Development of inhibitors of FACT function as a cancer treatment approach
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FAcilitates Chromatin Transcription (FACT) is a protein complex consisting of two subunits, Structure Specific Recognition Protein 1 (SSRP1) and Suppressor of Ty 16(SPT16). FACT is involved in chromatin remodeling during transcription, replication and DNA repair. The complex is not expressed in most differentiated cells, while it is only expressed in embryo and stem and undifferentiated cells of the adult organism. Moreover, it has been shown, that FACT is upregulated in undifferentiated aggressive cancers of several types and that tumor cells' survival depends on FACT expression or function, in contrast to normal cells which are not sensitive to FACT's inhibition. Therefore, FACT is suggested as a marker and target of several types of aggressive cancer. In this study we developed a High Throughput Screening (HTS) compatible system for the identification of small molecules that are able to inhibit FACT function by disrupting SSRP1 and Spt16 complex. This will inactivate FACT and unstabilize SSRP1 and SPT16, which stability depends on the binding of the two subunits together with their mRNAs. We generated a Spt16 mutant lacking Nuclear Localization Signal (NLS). Spt16 mutant localizes in the cytoplasm, where it binds to SSRP1 and inhibits its transportation to the nucleus. The two subunits are fused with green and red fluorescent proteins. When they form a complex the overlapping of the fluorescents produce a yellow signal. If a small molecule is able to inhibit their binding, SSRP1 will drive to the nucleus, resulting in the production of green signal in the nucleus and red in the cytoplasm. The inhibition of binding of FACT in this system can be monitored by Förster (Fluorescence) resonance energy transfer (FRET) or by quantification of fluorescent imaging overlap.