Natural Products Chemistry/Molecular Target Lead Discov.

ChemBridge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试验中筛选，旨在寻找突变Kit的抑制剂。在该测定中，命中被定义为对突变型KIT细胞与野生型KIT细胞产生超过30%差异生长抑制的化合物。迄今为止，已鉴定出145种化合物为命中物，相当于测定中约0.2%的命中率。到目前为止，所有的筛选都是以96孔/板的形式进行的。现在，过渡到常规384孔筛选似乎是可行的，并且ChemBridge库的其余部分将以这种高密度形式进行筛选。樱桃采摘，再确认，和剂量反应测试的所有化学桥命中将开始不久。一旦ChemBridge筛选完成，将开始在384孔板中对天然产物提取物进行中试筛选。来自维克托洛巴年科夫实验室的用于产生作为GST融合蛋白的BORIS的N-末端区域、C-末端区域和组合的N-和C-末端区域的大肠杆菌表达系统。已完成生长和表达优化研究。规模化生产提供了6.2 L N-末端BORIS和16.6 L C-末端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结构域的方法。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，ABCG 2多药转运蛋白抑制剂的初步测定开发正在进行中。已经获得过表达ABCG 2的三种细胞系，并正在进行评价，以评估HTS应用的最佳细胞系。计划利用脱镁叶绿酸a（PhA）进行基于荧光细胞的测定，脱镁叶绿酸a是ABCG 2特异性荧光底物，其通过ABCG 2转运出细胞。已知的抑制剂烟曲霉素C（FTC）将被用作阳性对照，阻断ABCG 2的作用，并允许PhA在细胞中积累。实验，以确定最佳的细胞生长条件，试剂参数，并测定格式和protocols.Assay开发工作正在进行中，以开发一个筛选HIF-2的抑制剂。HeLa和786- 0细胞被成功转染，然而来自原始GFP构建体的信号不够亮以支持高通量筛选形式。使用质粒pEGFP-C1并确认表达的EGFP足够明亮，选择能够表达半衰期为4小时的不稳定形式的EGFP的载体（pd 4 EGFP-N1）。然后用人VEGF启动子替换该载体中的CMV启动子。使用新构建体的转染实验是成功的，并且GFP亮度现在似乎足以用于使用786- 0细胞系的测定开发。目前，研究重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，随后记录生长曲线和最佳培养条件。我们目前正在寻找第二个HRE（GAPDH启动子），它是由HIF-2诱导的，但属于不同的信号通路，用作二次筛选。已经制备了含有长和短VHL形式的构建体，用于转染到786-O细胞中以用作对照。最后，我们试图鉴定一种可用作试验阳性对照的化合物。由于没有已知的HIF-2抑制剂，我们将从测试已知的HIF-2抑制剂开始，看看它们中是否有任何也抑制HIF-2。

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 effortsPreliminary 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 .