Characterization of the biological activities of novel epigenetic modulators

新型表观遗传调节剂的生物活性表征

基本信息

批准号：
8215846
负责人：
ELISABETH D MARTINEZ
金额：
$ 31.94万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8215846
关键词：
Acetylation Affect Animals Apoptosis Apoptotic Archives Basic Science Biological Biological Assay Cancerous Cell Cycle Cell Cycle Arrest Cell Death Cell Line Cell Proliferation Cells Chemicals Clinical Collaborations Depsipeptides Development Dose Down-Regulation Drug Delivery Systems Drug Exposure Drug effect disorder Enzymes Epigenetic Process Epithelial Cells Evaluation Event Family Fostering Foundations Future Gene Expression Gene Proteins Genes Genomics Goals Growth Histones Hour Human Implant Inhibition of Cancer Cell Growth Lung Malignant Neoplasms Malignant neoplasm of lung Mammalian Cell Mammary gland Measures Metastatic Lesion Methods Methylation Modification Mus Normal Cell Pathology Pathway interactions Patients Pharmaceutical Preparations Physiological Play Primary Lesion Proteins Regulation Right-On Site Small Interfering RNA Specificity Testing Therapeutic Titrations Transcriptional Regulation Tumor Burden Up-Regulation Work Xenograft procedure base cancer cell cell growth cell killing cellular targeting chromatin immunoprecipitation chromatin modification cyclin D3 design drug mechanism epigenomics gene repression histone modification in vivo killings malignant breast neoplasm novel promoter public health relevance research study response subcutaneous therapeutic development tumor tumor growth uncontrolled cell growth

项目摘要

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. PUBLIC HEALTH RELEVANCE: We have identified a drug that shuts down genes that foster uncontrolled cell growth and turns on genes that slow down tumor growth. This compound shows the ability to specifically kill cancer cells and to decrease tumor size in mice, without affecting normal cells. Here, we propose to define how this drug works and to further develop it for therapeutic applications.

描述（由申请人提供）：药理学剂即时需要有效有效地调节特定的表观遗传酶和效应子分子。我们已经设计，建立并成功地使用了基于哺乳动物的细胞测定法，以使用广泛而不是目标特定方法来识别表观遗传途径的化学调节剂，以最大程度地提高测定活性的机械多样性。使用此测定，我们最近使用滴定反应方法进行了定量HTS，以多种剂量评估300,000多种不同化合物。我们已经确定并独立确认了70多名活跃者。评估其中一种命中的抗癌潜力，发现该药物有选择地靶向肺癌细胞阻断其生长并触发其细胞死亡，而不会影响正常的人支气管上皮细胞。这种复合改变了转录途径并影响组蛋白的表观遗传修饰。在体内，它显示了小鼠异种移植物中的抗肿瘤活性。我们现在的目标是表征这一最佳命中，我们称之为药物4，确定其修饰的表观遗传事件和基因组部位，定义了其抗生长/抗癌活性的机制，并确定其靶向的细胞蛋白，并以这种方式确定我们可以在未来的研究中为我们的其他HIT来表征该管道。我们将使用从患者正常和癌性肺中得出的一对匹配的细胞系来表征该化合物对生理学与病理表观基因质/转录景观的影响，通过以下目的是：1。衡量药物4在正常细胞中对癌细胞的表达水平对控制细胞的表达水平的影响，并评估这些细胞的表达水平，并评估这些细胞的表达水平。癌症的药物4。我们将测量调节细胞生长/细胞死亡途径的基因水平，以鉴定受癌症药物有选择性影响的基因，但不能在正常细胞中识别那些。然后，我们将在这些基因子集的启动子处定义药物4诱导的表观遗传标记变化。 2.定义药物4的机制诱导生长抑制/癌细胞死亡，细胞和肿瘤中。我们将研究药物4触发的癌症特异性细胞周期停滞和细胞死亡是否取决于细胞周期蛋白D3/RB途径和EGLN3/KIF1B途径，如初步结果所示。 3.通过候选分子和无偏的方法识别我们化合物的细胞靶标。我们将确定药物4对纯化表观遗传酶和效应分子的活性以鉴定复合靶标的作用。如果需要，我们将执行生物素化的药物下拉和siRNA屏幕。这些目标将有助于我们阐明药物4的作用机理，尤其是对癌症的选择性。这里提出的研究还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发展奠定基础。这些化合物将对许多病理（包括癌症）的基础研究和临床治疗发育产生重大影响。公共卫生相关性：我们已经确定了一种关闭基因的药物，该药物促进了不受控制的细胞生长并开发了减慢肿瘤生长的基因。该化合物表明能够专门杀死癌细胞并减少小鼠的肿瘤大小，而不会影响正常细胞。在这里，我们建议定义该药物的工作原理并进一步开发用于治疗应用。