Screen for Small Molecule Antagonists of MBD2

描述(申请人提供)：本项目的重点是发现和表征由5-meCpG结合结构域(MBD)家族成员Mbd2介导的表观遗传基因沉默的小分子抑制剂。使用新的基于细胞的筛选来检测能够选择性激活GSTP1启动子/荧光素酶报告结构的小分子，该结构携带广泛的CpG岛甲基化，这种配置已知会导致前列腺癌、乳腺癌和肝癌的表观遗传沉默，我们从ChemBridge药效团集合中鉴定了13种新化合物(从筛选的20,000种化合物中)进行进一步评估。其中一种化合物能够在体外直接干扰Mbd2与含有5-meCpG的DNA的结合，其IC50为~7M，并在体内从癌细胞的染色质中释放Mbd2，该化合物能够重新激活癌细胞中表观遗传沉默的基因。一种可以直接对抗Mbd2抑制的小分子的发现，增加了越来越多的遗传证据，证明Mbd2是一个可行的表观遗传药物靶点。本项目的主要目标是进行基于目标的高通量筛选(HTS)，使用时间分辨荧光-荧光共振能量转移(TR-FRET)分析来监测Mbd2片段的克隆重组与含有5-meCpG的DNA的结合，以及约翰霍普金斯大学分子图书馆探针生产中心提供的各种化合物。当使用384孔板进行优化和调整以用于高通量筛选时，该方法的Z2值为0.59。希望有了这样的筛选，我们可以找到更有效、更水溶性的分子，对“铅”优化和结构研究有用。来自这种靶标HTS的“先导”化合物将接受二次筛选，以重新激活癌细胞中表观遗传沉默的GSTP1，并进行三次分析，以确定Mbd2与5-meCpG-DNA相互作用的“先导”的选择性。

DESCRIPTION (provided by applicant): 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.