Natural Products Chemistry/Molecular Target Lead Discov.

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试验中筛选，旨在寻找突变体Kit的抑制剂。在该试验中，hit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，145种化合物已被确定为命中，相当于在该分析中命中率约为0.2%。到目前为止，所有的筛选都是每板96孔的格式。过渡到常规的384口井筛选现在看来是可行的，ChemBridge库的其余部分将以这种高密度格式进行筛选。不久将开始对所有ChemBridge热门产品进行筛选、再确认和剂量反应测试。在ChemBridge筛选完成后，384孔形式的天然产物提取物的试点筛选将立即开始。我们已经从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、c端区以及N和c端组合区域作为GST融合蛋白。已完成生长和表达优化研究。规模化生产提供了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。目前正在评估从融合蛋白中高效切割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伙伴鉴定工作。ABCG2多药转运体抑制剂的初步检测开发正在进行中。已经获得了三种过表达ABCG2的细胞系，并正在进行评估，以评估用于HTS应用的最佳细胞系。计划利用phophide A (PhA)，一种ABCG2特异性荧光底物，由ABCG2运输出细胞，进行基于细胞的荧光检测。已知的抑制剂fumitremorgin C （FTC）将被用作阳性对照，阻断ABCG2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，化验格式和协议的计划。检测开发工作正在进行中，以开发HIF-2抑制剂的筛选。成功转染了HeLa和786-O细胞，但是来自原始GFP结构的信号不够亮，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，选择了一种载体（pd4EGFP-N1），该载体能够表达半衰期为4小时的不稳定形式的EGFP。然后将该载体中的CMV启动子替换为人vegf启动子。使用新结构的转染实验是成功的，GFP亮度现在看来足以用于786-O细胞系的检测开发。现在的焦点已经转移到单细胞克隆。一旦获得稳定的克隆培养，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，用作次要筛选。包含长和短VHL形式的构建体已经准备好转染到786-O细胞中作为对照。最后，我们正试图确定一种化合物，可以用作阳性对照试验。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，看看它们是否也能抑制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 .