Aurélie Mallinger

A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease

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 WNT-pathway inhibitor 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 CDK8/19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues alter WNT-pathway regulated gene expression and other on-target effects of modulating CDK8/19 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 demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

Discovery of Potent, Orally Bioavailable, Small-Molecule Inhibitors of WNT Signaling from a Cell-Based Pathway Screen

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine–piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.

Inhibitors of cyclin-dependent kinases as cancer therapeutics

&NA; Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin‐dependent kinases (CDKs). This attention initially stemmed from observations that different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non‐selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform‐selective inhibitors with accompanying biomarkers has re‐ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER +/HER2‐ advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies.

Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19

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.

Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

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. DOI: http://dx.doi.org/10.7554/eLife.20722.001

Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore

Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration.

2,8-Disubstituted-1,6-Naphthyridines and 4,6-Disubstituted-Isoquinolines with Potent, Selective Affinity for CDK8/19.

We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1SER727 phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing.

Evaluation of APOBEC3B recognition motifs by NMR reveals preferred substrates

APOBEC3B (A3B) deamination activity on ssDNA is considered a contributing factor to tumor heterogeneity and drug resistance in a number of human cancers. Despite its clinical impact, little is known about A3B ssDNA substrate preference. We have used nuclear magnetic resonance to monitor the catalytic turnover of A3B substrates in real-time. This study reports preferred nucleotide sequences for A3B substrates, including optimized 4-mer oligonucleotides, and reveals a breadth of substrate recognition that includes DNA sequences known to be mutated in drug-resistant cancer clones. Our results are consistent with available clinical and structural data and may inform the design of substrate-based A3B inhibitors.

Abstract 3869: Phosphoproteomic-based identification of CDK8/CDK19 substrates in colorectal cancer

Introduction CDK8 is an oncogenic cyclin-dependent kinase that exists as part of the kinase module within the Mediator complex. This complex interacts with the transcription machinery to regulate transcription; signal transduction pathways, including the WNT pathway; and biological processes, such as cell cycle progression. Recently, we identified a series of 3,4,5-trisubstituted pyridines as inhibitors of CDK8 and, its paralogue, CDK19 in colorectal cancer (CRC). Until now, there have been few validated substrates of CDK8/19. Here, we describe a motif-based phospho-proteomic approach, utilizing our 3,4,5-trisubstituted pyridine inhibitors that we have used to identify substrates of CDK8/19. These substrates could represent useful biomarkers for future drug discovery research. Experimental Outline We used a COLO205 cell line (COLO205 C4) carrying a TCF/LEF reporter construct responsive to CDK8/19 inhibition and CCT251545, a compound we have previously shown to be a potent and selective inhibitor of CDK8/19. Cells were treated with 350 nM CCT251545 (10 x EC50) for 2 or 6 hours. Proteins were then extracted from treated and control cells, trypsin-digested and immunoprecipitated for phospho-peptide enrichment using proline-directed motifs: PXS*P, S*PXR/K, PXS*PXR/K + T*PE + ST*P + K/HS*P. Potential substrates were identified by LC-MS/MS and validated by immunoprecipitation and western blotting. Substrates were validated in another CRC cell line, SW620, which harbors CRISPR knockouts for CDK8/19. Summary of Results LC-MS/MS analyses of COLO205 C4 cell extracts revealed a number of potential CDK8/19 substrates, including some Mediator complex subunits such as MED13, transcriptional coactivators such as HCFC1, and transcription factors such as STAT1. Phosphorylation of STAT1SER727 was the top ranked hit and follow-up studies, in COLO205 C4 cells and xenografts, confirmed repression of STAT1SER727 phosphorylation in the presence of CCT251545. An inactive analogue, CCT251099, and other kinase inhibitors (flavopiridol, KN-93, PD 0325901 and SB 202190) did not block STAT1SER727 phosphorylation. Repression of STAT1SER727 phosphorylation upon treatment with CCT251545 was also observed in SW620 and LS174T CRC cells and xenografts. Conclusion A motif-driven, mass spectroscopy-based phospho-proteomic study identified candidate substrates of CDK8/19. Phosphorylation of STAT1SER727 was validated as a useful marker of target engagement in CRC cell lines both in vitro and in vivo. Citation Format: Olajumoke O. Popoola, Rahul Samant, Maria-Jesus Ortiz-Ruiz, Aurelie Mallinger, Will Court, Steve Hobbs, Robert Te-Poele, Mark Stubbs, Rosemary Burke, Christina Esdar, Kai Schiemann, Dirk Wienke, Sue Eccles, Julian Blagg, Paul Workman, Paul Clarke. Phosphoproteomic-based identification of CDK8/CDK19 substrates in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3869.

