Screen for Small Molecule Antagonists of MBD2

MBD2 小分子拮抗剂的筛选

基本信息

批准号：
8409737
负责人：
WILLIAM George NELSON
金额：
$ 4.05万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-31 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8409737
关键词：
Alleles Antineoplastic Agents Binding Biological Assay Breast Cancer Cell Cells Chemicals Chimeric Proteins Chromatin Collection CpG Islands Credentialing DNA DNA Binding DNA Binding Domain Development Doctor of Philosophy Drug Delivery Systems Epigenetic Process Evaluation Exhibits Family Family member Fluorescence Fluorescence Resonance Energy Transfer GSTP1 gene Gene Expression Gene Silencing Genes Genetic Genetic Transcription Goals Histone Deacetylase Inhibitor Human In Vitro Inhibitory Concentration 50 Intestines Ion Channel Lead Luciferases MBD2 protein MCF7 cell Malignant Neoplasms Malignant neoplasm of liver Malignant neoplasm of prostate Mediating Methyl-CpG-Binding Protein 2 Methylation Molecular Bank Monitor Mus Pathway interactions Pharmaceutical Preparations Play Production Recombinant DNA Recombinant Proteins Recombinants Reporter Repression Reverse Transcriptase Polymerase Chain Reaction Role Screening procedure Secondary to Staging Tertiary Protein Structure Testing Time Water adenoma base cancer cell gene function gene repression high throughput screening in vivo inhibitor/antagonist mRNA Expression malignant breast neoplasm novel prevent promoter research study small molecule

项目摘要

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.

描述（由申请人提供）：该提出的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。 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.发现其中一种能够在癌细胞中重新激活表观遗传沉默的基因，它直接在体外与MBD2与含5-MECPG的DNA的结合与IC50的IC50约为7M。可以直接拮抗MBD2抑制的小分子的发现增加了累积的遗传证据，将MBD2作为可行的表观遗传药物靶标。当时该项目的主要目标是使用时间分辨的荧光荧光荧光共振能量转移（TR-FRET）测定法进行基于目标的高通量屏幕（HTS），以监测MBD2片段的克隆重组的结合，使得与5-MECPG的DNA与5-MECPG的DNA进行含量，并在约翰群落中均可使用莫雷克斯的培养基，并在约翰一小部分中均可使用。当对使用384孔板进行优化并适应用于高通量筛选时，该测定的Z2值为0.59。希望通过这样的屏幕，我们可以找到对“铅”优化和结构研究有用的更有效和更多的水溶性分子。该基于目标HTS的“铅”化合物将进行次级筛选，以在癌细胞中对表观遗传脱毛的GSTP1重新激活，以及第三级分析，以选择MBD2相互作用的“铅”与5-MECPG-DNA相互作用的选择性。