Characterization of small molecule inhibitors of HDACs and DNMTs

描述(申请人提供)：在过去的几年里，已经确定了一些药物可以抑制DNA甲基转移酶(DNMT)和组蛋白脱乙酰酶(HDAC)介导的甲基化或去乙酰化途径。这些化合物立即应用于癌症治疗，因为它们能够重新激活异常沉默的肿瘤抑制基因。确定更多针对这两个酶家族的小分子的必要性是显而易见的，而且在癌症生物学领域已经反复声明过。我们的总体目标是扩大现有的可以调节基因表达的药物库，并评估它们的基本作用机制。为了实现这一目标，我们最近开发了一种独特的基于哺乳动物细胞的系统，以一种可靠、简单、易于自动化的方式筛选这种小分子。与迄今使用的方法不同，我们的系统具有测量其寻求目标的确切生物事件的优势：哺乳动物细胞转录抑制的逆转。该分析已经在小分子文库的筛选中进行了测试，并成功地获得了8次匹配。对一种HIT化合物和一种类似物的初步评估显示了HDAC抑制剂的性能。由于这些化合物在结构上与已知的HDAC/DNMT抑制剂不同，它们将有助于提高这种药物类型的多样性。这项提案的资金将使我们能够确定已确定的化合物的作用机制，并评估它们的抗癌活性。我们的具体目标是：1：测量我们高含量筛选中确定的化合物在体外抑制HDAC或DNMT酶活性的能力；2：评估候选表观遗传调节剂抑制人类癌细胞生长和重新激活人类癌细胞系中异常沉默基因表达的能力；以及3：测量这些表观遗传调节剂对异种移植模型肿瘤生长的影响。这些研究将为进一步开发这些潜在的癌症治疗化合物提供基础，并使我们能够进入临床前药物评估。 与公共健康相关：我们已经开发出一种系统，可以识别能够打开癌细胞中异常关闭的基因的药物。使用这个系统，我们已经确定了八种候选药物，并建议对它们进行表征，以衡量它们阻止人类癌细胞生长的能力。

DESCRIPTION (provided by applicant): Over the last several years, a few drugs have been identified that inhibit the methylation or de-acetylation pathways mediated by DNA methyltransferases (DNMT) and histone deacetylases (HDAC). These compounds have had immediate application in the treatment of cancers because of their ability to reactivate aberrantly silenced tumor suppressor genes. The need to identify more small molecules that target these two enzyme families is clear and has been repeatedly stated in the field of cancer biology. Our overall goal is to expand the available repertoire of drugs that can modulate gene expression and to evaluate their basic mechanism of action. Toward this goal, we have recently developed a unique mammalian cell-based system to screen for such small molecules in a reliable, simple fashion that is amenable to automation. Unlike methods used to date, our system has the advantage of measuring the exact biological event it seeks to target: the reversal of transcriptional repression in mammalian cells. The assay has been tested in screens of small molecule libraries and has successfully resulted in eight hits. Preliminary evaluation of one hit compound and one analog has revealed HDAC inhibitor properties. Because these compounds are structurally distinct from known HDAC/DNMT inhibitors, they will contribute to improving the diversity of this drug type. The funding of this proposal will allow us to characterize the mechanism of action of the identified compounds and to evaluate their anti-cancer activity. Our specific aims are: 1: To measure the ability of the compounds identified in our high content screen to inhibit the enzymatic activity of HDAC or DNMT enzymes in vitro; 2: To evaluate the ability of the candidate epigenetic modulators to inhibit the growth of human cancer cells and to re-activate the expression of aberrantly silenced genes in human cancer cell lines; and 3: To measure the effects of these epigenetic modulators on tumor growth in xenograft models. These studies will provide the foundation for further work in the development of these potential cancer therapeutic compounds and allow us to move into pre-clinical drug evaluation. Public health relevance: We have developed a system to identify drugs with the ability to turn on genes that are abnormally turned off in cancer cells. Using this system, we have already identified eight candidate drugs and propose to characterize them measuring their ability to block the growth of human cancer cells.