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 .
