DeNovo DNA Methyltransferases as Anticancer Drug Targets

描述：（申请人提供） 我们的总体目标是重新验证DNA胞嘧啶-C5甲基转移酶 （MTases）作为抗癌药物开发的适当分子靶标， 提出并测试筛选特异性抑制配体的新方法， 这些酶。有很多证据表明DNA的改变 甲基化与致癌作用有关。全基因组减少5 mC 内容物在肿瘤发展的早期发生。然而，具体地区 “高甲基化”发生并与肿瘤的表观遗传沉默相关 抑制剂或参与DNA修复的酶。这导致了一个假设 阻断或逆转超甲基化将导致基因的重新激活， 对抑制进展、恢复正常表型或生长至关重要 调节或诱导细胞凋亡。掺入5-氮杂胞苷或5-氮杂-2 '- 脱氧胞苷（5azadC，Decitibine）进入DNA导致DNA MTase抑制， 细胞分裂过程中甲基化的被动丧失和 培养的人类癌细胞中的高甲基化基因。这两种药物都是 在各种临床试验中用作潜在的抗癌药物 治疗学然而，它们具有细胞毒性和遗传毒性。我们已经 开发并优化了无遗传毒性的寡脱氧核苷酸（ODN） DNMT 1的抑制剂，DNMT 1是体细胞中最丰富的DNA MTase形式。 DNMT 1在DNA合成过程中维持由从头MTase建立的甲基化模式。 发展它在体外具有有限的从头活性， 在体内无活性。因此，最有可能的是，最近的一些成员， 发现DNMT 3家族的DNA MTases，可以催化从头甲基化 在体内，负责进行甲基化的建立 模式和关键沉默的基因在肿瘤发生。的假设 我们将测试的是，抑制从头DNA MTases是必要的，不仅 用于有效降低甲基化和激活沉默基因， 也用于防止“重新再甲基化”。 我们的具体目标是：1）验证DNMT 3a/3b或尚未确定的类似物 a）设计和优化小ODN 对DNA MTases 3a、3b具有特异性且对DNMT 1无活性的抑制剂。他们 将含有5-azaC、5 FdC或脱碱基位点作为靶C的替代 在完全未甲基化的识别位点。B）确定/比较 DNMT 3a、3b抑制剂（单独和与5azadC或ODN组合）的作用 DNMT 1抑制剂）对培养的人类癌细胞中DNA甲基化的影响。这 将通过全基因组CpG岛筛选和亚硫酸氢盐 已知通过甲基化失活的基因中的CpG岛的测序。c）、 确定/比较DNMT 3a，3b抑制剂（单独和与药物组合）的作用。 与DNMT的5azadC或ODN抑制剂组合1）对基因表达的影响 使用表达阵列的已知肿瘤抑制因子、细胞周期 细胞凋亡的调节剂和介质。2)使用新型SDS-PAGE凝胶分析， 测量DNMT的抑制剂结合亲和力，以预测 DNMT 1、3a和3b的多种新的抑制性靶点。

DESCRIPTION: (provided by applicant) Our overall goal is to validate de novo DNA cytosine-C5 methyltransferases (MTases) as appropriate molecular targets for anti-cancer drug development and to propose and test new methods for screening specific inhibitory ligands of these enzymes. There is much evidence suggesting that alterations in DNA methylation are associated with carcinogenesis. Genome-wide decreases in 5mC content occur early in tumor development. However, specific regions of "hypermethylation" occur and are associated with epigenetic silencing of tumor suppressors or enzymes involved in DNA repair. This leads to the assumption that blocking or reversing hypermethylation will lead to reactivation of genes critical to inhibiting progression, restoring a normal phenotype or growth regulation or inducing apoptosis. Incorporation of 5-azacytidine or 5-aza-2'- deoxycytidine (5azadC, Decitibine) into DNA leads to DNA MTase inhibition with passive loss of methylation during cell division and activation of hypermethylated genes in cultured human cancer cells. Both drugs have been utilized in a variety of clinical trials as potential anti-cancer therapeutics. However, they are both cytotoxic and genotoxic. We have already developed and optimized non-genotoxic oligodeoxyribonucleotide (ODN) inhibitors of DNMT1, the most abundant form of DNA MTase in somatic cells. DNMT1 maintains methylation patterns established by de novo MTases during development. It has limited de novo activity in vitro and may be completely inactive in vivo. Thus, it is most probable that some member of the recently discovered DNMT3 family of DNA MTases, which can catalyze de novo methylation in vivo, is responsible for carrying out the establishment of methylation patterns and critical silencing of genes during tumorigenesis. The hypothesis we will test is that inhibition of de novo DNA MTases is necessary, not only for efficient reduction of methylation and activation of silenced genes but also for prevention of "de novo remethylation". Our specfic aims are: 1) To validate DNMT3a/3b or similar as yet unidentified de novo DNA MTases as targets by: a) Designing and optimizing small ODN inhibitors specific for DNA MTases 3a, 3b and inactive against DNMT1. They will contain 5-azaC, 5FdC, or abasic sites as replacements for the target C's in completely unmethylated recognition sites. b) Determining/comparing the effect of DNMT 3a, 3b inhibitors (alone and in combination with 5azadC or ODN inhibitors for DNMT1) on DNA methylation in cultured human cancer cells. This will be accomplished with a genome-wide CpG island screen and by bisulfite sequencing of CpG islands in genes known to be inactivated by methylation. c) Determining/comparing the effect of DNMT 3a, 3b inhibitors (alone and in combination with 5azadC or ODN inhibitors of DNMT1) on gene expression profiles using expression arrays for know tumor suppressors, cell cycle regulators and mediators of apoptosis. 2) Use a novel SDS-PAGE gel assay that measures inhibitor binding affinity for DNMTs to predict the potency of a variety of new inhibitory targets for DNMTs 1, 3a and 3b.