Abstract 3025: Discovery of preclinical development candidate inhibitors of the mediator complex-associated kinases CDK8 and CDK19 and evaluation of their therapeutic potential

Background The Mediator complex-associated kinases CDK8 and CDK19 are cyclin C-dependent enzymes that, with MED12 and MED13, form the kinase module of the Mediator complex. CDK8 expression correlates with activation of β-catenin in colon and gastric cancers and has also been associated with increased mortality in colorectal, breast and ovarian cancers. CDK8 is located in a region of chromosome 13 known to undergo copy number gain in ∼60% of colorectal cancers and inducible shRNA-mediated knockdown of CDK8 protein reduces the growth of colorectal cancer human tumor xenograft animal models harboring CDK8 gene amplification. Results Here we report the discovery and evaluation of CCT251545, a potent, selective and orally bioavailable small molecule chemical probe for CDK8 and CDK19 that we identified from a cell-based WNT pathway screen [1]. We also report a structure-based design approach to the discovery of CCT251921, a potent, selective and orally bioavailable inhibitor of CDK8, with equipotent affinity for CDK19, that has optimised pharmacokinetic and pharmaceutical properties suitable for preclinical development. Furthermore, we describe the discovery of MSC2530818, a structurally differentiated back-up candidate with equivalent pharmacological profile to CCT251921, from a high throughput screen versus CDK8 and subsequent structure-based design. Taking advantage of these two structurally distinct and highly selective dual CDK8/19 modulators we were able to reliably define on-target effects of targeting both CDK8 and CDK19 in the cellular context and in in vivo animal models. We describe gene expression profiles resulting from dual inhibition of CDK8 and CDK19 to demonstrate robust modulation of WNT signalling and additional pathways, including stress and immune response, consistent with the multiple contexts in which Mediator complex is known to regulate gene transcription. We show that both CCT251921 and MSC2530818 exhibit potent cell-based and in vivo inhibition of STAT1SER727 phosphorylation, a target engagement biomarker of CDK8 inhibition, and further demonstrate in vivo antiproliferative activity in human tumour xenograft animal models of colorectal cancer and acute myeloid leukaemia at exposures where pharmacodynamics biomarker modulation is evident. Recent observations suggest CDK8 as a novel anticancer therapeutic target; here we will disclose, for the first time, comprehensive preclinical efficacy, toleration and safety findings for both CCT251921 and MSC2530818 which will inform on the potential for dual CDK8/19 inhibition in the clinical setting. References 1. Dale, T. et. al. Identification of a potent and selective chemical probe for exploring the role of Mediator complex-associated protein kinases CDK8 and CDK19 in human disease. 2015, Nat. Chem. Biol., 11, 973-980. Citation Format: Paul Clarke, Christina Esdar, Aurelie Mallinger, Kai Schiemann, Dennis Waalboer, Simon Crumpler, Christian Rink, Frank Stieber, Michel Calderini, Olajumoke Adeniji-Popoola, Maria-Jesus Ortiz-Ruiz, Rahul S. Samant, Paul Czodrowski, Djordje Musil, Daniel Schwarz, Klaus Schneider, Michael Busch, Mark Stubbs, Rosemary Burke, Robert TePoele, Sharon Gowan, Felix Rohdich, Florence Raynaud, Richard Schneider, Oliver Poeschke, Andree Blaukat, Klaus Urbahns, Paul Workman, Wolfgang Kaufmann, Stephanie Simon, Suzanne A. Eccles, Trevor Dale, Dirk Wienke, Julian Blagg. Discovery of preclinical development candidate inhibitors of the mediator complex-associated kinases CDK8 and CDK19 and evaluation of their therapeutic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3025.