Screening for inhibitors of the Wnt signalling pathway

The Wnt pathway is a highly conserved signalling pathway which controls many cellular processes in normal development and disease. In many tumors, genetic or epigenetic alterations in key components of the pathway have been described. In addition, new roles of Wnt signalling in neurodegenerative diseases, diabetes and osteoporosis are emerging. Thus, the Wnt signalling pathway is an important therapeutic target.

In the absence of a Wnt signal, β-catenin, the central player of the pathway, is continuously phosphorylated by glycogen synthase kinase 3 beta (GSK3 beta) in the so-called destruction complex (see Fig. 1). The destruction complex consists of at least the adenomatous polyposis coli protein, axin and GSK3 beta. Phosphorylated β-catenin is degraded by the proteasome and Wnt target genes are not expressed because ß-catenin does not interact with its targets, the TCF/LEF transcription factors, which function as transcriptional repressors by interacting with HDACs. In a Wnt activated cell, the destruction complex is inactivated, β-catenin accumulates in the cytoplasm and translocates to the nucleus. Nuclear β-catenin associates with transcription factors of the TCF/LEF family and recruits transcriptional co-activators and chromatin remodeling complexes which in concert drive the expression of target genes. Wnt target genes include CCND1, MMP7, VEGF, MYC and also genes involved in feed-back regulation like TCF7L2 or LEF1. New chemotypes for Wnt inhibition are needed as existing leads either lack specificity or act at a distance from the true driver of pathway activation.

Wnt activity was analysed by means of a Wnt responsive luciferase reporter gene assay (TOPflash) using a human embryonic kidney cell line stably overexpressing the frizzled receptor. Cells were activated by addition of Wnt conditioned medium which has been harvested from L-Cells overexpressing the Wnt protein. After compound treatment for six hours, the level of the luciferase reporter was determined using OneGlo reagent (Promega). The Wnt screening pipeline also includes counterscreens for cell viability and transcriptional/ translational inhibition of the luciferase protein (Fig. 25). Cell viability was measured with a commercial reagent for the quantification of cellular ATP. For the luciferase assay, a HEK293 cell line with constitutive expression of the luciferase protein was used.

Fig. 1: Wnt signalling pathway.