High throughput screen for regulators of globin

球蛋白调节剂的高通量筛选

• 批准号: 7060220
• 负责人: Benjamin Levine Ebert
• 金额: $ 12.59万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-26 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7060220
• 关键词: amidohydrolases; biotechnology; bone marrow; clinical research; drug screening /evaluation; enzyme inhibitors; erythroid stem cell; gene expression; gene expression profiling; genetic regulation; globin; hemoglobin F; high throughput technology; messenger RNA; microarray technology; sickle cell anemia; small molecule; technology /technique development

DESCRIPTION (provided by applicant): Sickle cell disease (SCD) is a common disease caused by a single amino acid substitution, ValSGIn, in the beta globin gene. Due to tremendous scientific advances over the past 60 years, we now understand the molecular pathophysiology of SCD in exquisite detail. Unfortunately, progress in the development of novel therapies has been slower. Prospects for an effective therapy appear promising as extensive evidence demonstrates that reactivation of fetal hemoglobin prevents polymerization of deoxygenated hemoglobin, the central abnormality in SCD. In the past, a high throughput screen for multiple transcripts in each well of a multiwell plate has not been feasible. We have developed an assay that detects seven distinct globin transcripts and GAPDH expression in each well of a multiwell plate with high fidelity and at low cost, enabling us to screen libraries of small molecules in high throughput for compounds that differentially regulate expression of the globin genes. We have designed our assay to analyze expression of globin genes in primary human erythroid progenitor cells cultured in vitro. RNA is captured on oligo-dT coated 384-well plates (RNAture) and reverse transcribed. The cDNA for each gene of interest is amplified by ligation mediated PCR, labeled amplicons are captured on fluorescent microspheres (Luminex), and the relative abundance of different amplicons is detected by high speed flow cytometry. The entire process of RNA purification, ligation mediated amplification, fluorescent bead detection, and data acquisition is amenable to automation. To date, we have demonstrated that we can measure expression of all seven globin transcripts in multiwell format, and we can detect the relative induction of gamma globin expression by known inducers of fetal hemoglobin. In this proposal, we describe the optimization, validation, and automation of this assay as well as the performance of a pilot small molecule screen. In collaboration with the laboratory of Dr. Stuart Schreiber at the Broad Institute of Harvard and MIT, we propose to screen a library of small molecules enriched in FDA approved and other known bioactive compounds and a library of collated HDAC inhibitors. Upon completion of these studies, we hope to have identified a small number of compounds that activate fetal hemoglobin for further investigation. Furthermore, we hope to have validated the assay in a pilot screen in preparation for more extensive screens of small molecule libraries.

描述（由申请人提供）：镰状细胞病（SCD）是一种常见疾病，由β -珠蛋白基因中的单个氨基酸替换（ValSGIn）引起。由于过去60年来科学的巨大进步，我们现在对SCD的分子病理生理学有了详细的了解。不幸的是，开发新疗法的进展一直比较缓慢。广泛的证据表明，胎儿血红蛋白的再激活可以阻止脱氧血红蛋白的聚合，这是SCD的中心异常，因此有效治疗的前景似乎很有希望。在过去，在多孔板的每口井中对多个转录本进行高通量筛选是不可行的。我们开发了一种检测方法，可以在多孔板的每孔中以高保真度和低成本检测七种不同的珠蛋白转录本和GAPDH表达，使我们能够以高通量筛选小分子文库，以区分调节珠蛋白基因表达的化合物。我们设计了我们的实验来分析球蛋白基因在体外培养的原代人红细胞祖细胞中的表达。RNA被捕获在寡聚dt包被的384孔板（RNAture）上并进行逆转录。每个目标基因的cDNA通过连接介导PCR扩增，标记的扩增子在荧光微球（Luminex）上被捕获，并通过高速流式细胞术检测不同扩增子的相对丰度。RNA纯化、连接介导扩增、荧光头检测和数据采集的整个过程都可以实现自动化。到目前为止，我们已经证明，我们可以在多孔格式中测量所有七种珠蛋白转录本的表达，并且我们可以检测已知的胎儿血红蛋白诱导剂对γ珠蛋白表达的相对诱导。在本提案中，我们描述了该分析的优化，验证和自动化以及试点小分子筛选的性能。与哈佛大学Broad研究所和麻省理工学院的Stuart Schreiber博士的实验室合作，我们建议筛选富含FDA批准和其他已知生物活性化合物的小分子文库和整理的HDAC抑制剂文库。在完成这些研究后，我们希望能够确定少量激活胎儿血红蛋白的化合物，以供进一步研究。此外，我们希望在试点筛选中验证该分析，为更广泛的小分子文库筛选做准备。