Rapid Screening Assay for Novel Epigenetic Drugs

新型表观遗传药物的快速筛选分析

• 批准号: 8780028
• 负责人: Michael Rountree
• 金额: $ 22.5万
• 依托单位: NZUMBE EPIGENETICS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-22 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780028
• 关键词: Accounting; Affect; BRCA1 gene; Back; Biological Assay; Calibration; Cancer Etiology; Cell Cycle; Cell Density; Cell Line; Cells; Cessation of life; Colon Carcinoma; Custom; DNA Methylation; Development; Disease; Ensure; Enzymes; Epigenetic Process; Exhibits; Failure; Fluorescence; Gene Expression; Gene Silencing; Genes; Genome Stability; Goals; Green Fluorescent Proteins; Growth; Health; Human; Knowledge; Laboratories; Lead; Legal patent; Letters; Libraries; Light; Link; Luciferases; MLH1 gene; Malignant Neoplasms; Measures; Mission; Modeling; Modification; Mutate; Normal Cell; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plant Roots; Plasmids; Play; Preclinical Drug Evaluation; Process; Proteins; Publishing; Reproducibility; Role; Sensitivity and Specificity; Services; Specificity; Testing; Time; Transcript; Treatment Protocols; Tumor Suppressor Genes; Tumor Suppressor Proteins; Validation; Work; base; cancer cell; cancer therapy; clinically relevant; drug development; drug discovery; inhibitor/antagonist; interest; luminescence; malignant breast neoplasm; malignant phenotype; novel; novel strategies; promoter; prototype; screening; theories

DESCRIPTION (provided by applicant): A hallmark of virtually every cancer is aberrant epigenetic silencing of vital genes required for a normal cell cycle and genome stability (e.g. MLH1, BRCA1, VHL, p16, etc.). Loss of expression of these tumor suppressor genes plays a critical role in cancer formation and growth. Thus, targeting this root cause of cancer has significant potential to treat and potentially cure a host of cancers. Indeed, a major initiative i essentially every major pharmaceutical company is to investigate epigenetic cancer therapies targeting the reactivation of these key genes. However, despite its promise, at this time drug-induced gene reactivation does not work therapeutically because reactivation is invariably unstable. One reason for this failure is that current approaches consider silencing as an end point instead of a process, and as a result do not strategically target epigenetic modifications that occur at different times during the silencing process. We believe achieving stable reactivation of tumor suppressor genes requires not only targeting the end points of silencing (e.g., DNA methylation), but also targeting the early steps of the silencing process. Nzumbe's long-term goal is to develop screening assays using our patent pending platform to induce epigenetic silencing and then identify compounds that inhibit the start of the silencing process. No other company has the ability to perform this type of screen. For this Phase I application, we will develop screens for two clinically relevant tumor suppressor genes that undergo epigenetic silencing in human cancers; BRCA1, which is silenced in breast cancer, and MLH1, which is silenced in colon cancer. Combined these two cancers account for approximately 370,000 new patients and 90,000 deaths every year. Briefly, our primary screen will involve expressing green fluorescent protein (GFP) and luciferase (Luc) from the MLH1 and BRCA1 promoters, inducing gene silencing, and then measuring changes in fluorescence intensity in microtiter plates that will indicate whether test compounds exhibit inhibitory activities. Two specific aims are proposed. For the first aim, we will develop the BRCA1 and MLH1 screens in breast and colon cancer cells, respectively, and calibrate the assays to ensure reproducibility and low levels of variability. For the second aim, we validate the screening assays, first with known positive and negative controls and then with a set of 11 test compounds exhibiting known and distinct epigenetic modifying activities. Successful completion of the proposed work will provide commercially viable assays to screen small and large molecule libraries. Ultimately, compounds identified with the BRCA1 and MLH1 assays can be used as part of treatment regimens for breast and colon cancer. Because our approach can be modified for a variety of tumor suppressor gene promoters, we will propose in a Phase II application to develop a highly desirable and commercially viable drug screening service that is applicable to a variety of cancers

描述(申请人提供)：几乎每一种癌症的一个特征是正常细胞周期和基因组稳定所需的重要基因的异常表观遗传沉默(例如MLH1、BRCA1、VHL、p16等)。这些抑癌基因的表达缺失在肿瘤的形成和生长中起着至关重要的作用。因此，针对这种癌症的根本原因具有治疗和治愈多种癌症的巨大潜力。事实上，我几乎每个主要制药公司的一个主要倡议是研究以重新激活这些关键基因为目标的表观遗传学癌症疗法。然而，尽管有希望，目前药物诱导的基因重新激活在治疗上并不起作用，因为重新激活总是不稳定的。这一失败的一个原因是，目前的方法将沉默视为终点而不是过程，因此没有战略性地针对沉默过程中不同时间发生的表观遗传修饰。我们认为，要稳定地重新激活肿瘤抑制基因，不仅需要针对沉默的终点(例如DNA甲基化)，还需要针对沉默过程的早期步骤。Nzumbe的长期目标是利用我们正在申请专利的平台开发筛选分析，以诱导表观遗传沉默，然后识别抑制沉默过程开始的化合物。没有其他公司有能力执行这种类型的屏幕。对于这个第一阶段的应用，我们将开发两个在人类癌症中经历表观遗传沉默的临床相关肿瘤抑制基因的筛查：BRCA1，它在乳腺癌中沉默，以及MLH1，它在结肠癌中沉默。这两种癌症加在一起，每年约有37万新患者和9万人死亡。简而言之，我们的初步筛选将包括表达来自MLH1和BRCA1启动子的绿色荧光蛋白(GFP)和荧光素酶(Luc)，诱导基因沉默，然后测量微滴定板中荧光强度的变化，以指示测试化合物是否具有抑制活性。提出了两个具体目标。对于第一个目标，我们将分别开发乳腺癌和结肠癌细胞的BRCA1和MLH1筛查，并校准检测以确保重复性和低水平的变异性。对于第二个目的，我们首先用已知的阳性和阴性对照，然后用一组11个表现出已知和独特的表观遗传修饰活性的测试化合物来验证筛选试验。拟议工作的成功完成将为筛选小分子和大分子文库提供商业上可行的分析方法。最终，BRCA1和MLH1检测确定的化合物可以用作乳腺癌和结肠癌治疗方案的一部分。由于我们的方法可以针对多种肿瘤抑制基因启动子进行修改，我们将在第二阶段的应用中提议开发一种非常可取的、商业上可行的、适用于各种癌症的药物筛选服务。