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DeNovo DNA Methyltransferases as Anticancer Drug Targets

DeNovo DNA 甲基转移酶作为抗癌药物靶点

基本信息

批准号：
6515085
负责人：
JUDITH K CHRISTMAN
金额：
$ 13.52万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-04-01 至 2004-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6515085
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

项目摘要

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：(申请人提供) 我们的总体目标是验证从头DNA胞嘧啶-C5甲基转移酶 (MTase)作为抗癌药物开发的合适分子靶点提出并测试筛选特异性抑制配体的新方法。这些酶。有许多证据表明，DNA的变化甲基化与癌症的发生有关。全基因组下降5mC 内容物出现在肿瘤发展的早期。然而，特定的区域 “高甲基化”发生并与肿瘤的表观遗传沉默有关参与DNA修复的抑制子或酶。这导致了这样的假设阻断或逆转超甲基化将导致基因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。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激活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，它们都具有细胞毒性和遗传毒性。我们已经这么做了开发和优化非遗传毒性寡核苷酸(ODN) DNMT1的抑制剂，它是体细胞中最丰富的DNA MTase形式。 DNMT1维持从头开始的MTase在发展。它在体外具有有限的从头活性，并可能完全活体内不活跃。因此，最有可能的是最近的一些成员发现可催化从头甲基化的DNA MTase DNMT3家族在体内，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。假说我们将测试的是，抑制从头DNA MTase是必要的，不仅是有效地减少甲基化和激活沉默基因，但是也是为了防止“从头再甲基化”。我们的具体目标是：1)验证DNMT3a/3b或类似的尚未确定的作为靶点的从头DNA转移酶：a)设计和优化小的ODN 专用于DNA MTase 3a、3b的抑制剂，对DNMT1无效。他们将包含5-azaC、5FdC或碱性位点作为目标C的替代在完全未甲基化的识别部位。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的抑制剂)对培养的人类癌细胞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完成已知甲基化失活的基因中CpG岛的测序。c) 测定/比较DNMT 3a、3b抑制剂的效果(单独和在与DNMT1的5氮杂C或ODN抑制剂合用对基因表达的影响使用表达阵列了解肿瘤抑制因子、细胞周期细胞凋亡的调节者和中介者。2)使用一种新的SDS-PAGE凝胶分析测定DNMT的抑制剂结合亲和力以预测A DNMTs1、3a和3b的多种新的抑制靶点。