Fluorescence-Based Screen for Human DNA 5-Cytosine-methyltransferase 1

基于荧光的人类 DNA 5-胞嘧啶甲基转移酶 1 筛选

基本信息

批准号：
8010339
负责人：
JAMES T. STIVERS
金额：
$ 4.1万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-15 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8010339
关键词：
Adenosine Antineoplastic Agents Biological Assay Cell Culture Techniques CpG Islands CpG dinucleotide Custom Cytosine DNA DNA Methylation DNA Methyltransferase DNA Modification Methylases Development Drug Delivery Systems Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Epigenetic Process Event Fluorescence Future Gene Expression Gene Expression Regulation Gene Silencing Goals Growth Hand Human Hypermethylation In Vitro Intervention Laboratories Length Libraries Malignant Neoplasms Methionine Methylation Methyltransferase Mutation Pathway interactions Pharmaceutical Preparations Phenotype Promoter Regions Public Health Reading Screening procedure Site System Technology Testing Time Tumor Suppressor Genes base cancer therapy cost flexibility high throughput screening human DNA inhibitor/antagonist loss of function member novel novel strategies overexpression public health relevance restriction enzyme small molecule small molecule libraries therapeutic development

项目摘要

DESCRIPTION (provided by applicant): Human DNA (cytosine-5)-methyltransferases (C-5-MTase) are key regulators in the epigenetic control of gene expression through the S-adenosyl methionine(SAM)- dependent methylation of the DNA base cytosine. Hypermethylation of CpG islands occurs in nearly all cancers, often resulting in transcriptional silencing of tumor suppressor genes. Unlike mutations that promote cancer, such epigenetic events are reversible and amenable to pharmacological intervention, making human MTases attractive anticancer drug targets. We have fully developed the first robust, efficient and economical fluorescence assay suitable for in vitro high-throughput (HTP) screening of human DNA methyltransferase I (DNMT1). In this proposal we propose to use this assay to screen a custom library of MTase-directed compounds that will be constructed using our "substrate fragment tethering" (SFT) technology. Our future plan is to use this assay to rapidly evaluate various substrate fragment tethering strategies with the goal of discovering novel MTase inhibitors that will be useful for studying epigenetic regulation of gene expression or as leads for therapeutic development. PUBLIC HEALTH RELEVANCE: Hypermethylation of CpG dinucleotides in promoter regions of tumor suppressor genes by DNA cytosine-5-methyltransferase (MTase) enzymes is an important hallmark of human cancers. Such reversible methylation, known as epigenetic silencing, is a key pathway resulting in loss-of-function phenotypes that promote the growth of many cancers. Thus, inhibitors of MTase enzymes have the potential for reversing hypermethylation-induced gene silencing and are exciting new targets for epigenetic cancer therapies. This study describes new approaches to the discovery of such small molecule inhibitors.

描述（由申请人提供）：人类 DNA (胞嘧啶-5)-甲基转移酶 (C-5-MTase) 是通过 DNA 碱基胞嘧啶的 S-腺苷甲硫氨酸 (SAM) 依赖性甲基化对基因表达进行表观遗传控制的关键调节因子。 CpG 岛的高甲基化发生在几乎所有癌症中，通常导致肿瘤抑制基因的转录沉默。与促进癌症的突变不同，此类表观遗传事件是可逆的，并且易于药物干预，使人类 MTase 成为有吸引力的抗癌药物靶点。我们已经完全开发出第一个稳健、高效且经济的荧光检测方法，适用于人类 DNA 甲基转移酶 I (DNMT1) 的体外高通量 (HTP) 筛选。在本提案中，我们建议使用此测定法来筛选 MTase 定向化合物的定制文库，该文库将使用我们的“底物片段束缚”（SFT）技术构建。我们未来的计划是使用这种测定法快速评估各种底物片段束缚策略，目标是发现新型 MTase 抑制剂，这些抑制剂将有助于研究基因表达的表观遗传调控或作为治疗开发的先导。公共健康相关性：DNA 胞嘧啶 5-甲基转移酶 (MTase) 导致肿瘤抑制基因启动子区域的 CpG 二核苷酸高度甲基化，这是人类癌症的一个重要标志。这种可逆甲基化被称为表观遗传沉默，是导致功能丧失表型的关键途径，从而促进许多癌症的生长。因此，MTase 酶抑制剂具有逆转高甲基化诱导的基因沉默的潜力，并且是表观遗传癌症治疗的令人兴奋的新靶标。这项研究描述了发现此类小分子抑制剂的新方法。