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Screen for Small Molecule Antagonists of MBD2

MBD2 小分子拮抗剂的筛选

基本信息

批准号：
8519572
负责人：
WILLIAM George NELSON
金额：
$ 3.93万
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/8519572
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 Targeting 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 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 screening 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）：本项目的重点是发现和表征由5-meCpG结合域（MBD）家族成员MBD2介导的表观遗传基因沉默的小分子抑制剂。利用新的基于细胞的筛选技术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，这些小分子携带广泛的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药效团收集（从筛选的20万种化合物中）中鉴定出13种新化合物，用于进一步评估。其中一种化合物能够重新激活癌细胞中表观遗传沉默的基因，在体外直接干扰MBD2与含有5- mecpg的DNA的结合，IC50约为7 M，并在体内从癌细胞的染色质中释放MBD2。发现一种可以直接拮抗MBD2抑制的小分子增加了越来越多的遗传证据，证明MBD2是一种可行的表观遗传药物靶点。当前项目的主要目标是进行基于靶标的高通量筛选（HTS），使用时间分辨荧光-荧光共振能量转移（TR-FRET）试验来监测克隆的重组MBD2片段与含有5- mecpg的DNA的结合，并在约翰霍普金斯大学分子图书馆探针生产中心提供多种化合物。经过优化并适应于384孔板高通量筛选后，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和更水溶性的分子，用于“铅”优化和结构研究。从这种基于靶标的HTS中获得的“先导”化合物将进行二次筛选，以重新激活癌细胞中表观遗传沉默的GSTP1，并进行第三次分析，以确定MBD2与5-meCpG- DNA相互作用的“先导”化合物的选择性。