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

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s -腺苷蛋氨酸（SAM）依赖的DNA碱基胞嘧啶甲基化来调控基因表达的表观遗传调控的关键调控因子。CpG岛的高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，并且可以进行药物干预，这使得人类MTases成为抗癌药物的重要靶点。我们已经完全开发了第一个适用于体外高通量（HTP）筛选人类DNA甲基转移酶I （DNMT1）的强大，高效和经济的荧光检测方法。在本提案中，我们建议使用该方法筛选mase导向化合物的定制文库，该文库将使用我们的“底物片段捆绑”（SFT）技术构建。我们未来的计划是使用该方法快速评估各种底物片段系缚策略，目的是发现新的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控或作为治疗开发的线索。

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.