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

描述(申请人提供)：人类脱氧核糖核酸(胞嘧啶-5)-甲基转移酶(C-5-MTase)是通过依赖S腺苷甲基化的脱氧核糖核酸碱基胞嘧啶甲基化来控制基因表达的关键调控因子。几乎所有的癌症都会发生CpG岛的高甲基化，通常会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，可以进行药物干预，使人类MTase成为具有吸引力的抗癌药物靶点。我们已经完全建立了第一个可靠、高效和经济的适合于体外高通量(HTP)筛选人DNA甲基转移酶I(DNMT1)的荧光分析方法。在这项提案中，我们建议使用这一测试来筛选一个定制的MTase导向化合物文库，该文库将使用我们的“底物片段拴系”(SFT)技术构建。我们未来的计划是使用这种测试来快速评估各种底物片段拴系策略，目标是发现新的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节或作为治疗开发的先导。 公共卫生相关性：肿瘤抑制基因启动子区的CpG二核苷酸被DNA胞嘧啶-5-甲基转移酶(MTase)高甲基化是人类癌症的一个重要标志。这种可逆的甲基化，称为表观遗传沉默，是导致功能丧失的表型的关键途径，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，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。

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.