Natural Products Chemistry and Molecular Target Lead Dis

天然产物化学和分子靶标先导化合物

• 批准号: 6952059
• 负责人: michael lee
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6952059
• 关键词: DNA binding protein; HeLa cells; affinity chromatography; antineoplastics; biological products; cell cycle; cell growth regulation; chimeric proteins; drug screening /evaluation; growth inhibitors; high throughput technology; human tissue; inhibitor /antagonist; membrane transport proteins; microorganism mass culture

Greater than 85,000 synthetic compounds in the ChemBridge library have been screened in the cell-based differential cytotoxicity assay designed to find inhibitors of mutant Kit. In this assay, a hit has been defined as a compound that produces greater than 30% differential growth inhibition of mutant KIT cells vs wild type KIT cells. To date, 145 compounds have been identified as hits which equates to a hit rate of approximately 0.2% in the assay. All of the screening so far has been in 96-well per plate format. Transition to routine 384-well screening now appears feasible and the remainder of the ChemBridge library will be screened in this high density format. Cherry picking, reconfirmation, and dose-response testing of all of the ChemBridge hits will begin shortly. Pilot screening of natural products extracts in 384-well format will begin as soon as the ChemBridge screening is complete. We have obtained E. coli expression systems for producing the N-terminal region, C-terminal region and the combined N- and C-terminal regions of BORIS as GST fusion proteins from Victor Lobanenkov's laboratory. Growth and expression optimization studies have been completed. Scaled up production has provided 6.2 L of N-terminal BORIS and 16.6 L of C-terminal BORIS cultures. Affinity purification of the GST fusion proteins is currently underway. Procedures for efficient cleavage of the GST domain from the fusion proteins are being evaluated. Purified BORIS domains will be used for antibody production, protein structural studies, and binding partner identification efforts Preliminary assay development for inhibitors of the ABCG2 multidrug transporter are ongoing. Three cell lines that over express ABCG2 have been acquired and are being evaluated to assess the optimal cell line for HTS applications. A fluorescent cell-based assay is planned utilizing Pheophorbide a (PhA), an ABCG2-specific fluorescent substrate which is transported out of the cell by ABCG2. The known inhibitor fumitremorgin C (FTC) will be used as a positive control, blocking the action of ABCG2 and allowing PhA to accumulate in the cell. Experiments to determine optimal cellular growth conditions, reagent parameters, and assay format and protocols are planned. Assay development efforts are underway to develop a screen for inhibitors of HIF-2 . HeLa and 786-O cells were successfully transfected ,however the signal from the original GFP construct was not bright enough to support high throughput screening formats. Using the plasmid pEGFP-C1 and confirming that the EGFP expressed was bright enough, a vector (pd4EGFP-N1) that enables expression of a destabilized form of EGFP with a half-life of four hours was chosen. The CMV promoter in this vector was then replaced with the human VEGF-promoter. Transfection experiments using the new construct were a success and the GFP brightness now appears adequate for assay development using the 786-O cell line. The focus has now shifted to single cell cloning. Once a stable cloned culture is obtained, the actual GFP t1/2 will be determined, followed by documentation of growth curves and optimal culture conditions. We are currently looking at a second HRE (of the GAPDH promoter) that is induced by HIF-2 but belongs to a different signaling pathway to use as a secondary screen. Constructs containing the long and short VHL forms have been prepared for transfection into 786-O cells to be used as controls. Finally, we are trying to identify a compound that can be used as a positive control in the assay. Since no HIF-2 inhibitors are known, we will start by testing known HIF-2 inhibitors to see if any of them also inhibit HIF-2 .

ChemBridge 文库中超过 85,000 种合成化合物已在基于细胞的差异细胞毒性测定中进行了筛选，该测定旨在寻找突变 Kit 的抑制剂。在此测定中，命中被定义为对突变型 KIT 细胞与野生型 KIT 细胞产生大于 30% 差异生长抑制的化合物。迄今为止，已鉴定出 145 种化合物为命中化合物，相当于测定中的命中率约为 0.2%。迄今为止，所有筛选均采用每板 96 孔的形式。过渡到常规 384 孔筛选现在看来是可行的，ChemBridge 文库的其余部分将以这种高密度形式进行筛选。所有 ChemBridge 热门产品的筛选、再确认和剂量反应测试将很快开始。 ChemBridge 筛选完成后，将立即开始以 384 孔形式对天然产物提取物进行试点筛选。 我们从Victor Lobanenkov实验室获得了大肠杆菌表达系统，用于生产BORIS的N端区域、C端区域以及N端和C端组合区域作为GST融合蛋白。生长和表达优化研究已经完成。扩大生产已提供 6.2 L N 端 BORIS 和 16.6 L C 端 BORIS 培养物。 GST 融合蛋白的亲和纯化目前正在进行中。正在评估从融合蛋白中有效切割 GST 结构域的程序。纯化的 BORIS 结构域将用于抗体生产、蛋白质结构研究和结合伴侣鉴定工作 ABCG2 多药物转运蛋白抑制剂的初步检测开发正在进行中。已经获得了三种过度表达 ABCG2 的细胞系，并正在对其进行评估，以评估适合 HTS 应用的最佳细胞系。计划利用脱镁叶绿酸 a (PhA) 进行基于细胞的荧光测定，Pheophorbide a 是一种 ABCG2 特异性荧光底物，由 ABCG2 转运出细胞。已知的抑制剂fumitremorgin C (FTC)将用作阳性对照，阻断ABCG2的作用并允许PhA在细胞中积累。计划进行实验以确定最佳细胞生长条件、试剂参数以及测定格式和方案。 正在进行检测开发工作，以开发 HIF-2 抑制剂的筛选。 HeLa 和 786-O 细胞已成功转染，但原始 GFP 构建体的信号不够明亮，无法支持高通量筛选格式。使用质粒 pEGFP-C1 并确认表达的 EGFP 足够亮，选择了能够表达半衰期为 4 小时的不稳定形式的 EGFP 的载体 (pd4EGFP-N1)。然后将该载体中的 CMV 启动子替换为人 VEGF 启动子。使用新构建体的转染实验取得了成功，并且 GFP 亮度现在似乎足以使用 786-O 细胞系进行测定开发。现在焦点已转移到单细胞克隆。一旦获得稳定的克隆培养物，将确定实际的 GFP t1/2，然后记录生长曲线和最佳培养条件。我们目前正在研究第二个 HRE（GAPDH 启动子），它由 HIF-2 诱导，但属于不同的信号通路，可用作辅助筛选。含有长和短 VHL 形式的构建体已准备好转染到 786-O 细胞中用作对照。最后，我们试图确定一种可用作检测中阳性对照的化合物。由于尚无已知的 HIF-2 抑制剂，我们将首先测试已知的 HIF-2 抑制剂，看看它们是否也能抑制 HIF-2。