DeNovo DNA Methyltransferases as Anticancer Drug Targets

DeNovo DNA 甲基转移酶作为抗癌药物靶点

• 批准号: 6333986
• 负责人: JUDITH K CHRISTMAN
• 金额: $ 14.75万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6333986
• 关键词: CpG islands; DNA methylation; SDS polyacrylamide gel electrophoresis; Sf9 cell line; apoptosis; azacitidine; breast neoplasms; colon neoplasms; deoxycytidine; drug screening /evaluation; enzyme activity; enzyme inhibitors; gel electrophoresis; methyltransferase; neoplasm /cancer pharmacology; oligonucleotides; prostate neoplasms; tumor suppressor genes

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.

描述：（由申请人提供） 我们的总体目标是从头验证 DNA 胞嘧啶-C5 甲基转移酶 （MTase）作为抗癌药物开发的适当分子靶标 提出并测试筛选特定抑制配体的新方法 这些酶。有大量证据表明DNA的改变 甲基化与癌变有关。全基因组降低 5mC 内容发生在肿瘤发展的早期。然而，特定地区 “高甲基化”的发生与肿瘤的表观遗传沉默有关 参与 DNA 修复的抑制剂或酶。这导致了假设 阻断或逆转高甲基化将导致基因重新激活 对于抑制进展、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-aza-2'- 脱氧胞苷（5azadC、地西他滨）进入 DNA 会导致 DNA MTase 抑制 细胞分裂和激活过程中甲基化的被动丧失 培养的人类癌细胞中的高甲基化基因。两种药物均已 在各种临床试验中用作潜在的抗癌药物 疗法。然而，它们都具有细胞毒性和基因毒性。我们已经 开发和优化的非基因毒性寡脱氧核糖核苷酸（ODN） DNMT1 抑制剂，DNMT1 是体细胞中最丰富的 DNA MTase 形式。 DNMT1 维持从头 MTase 建立的甲基化模式 发展。它在体外的从头活性有限，可能完全 在体内无活性。因此，最近的一些成员很可能 发现了 DNA MTase 的 DNMT3 家族，可以催化从头甲基化 在体内，负责进行甲基化的建立 肿瘤发生过程中基因的模式和关键沉默。假设 我们将测试的是，抑制从头 DNA 转移酶是必要的，不仅 用于有效减少甲基化和激活沉默基因，但是 也用于预防“从头再甲基化”。 我们的具体目标是： 1) 验证 DNMT3a/3b 或尚未确定的类似目标 de novo DNA MTases 作为靶标： a) 设计和优化小型 ODN 对 DNA MTase 3a、3b 具有特异性且对 DNMT1 无活性的抑制剂。他们 将包含 5-azaC、5FdC 或脱碱基位点作为目标 C 的替代品 完全未甲基化的识别位点。 b) 确定/比较 DNMT 3a、3b 抑制剂（单独或与 5azadC 或 ODN 联合使用）的作用 DNMT1 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来完成 对已知因甲基化而失活的基因中的 CpG 岛进行测序。 c) 确定/比较 DNMT 3a、3b 抑制剂的效果（单独使用和单独使用） 与 5azadC 或 DNMT1 的 ODN 抑制剂组合）对基因表达的影响 使用表达阵列分析已知肿瘤抑制因子、细胞周期 细胞凋亡的调节剂和介质。 2) 使用新型 SDS-PAGE 凝胶测定法 测量抑制剂与 DNMT 的结合亲和力来预测 DNMT 的效力 DNMT 1、3a 和 3b 的多种新抑制靶点。