Characterization of small molecule inhibitors of HDACs and DNMTs

描述（由申请人提供）：在过去的几年中，已经确定了一些药物可以抑制DNA甲基转移酶（DNMT）和组蛋白去乙酰化酶（HDAC）介导的甲基化或去乙酰化途径。由于这些化合物能够重新激活异常沉默的肿瘤抑制基因，因此它们在癌症治疗中有直接的应用。确定更多靶向这两种酶家族的小分子的必要性是明确的，并且在癌症生物学领域已反复声明。我们的总体目标是扩大现有的药物库，可以调节基因表达和评估其基本的作用机制。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，以可靠、简单的方式筛选这种小分子，并且可以自动化。与迄今为止使用的方法不同，我们的系统具有测量其寻求目标的确切生物学事件的优势：逆转哺乳动物细胞中的转录抑制。该方法已经在小分子文库中进行了筛选测试，并成功地产生了8个结果。对一种命中化合物和一种类似物的初步评价揭示了HDAC抑制剂的性能。由于这些化合物在结构上不同于已知的HDAC/DNMT抑制剂，它们将有助于改善这种药物类型的多样性。这项提案的资助将使我们能够确定已确定化合物的作用机制并评估其抗癌活性。我们的具体目标是：1 .测量在高含量筛选中鉴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中异常沉默基因表达的能力；3 .测量这些表观遗传调节剂对异种移植瘤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。

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.