Natural Products Chemistry and Molecular Target Lead Dis

ChemBridge文库中的超过85,000种合成化合物已经在基于细胞的差异细胞毒性试验中进行了筛选，该试验旨在寻找突变Kit的抑制剂。在本试验中，HIT被定义为对突变KIT细胞和野生型KIT细胞产生30%以上差异生长抑制的化合物。到目前为止，已有145种化合物被确定为命中，这相当于在检测中的命中率约为0.2%。到目前为止，所有的筛选都是96孔/板的形式。过渡到常规384井筛选现在看起来是可行的，并且ChemBridge文库的其余部分将以这种高密度格式进行筛选。对ChemBridge的所有热门产品进行樱桃采摘、重新确认和剂量反应测试将很快开始。一旦ChemBridge筛选完成，就将开始对384孔形式的天然产品提取物进行试点筛选。 我们从Victor Lobanenkov的实验室获得了表达Boris N-末端、C-末端以及N-和C-末端的融合蛋白的大肠杆菌表达系统。生长和表达优化研究已经完成。扩大生产已经提供了6.2N端鲍里斯L和16.6C端鲍里斯L的培养。GST融合蛋白的亲和纯化目前正在进行中。从融合蛋白中有效切割GST结构域的程序正在评估中。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多药转运蛋白抑制剂的初步检测正在进行中。已经获得了三个过表达ABCG2的细胞系，并正在进行评估，以评估适合高温超导应用的最佳细胞系。计划利用脱镁叶绿酸a(PHA)进行基于荧光细胞的检测，PHA是一种ABCG2特异的荧光底物，由ABCG2转运出细胞。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在细胞内积累。计划进行实验，以确定最佳的细胞生长条件、试剂参数以及分析形式和方案。 目前正在努力开发一种筛选HIF-2抑制剂的检测方法。HeLa和786-O细胞被成功地转染，但来自原始GFP构建的信号不够明亮，不足以支持高通量筛选形式。使用pEGFP-C1并确认表达的EGFP足够明亮，选择了能够表达半衰期为4小时的失稳形式的EGFP的载体(pd4EGFP-N1)。将载体中的巨细胞病毒启动子替换为人血管内皮生长因子启动子。使用新构建物的转基因实验取得了成功，GFP亮度现在似乎足以用于786-O细胞系的分析开发。现在的重点已经转移到了单细胞克隆上。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启动子的)，它是由HIF-2诱导的，但属于另一种信号通路，用作辅助筛选。含有长VHL形式和短VHL形式的构建体已经准备好用于转染786-O细胞作为对照。最后，我们正试图确定一种可以作为阳性对照的化合物。由于尚不知道HIF-2抑制剂，我们将从检测已知的HIF-2抑制剂开始，看看它们中是否有抑制HIF-2的药物。

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 .