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DNA（胞质5） - 甲基转移酶（C-5-mTase）是通过S-腺苷蛋氨酸（SAM） - 依赖性甲基化DNA碱基胞质的依赖性甲基化基因表达的表观遗传控制的关键调节剂。 CpG岛的高甲基化几乎在所有癌症中都发生，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，这种表观遗传事件是可逆的，并且可以接受药理学干预，从而使人类MTases有吸引力的抗癌药物靶标。我们已经完全开发了适用于人类DNA甲基转移酶I（DNMT1）的第一个适合体外高通量（HTP）筛选的强大，有效和经济的荧光测定。在此提案中，我们建议使用此测定法筛选以MTase定向化合物的自定义库，该化合物将使用我们的“底物碎片束缚”（SFT）技术构建。我们未来的计划是使用该测定法快速评估各种底物碎片束缚策略，目的是发现新型的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节或作为治疗性发育的铅。公共卫生相关性：通过DNA胞质-5-甲基转移酶（MTase）酶在肿瘤抑制基因的启动子区域中CPG二核苷酸的高甲基化是人类癌症的重要标志。这种可逆的甲基化（称为表观遗传沉默）是一个关键途径，导致功能障碍表型促进许多癌症的生长。因此，MTase酶的抑制剂具有逆转高甲基化引起的基因沉默的潜力，并且是表观遗传癌症疗法的令人兴奋的新靶标。这项研究描述了发现这种小分子抑制剂的新方法。