High Throughput Molecular Screening for Compounds(RMI)

描述(申请人提供)：转化生长因子β1(Tgfb1)是一种有效的多效性细胞因子，参与广泛的生物学过程。过量的Tgfbl活性会导致肺、肾和肝脏的纤维化。Tgfb1的细胞内信号主要是通过一类被称为Smad蛋白的分子来调节的。Smad蛋白，Smad2或Smad3，被Tgfb1受体复合体磷酸化，转位到细胞核，启动核信号。干扰Smads介导的Tgfb1信号可阻止许多器官组织纤维化的发展。调节这一途径的能力具有明显的治疗复制作用。为了便于筛选这些化合物，我们开发了对Tgfb1刺激做出反应的成纤维细胞，它们选择性、灵敏和定量地稳定表达Firefv荧光素酶。我们建议通过4个季度里程碑的方式来优化该系统，以实现高通量化合物筛选。首先，我们将在384孔格式下确定该分析的稳健性和可靠性；它对常见有机溶剂二甲基亚砜(DMSO)的影响的耐受性；最佳的培养和培养条件；以及优化分析的时间和产量。其次，我们将开发一种反筛选，使用细胞增殖和活性的测量来确保没有毒性，并通过测量化合物影响Tgfb1对成纤维细胞的已知有丝分裂活性的能力来独立验证化合物的生物学功效。我们还将筛选一个小的但化学成分多样化的文库，来自MicroSource的960化合物GenPlus文库，一式两份。检测性能和反筛选特性将在此筛选的基础上进行优化。第三，我们将使用耶鲁化学基因组学筛选设施拥有的20,000个化合物多样性文库(Maybridge筛选库)进行更大的筛选。根据这20,000个复合筛选的点击数和质量，我们将选择大约1000个化合物进行第二轮测试，一式两份。通过重复测试和反筛选确定并确认的前100种化合物将进一步进行药理学表征。第四，我们将开始开发一种策略来评估这100个候选基因的生物学机制，方法是使用高通量荧光显微镜方法，使用In Cell Analyzer 1000研究前100个激动剂/拮抗剂HITS对磷酸化Smad2/3/4核转位的影响。这些研究对于开发强大和可靠的分析方法，以从调节重要细胞过程活动的不同集合中识别生物活性分子至关重要。由于制药行业潜在的商业重要性，目前这类试剂的公开可获得性相当有限。

DESCRIPTION (provided by applicant): The potent and pleiotropic cytokine, transforming growth factor beta1 (Tgfb1), is involved in a broad range of biological processes. Excessive Tgfbl activity causes fibrosis in the lungs, kidney and liver. Intracellular signaling by Tgfb1 is mediated largely through a class of molecules known as Smad proteins. The Smad proteins, Smad2 or Smad3, are phosphorylated by the Tgfb1 receptor complex, translocate to the nucleus and initiate nuclear signaling. Interference with SmadS-mediated Tgfb1 signaling prevents the development of tissue fibrosis in many organs. The the ability to modulate this pathway has obvious therapeutic replications. To facilitate screen of these compounds, we have developed fibroblasts that stably express fireflv luciferase selectively, sensitively, and quantitatively in response to Tgfb1 stimulation. We propose to optimize this system for high-throughput compound screening by way of 4 quarterly milestones. First, we will establish the robustness and reliability of the assay in a 384 well format; its tolerance of the effects of dimethyl sulfoxide (DMSO), a common organic solvent; optimal plating and culture conditions; and optimize assay timing and throughput. Secondly, we will develop a counterscreen using a measure of cell proliferation and viability to assure the absence of toxicity and to independently verify the biological efficacy of compounds by measuring their ability to influence the known mitogenic activity of Tgfb1 for fibroblasts. We will also screen a small but chemically diverse library, the 960 compound GenPlus library from MicroSource, in duplicate. Assay performance and counter-screening characteristics will be optimized based on this screen. Thirdly, we will perform a larger screen using a 20,000 compound diversity library (the Maybridge Screening collection) owned by the Yale Chemical Genomics Screening facility. Based on the number and quality of hits from this 20,000 compound screen, performed as singlet measures, we will choose approximately 1000 compounds for a second round of testing in duplicate. The top 100 compounds identified and confirmed through duplicate testing and counter-screening, will be further characterized pharmacologically. Fourth, we will begin development of a strategy for assessing the biological mechanism of these top 100 candidates by using a high-throughput fluorescence microscopy method to study the effect of the top 100 agonist/antagonist hits on phospho-Smad2/3/4 nuclear translocation using the In Cell Analyzer 1000. These studies are critical to develop robust and reliable assays to identify biologically active molecules from diverse collections that regulate the activity of important cellular processes. The current public availability of reagents of this sort is quite limited due to high potential commercial importance in the pharmaceutical industry.