacodynamic markers are altered in vitro and in vivo, consistent with the inhibition of cyclin-dependent kinases. Such markers may be potentially useful in the clinical development of CYC202 and other cyclin-dependent kinase inhibitors.