The focus of this proposed Project is the discovery and characterization of small molecule inhibitors of epigenetic gene silencing mediated by the 5-meCpG binding domain (MBD) family member MBD2. Using novel cell-based screens to detect small molecules capable of selective activation of a GSTP1 promoter/luciferase reporter construct carrying extensive CpG island methylation, a configuration known to cause epigenetic silencing in prostate, breast, and liver cancers, we identified 13 new compounds from the ChemBridge PHARMACophore collection (from >20,000 compounds screened) for further evaluation. One of the compounds, which was able to reactivate epigenetically silenced genes in cancer cells, was found to directly interfere with the binding of MBD2 to 5-meCpG-containing DNA in vitro, with an IC50 of ~7 M., and to release MBD2 from chromatin in cancer cells in vivo. The finding of a small molecule that can directly antagonize MBD2 repression adds to an accumulating body of genetic evidence credentialing MBD2 as a viable epigenetic drug target. The major goal of this current Project then is to conduct a target-based high-throughput screen (HTS), using a time resolved fluorescence-fluorescence resonance energy transfer (TR-FRET) assay to monitor the binding of a cloned recombinant of MBD2 fragment to 5-meCpG-containing DNA, with a diverse collection of compounds available at the Molecular Libraries Probe Production Center at Johns Hopkins. When optimized and adapted for use in high-throughput screening using 384-well plates, the assay exhibited a Z2-value of 0.59. Hopefully, with such a screen, we can find more potent and more water-soluble molecules useful for "lead" optimization and structural studies. "Lead" compounds from this target-based HTS will be subjected to secondary screening, for reactivation of epigenetically-silenced GSTP1 in cancer cells, and tertiary analyses, for selectivity of the "leads" for MBD2 interactions with 5-meCpG- DNA.
This application focuses on the discovery of anti-cancer drugs which act by a new mechanism. As many as 500 or more genes are epigenetically silenced in most human cancers. The new drugs to be identified in a proposed screen of a hundred thousand or more chemical compounds will be able to reactivate the silenced genes in cancer cells, restoring gene function.