Other Drug Targets

HCS can also be applied to the use of primary human cells, as shown in a report demonstrating the assay development and compound screening for the identification of activators of the Wnt/Frizzled (Fzd) pathway. Using primary human bone cells (preosteoblasts) as a model, activation of the Wnt/Fzd pathway was determined by immunofluorescent staining of translocation of the transcription factor beta-catenin. A library screen of 51 000 compounds yielded a hit rate of 1.4%, which was reduced to 0.6% after the removal of false-positive compounds, whether these were autofluorescent or cytotoxic. A major benefits of HCS was demonstrated by this approach, in that the assay requires only a limited cell number per well, thus eliminating the bottleneck in cell supply [104].

As HCS measures compound activities in a cellular context, it should be - to some extent - predictive with regard to compound activities in vivo. This was exemplified in a report where inhibitors of a MAPK phosphatase were found. Using a HCS assay based on phosphorylation of the kinase Erk, a compound with in-vivo activity was identified which was shown not to be active in a biochemical screening assay [105].

By using a translocation assay which measures the cytoplasm to nuclear localization of a GFP-tagged forkhead transcription factor, a signaling pathway inhibitor was identified which demonstrated anti-tumorous activity in a breast cancer xenograft model [101].

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