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

Abstract

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.

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

“…Compound 3 binds in a twisted conformation, as previously described for 1, with the indazole substituent at C5 of the pyridine ring forming a pi-cation interaction with Arg356 (Figure 1B) (Mallinger et al, 2015, 2016a). Compound 4 forms similar interactions with the hinge region and with the catalytic Lys52 to those observed for compound 3, and its p -chlorophenyl substituent occupies the same region as the indazole subsituent of compound 3; however, the scaffold architecture of the two compounds is entirely different (Figure 1B).…”

“…All four compounds had single digit nanomolar binding affinities for CDK8 and 19, and were very highly selective with little evidence for off-target activity in extended protein kinase panels (Figure 1—source data 1). Our compounds also potently inhibited inducible (7dF3; Ewan et al, 2010) or basal (LS174T; Dale et al, 2015; Mallinger et al, 2015) WNT-pathway luciferase-reporter expression together with STAT1 SER727 phosphorylation ‑ a target-engagement biomarker ‑ at low nanomolar concentrations (Figure 1A and Figure 1—source data 1) (Bancerek et al, 2013; Dale et al, 2015).

10.7554/eLife.20722.003Figure 1.Optimised compounds for exploring CDK8 and CDK19 function.( A ) Chemical structure and activity of compounds 2, 3 and 4 (n > 2, mean ± s.d.).

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“…In addition to two tool compounds, 1 (CCT251545; Mallinger et al, 2015) and 2 (compound 42;Mallinger et al, 2016a), that fulfill all of the criteria set out for chemical probes (Frye, 2010), the lead compounds from each of the chemical series, 3 (CCT251921; Mallinger et al, 2016a) and 4 (MSC2530818; Czodrowski et al, 2016) had optimal pharmacological and pharmaceutical properties that made them suitable for further progression to preclinical studies (Figure 1A and Figure 1—source data 1). All four compounds had single digit nanomolar binding affinities for CDK8 and 19, and were very highly selective with little evidence for off-target activity in extended protein kinase panels (Figure 1—source data 1).…”

CDK8 Kinase Activity Promotes Glycolysis

“…Despite the obvious importance of Mediator in gene expression control, little is known about shared and unique functions of the CDK-module paralogs. Recently, a number of dual inhibitors of CDK8 and CDK19 have been developed, including Cortistatin A, Senexin A, CCT251921 and MSC2530818 (Cee et al, 2009; Clarke et al, 2016; Czodrowski et al, 2016; Dale et al, 2015; Koehler et al, 2016; Mallinger et al, 2015; Mallinger et al, 2016a; Mallinger et al, 2016b; Porter et al, 2012; Schiemann et al, 2016). While many of these compounds appear to have efficacy against cancer cell lines in vitro , it remains unclear whether the effects are due to inhibition of CDK8, CDK19 or both.…”

The pathomorphologist’s role in the era of personalised therapy regarding the case of colorectal tumours

“…The loss of function of the APC protein impairs b-catenin phosphorylation, and this substance, accumulated in the cell nucleus, potentiates the expression of the genes stimulating the proliferation processes [41]. Currently there are attempts made at inhibiting this pathway with the use of the available therapeutic agents, yet no efficient inhibitors have been found.…”