Characterization of the biological activities of novel epigenetic modulators

描述（由申请人提供）：迫切需要能够有效调节特定表观遗传酶和效应分子的药物。我们已经设计、建立并成功地使用了一种基于哺乳动物细胞的检测方法来识别表观遗传途径的化学调节剂，使用广泛而不是特定靶标的方法来最大化检测活性的机制多样性。使用这种方法，我们最近使用滴定反应方法进行了定量HTS，以评估超过30万种不同剂量的化合物。我们已经确认并独立证实了70多名活跃分子。对其中一种药物的抗癌潜力进行了评估，发现这种药物选择性地靶向肺癌细胞，阻断其生长并引发细胞死亡，而不影响正常的人类支气管上皮细胞。这种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个最受欢迎的药物，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热门药物。我们将使用来自正常和癌肺患者的配对细胞系来表征该化合物对生理和病理表观基因组/转录景观的影响，目的如下：测量药物4在正常细胞和癌细胞中对控制细胞增殖途径的基因表达水平的影响，并评估癌症细胞和肿瘤中这些基因座亚群中被药物4选择性改变的表观遗传标记。我们将测量调节细胞生长/细胞死亡途径的基因水平，以识别那些在癌症中而在正常细胞中被我们的药物选择性影响的基因。然后，我们将定义药物4在这些基因子集的启动子上引起的表观遗传标记变化。2.确定药物4在细胞和肿瘤中诱导生长抑制/癌细胞死亡的机制。我们将研究药物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cyclin D3/Rb途径和Egln3/KIF1B途径，正如初步结果所提示的那样。3.通过候选分子和无偏方法确定我们化合物的细胞靶标。我们将确定药物4对纯化的表观遗传酶和效应分子活性的影响，以确定化合物靶点。如果需要，我们将进行生物素化药物下拉和siRNA筛选。这些目标将有助于我们阐明药物4的作用机制，特别是它对癌症的选择性。这里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。这些化合物将对包括癌症在内的许多疾病的基础研究和临床治疗发展产生重大影响。

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.