Characterization of the biological activities of novel epigenetic modulators

描述（由申请人提供）：迫切需要药理学试剂来强力和有效地调节特定的表观遗传酶和效应分子。我们设计、建立并成功使用了基于哺乳动物细胞的测定法，以使用广泛而非靶特异性方法来鉴定表观遗传途径的化学调节剂，以最大化测定活性物质的机制多样性。使用这种检测方法，我们最近使用滴定响应方法进行了定量HTS，以评估多个剂量的30多万种不同化合物。我们已经确定并独立确认了70多名活跃分子。对其中一种药物的抗癌潜力的评估发现，这种药物选择性地靶向肺癌细胞，阻断其生长并引发其细胞死亡，而不影响正常的人支气管上皮细胞。该化合物改变转录途径并影响组蛋白的表观遗传修饰。在体内，它在小鼠异种移植物中显示出抗肿瘤活性。我们现在的目标是表征这种热门药物，我们称之为药物4，确定其修饰的表观遗传事件和基因组位点，定义其抗生长/抗癌活性的机制，并确定其靶向的细胞蛋白质，通过这种方式，建立一个管道，通过它我们可以在未来的研究中表征我们的其他命中。我们将使用来自患者正常和癌性肺的成对匹配细胞系，通过以下目的来表征该化合物对生理学与病理学表观基因组/转录景观的影响：1.测量药物4在正常细胞与癌细胞中对控制细胞增殖途径的基因表达水平的影响，并评估癌细胞和肿瘤中的表观遗传标记在癌症中被药物4选择性改变的这些基因座的子集上。我们将测量调节细胞生长/细胞死亡途径的基因水平，以确定在癌症中而不是在正常细胞中受我们药物选择性影响的基因。 然后，我们将定义药物4诱导的表观遗传标记在这些基因的一个子集的启动子的变化。2.在细胞和肿瘤中定义药物4诱导生长抑制/癌细胞死亡的机制。 我们将研究由药物4触发的癌症特异性细胞周期阻滞和细胞死亡是否分别依赖于细胞周期蛋白D3/Rb通路和Egln 3/KIF 1B通路，正如初步结果所表明的那样。3.通过候选分子和无偏方法确定我们化合物的细胞靶点。 我们将确定药物4对纯化的表观遗传酶和效应分子的活性的影响，以鉴定化合物靶标。如果需要，我们将进行生物素化药物唐斯和siRNA筛选。这些目标将帮助我们阐明药物4的作用机制，特别是其对癌症的选择性。本文提出的研究也将为我们已经确定的其他潜在表观遗传调节剂的未来表征和开发奠定基础。这些化合物将对基础研究和包括癌症在内的许多病理学的临床治疗开发产生重大影响。

DESCRIPTION (provided by applicant): There is an immediate need for pharmacological agents to potently and effectively modulate specific epigenetic enzymes and effector molecules. We have designed, established and successfully used a mammalian cell- based assay to identify chemical modulators of epigenetic pathways using a broad rather than a target specific approach to maximize the mechanistic diversity of assay actives. Using this assay we have recently conducted quantitative HTS using a titration-response method to evaluate more than 300,000 diverse compounds at multiple doses. We have identified and independently confirmed more than 70 actives. Evaluation of the anti- cancer potential of one of these hits, uncovered that this drug selectively targets lung cancer cells blocking their growth and triggering their cell death, without affecting normal human bronchial epithelial cells. This compound alters transcriptional pathways and affects epigenetic modifications of histones. In vivo, it shows anti-tumor activity in mouse xenografts. Our goal now is to characterize this top hit, which we call Drug 4, determining the epigenetic events and genomic sites it modifies, defining the mechanism of its anti-growth/anti- cancer activity and identifying the cellular proteins it targets, and in this way, to set up a pipeline through which we can characterize our other hits in future studies. We will use pairs of matched cell lines derived from the normal and the cancerous lung of patients to characterize the effects of this compound on the physiological vs. the pathological epigenomic/transcriptional landscape through the following aims: 1.Measure the effects of Drug 4 in normal vs. cancer cells on the expression level of genes that control cell proliferation pathways and evaluate epigenetic marks in cancer cells and in tumors at a subset of these loci selectively altered by Drug 4 in cancer. We will measure levels of genes that regulate cell growth/cell death pathways to identify those selectively affected by our drug in cancer but not in normal cells. We will then define drug 4-induced changes in epigenetic marks at the promoters of a subset of these genes. 2.Define the mechanism of Drug 4 induced growth inhibition/cancer cell death, in cells and in tumors. We will investigate if the cancer-specific cell cycle arrest & cell death triggered by Drug 4 depend on the cyclin D3/Rb pathway & the Egln3/KIF1B pathways, respectively, as suggested by preliminary results. 3.Identify the cellular targets of our compound through candidate molecule and unbiased approaches. We will determine the effect of Drug 4 on the activity of purified epigenetic enzymes and effector molecules to identify compound targets. We will perform biotinylated-drug pull downs and siRNA screens if needed. These aims will help us elucidate the mechanism of action of Drug 4, and in particular, its selectivity against cancer. The studies proposed here will also lay the foundation for the future characterization and development of the other potential epigenetic modulators we have already identified. These compounds will have significant impact on basic research and on clinical therapeutic development for many pathologies, including cancer.