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

描述(申请人提供)：我们提出了一个HTS多重筛选项目，使用小分子来探测复杂的、高度保守的生物途径。该项目利用酵母GFP菌株收集来探索TOR途径，这是一条在人类身上具有治疗意义的途径[1]。雷帕霉素的靶标TOR是一种必需的丝氨酸/ThR蛋白激酶，它在两个不同的多蛋白复合体TOR复合体1和2中发挥作用。这些复合体的结构和功能从酵母到人类都是保守的。TOR复合体1被雷帕霉素抑制，被认为是将生长信号与细胞代谢相结合；TOR复合体2不被雷帕霉素抑制，似乎调节生长的空间方面，如细胞极性[2，3]。雷帕霉素是一种从拉帕努伊岛土壤中发现的细菌中分离出来的抗真菌化合物[4]，最初用于通过鉴定对雷帕霉素敏感或耐药的突变株来表征萌芽酵母(酿酒酵母)中的TOR途径[5]。雷帕霉素在真菌中起到细胞抑制剂的作用，阻止细胞处于类G0状态。作为一种药物，它被广泛用作免疫抑制剂，雷帕霉素和这种大环内酯的衍生物正在评估一些临床应用。由于其多效性，雷帕霉素在制药行业被认为是一种“肮脏的药物”。为了确定更特异的TOR抑制剂和激活剂，我们提出了一种基于细胞的多重高通量流式细胞术来筛选MLSMR，以确定针对TOR途径中特定蛋白的化学物质。化学筛选应该产生调节这一途径的特定分支的小分子，这些分子可能具有显著的治疗潜力和较少的副作用。我们将在雷帕霉素治疗前后以多重形式筛选酵母GFP融合菌株集(4,159株)[6]，因为融合文库对基于荧光的流式细胞仪读数具有高度的适应性。荧光的变化表明一种化学物质已经影响了目标绿色荧光蛋白融合蛋白的生产(S)。我们确定的模仿或抑制雷帕霉素激活的化学物质可能在其他生物体中有靶点，因为TOR途径成分高度保守。我们已经成功地在两个不同的生长条件下筛选了这个集合，并有能力识别影响一个或多个多重目标的化学物质。我们将进行初步筛选，以检测TOR途径的激动剂和拮抗剂。作为二次筛选，我们将：1)比较新分子对GFP收集中蛋白质表达的影响，以及2)通过分析细胞周期和/或生长停滞来评估小分子影响雷帕霉素的细胞抑制潜力的能力。预期结果：在没有雷帕霉素的情况下，筛查将揭示模拟雷帕霉素或以其他方式影响TOR途径的分子。在雷帕霉素存在的情况下进行筛选，将揭示对抗雷帕霉素的分子。 公共卫生相关性：我们提出了一个HTS多重筛查项目，利用小分子来探索一条复杂的、高度保守的生物途径。该项目利用酵母GFP菌株收集来探索雷帕霉素途径的TOR靶点，这是一条具有治疗意义的人类途径。

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.