Chemical Screen of TOR pathway GFP fusion proteins in S. cerevisiae

酿酒酵母 TOR 途径 GFP 融合蛋白的化学筛选

• 批准号: 7692448
• 负责人: MARGRET C. WERNER-WASHBURNE
• 金额: $ 2.5万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-16 至 2011-04-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7692448
• 关键词: Adverse effects; Affect; Agonist; Antifungal Agents; Bacteria; Biological; Biological Assay; Cell Cycle; Cell Polarity; Cells; Chemicals; Chimeric Proteins; Collection; Complex; Cues; Cytostatics; Drug Industry; Flow Cytometry; Fluorescence; Growth; Immunosuppressive Agents; Island; Lactones; Libraries; Metabolism; Multiprotein Complexes; Organism; Pathway interactions; Pharmaceutical Preparations; Production; Protein Kinase; Proteins; Resistance; Saccharomyces cerevisiae; Saccharomycetales; Screening Result; Screening procedure; Sirolimus; Soil; Structure; Therapeutic; Yeasts; base; clinical application; cytotoxic; follow-up; fungus; inhibitor/antagonist; man; mutant; novel; protein expression; public health relevance; small molecule

DESCRIPTION (provided by applicant): We propose an HTS multiplex screening project to use small molecules to probe a complex, highly conserved, biological pathway. The project takes advantage of the yeast GFP strain collection to probe the TOR pathway, a pathway with therapeutic implications in man [1]. The target of rapamycin, TOR, is an essential ser/thr protein kinase that functions in two distinct multiprotein complexes, TOR complexes 1 and 2. The structure and functions of these complexes have been conserved from yeast to man. TOR complex 1 is inhibited by rapamycin and is thought to couple growth cues to cellular metabolism; TOR complex 2 is not inhibited by rapamycin and appears to regulate spatial aspects of growth, such as cell polarity [2, 3]. Rapamycin, an antifungal compound isolated from a bacterium found in soil on the island of Rapa Nui [4], was the drug originally used to characterize the TOR pathway in budding yeast (S. cerevisiae) by identifying mutants that were rapamycin sensitive or resistant [5]. Rapamycin acts as a cytostatic agent in fungi, arresting cells in a G0-like state. As a drug it is widely used as an immunosuppressant and rapamycin and derivatives of this macrocyclic lactone are being evaluated for a number of clinical applications. Because of its pleiotropic effects, rapamycin is thought of as a "dirty drug" in the pharmaceutical industry. To identify more specific TOR inhibitors and activators, we propose a cell-based multiplex high throughput flow cytometry assay to screen the MLSMR to define chemicals that target specific proteins in the TOR pathway. A chemical screen should yield small molecules that modulate specific branches of this pathway and these molecules may have significant therapeutic potential and fewer side effects. We will screen the yeast GFP-fusion strain set (4,159 strains) [6] prior to and post-rapamycin treatment in multiplex format because the fusion library is highly amenable to fluorescence-based flow cytometric readout. Change in fluorescence indicates that a chemical has affected production of the target GFP-fusion protein(s). Chemicals we identify that mimic or inhibit rapamycin activation will likely have targets in other organisms because TOR pathway components are highly conserved. We have already successfully screened this collection under two different growth conditions and have the capacity to identify chemicals affecting one or more than one of the multiplexed targets. We will conduct primary screens to detect both agonists and antagonists of the TOR pathway. As secondary screens, we will: 1) compare the impact of the novel molecules on protein expression in the GFP collection and 2) evaluate the ability of the small molecules to impact the cytostatic potential of rapamycin through analysis of cell cycle and/or growth arrest. Expected Results: The screen in the absence of rapamycin will reveal molecules that mimic rapamycin or otherwise impact the TOR pathway. The screen in the presence of rapamycin will reveal molecules that antagonize rapamycin. PUBLIC HEALTH RELEVANCE: We propose an HTS multiplex screening project to use small molecules to probe a complex, highly conserved, biological pathway. The project takes advantage of the yeast GFP strain collection to probe the TOR - target of rapamycin - pathway, a pathway with therapeutic implications in man.

描述（由申请人提供）：我们提出了一个HTS多重筛选项目，以使用小分子来探测复杂，高度保守的生物途径。该项目利用酵母GFP菌株收集来探测TOR途径，这是人类中具有治疗意义的途径[1]。雷帕霉素Tor的靶标是一种必不可少的Ser/Thr蛋白激酶，在两个不同的多蛋白络合物中起作用，Tor Complexches 1和2。这些络合物的结构和功能已经从酵母到人体内保存。雷帕霉素抑制了Tor复合物1，并被认为将生长线索与细胞代谢相结合。雷帕霉素不会抑制Tor Complyst 2，并且似乎调节生长的空间方面，例如细胞极性[2，3]。雷帕霉素是一种从Rapa Nui岛上发现的细菌中分离出的抗真菌化合物[4]，是该药物最初用于表征出现酵母（S. cerevisiae）中TOR途径的药物，通过鉴定Rapamycin敏感或抗药性的突变体[5]。雷帕霉素在真菌中充当胞浆剂，以G0样状态阻止细胞。作为一种药物，它被广泛用作这种大环内酯的免疫抑制剂和雷帕霉素以及该大量临床应用的衍生物。由于其多效性作用，雷帕霉素被认为是制药行业的“脏药”。为了识别更特定的TOR抑制剂和激活剂，我们提出了基于细胞的多重吞吐量流式细胞仪测定法，以筛选MLSMR，以定义靶向TOR途径中特定蛋白的化学物质。化学筛选应产生小分子，以调节该途径的特定分支，并且这些分子可能具有明显的治疗潜力和更少的副作用。我们将在酵母GFP融合菌株集（4,159株）[6]筛选和宽霉素后的多重形式，因为融合库非常适合基于荧光的基于荧光的流式细胞量读数。荧光的变化表明，化学物质影响了靶GFP融合蛋白的产生。化学物质我们发现，由于TOR途径成分是高度保守的，因此模仿或抑制雷帕霉素的激活可能会在其他生物体中具有靶标。我们已经在两个不同的生长条件下成功筛选了该集合，并具有识别影响一个或多个多路复用靶标的化学物质。我们将进行主要筛选，以检测TOR途径的激动剂和拮抗剂。作为次要筛选，我们将：1）比较新分子对GFP集合中蛋白质表达的影响，并比较小分子通过分析细胞周期和/或生长滞留的分析来评估小分子影响雷帕霉素的细胞抑制潜力的能力。预期的结果：在没有雷帕霉素的情况下，屏幕会揭示模仿雷帕霉素或以其他方式影响TOR途径的分子。雷帕霉素存在的屏幕将揭示与雷帕霉素拮抗的分子。 公共卫生相关性：我们提出了一个HTS多重筛查项目，以使用小分子来探测复杂，高度保守的生物学途径。该项目利用酵母GFP菌株收集来探测雷帕霉素 - 途径的靶标，这是一种在人类中具有治疗意义的途径。