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

• 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
• 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

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（由申请方提供）：人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）依赖性甲基化进行基因表达表观遗传控制的关键调节因子。CpG岛的超甲基化发生在几乎所有的癌症中，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，这种表观遗传事件是可逆的，并且适合于药理学干预，使得人类MT酶成为有吸引力的抗癌药物靶标。我们已经完全开发了第一个强大的，有效的和经济的荧光检测适用于体外高通量（HTP）筛选人DNA甲基转移酶I（DNMT 1）。在这个提议中，我们建议使用这种检测来筛选MTase指导的化合物的定制库，该库将使用我们的“底物片段连接”（SFT）技术来构建。我们未来的计划是使用这种方法来快速评估各种底物片段的拴系策略，目的是发现新的MTase抑制剂，这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线索。 公共卫生相关性：DNA胞嘧啶-5-甲基转移酶（MTase）引起的抑癌基因启动子区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，是导致功能丧失表型的关键途径，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点。 这项研究描述了发现这种小分子抑制剂的新方法。