Preparation of compounds for screening The Maybridge Hitskit 3000 library con sists of 3000 chemically diverse compounds. The library was delivered find more info in 36 racks each containing 80 compounds dissolved in DMSO to 10 mg/ml. For the screening, ali quots of the DMSO solutions were transferred to 96 well plates and were further diluted with PBS to Inhibitors,Modulators,Libraries obtain stock solutions of 100 ug/ml from which four different 384 well plates for screening were prepared with final test concen trations of 1 ug/ml. In all steps, the Biomek 2000 pipetting station connected to a plate stacker carousel in a safety cabinet was used. For dose response studies, plates containing VLX40 and other compounds were prepared by 10 fold serial dilu tions in the concentrations 0. 004 to 40 uM using the same robotic system.

The plates were stored at 70 C until further use. The screening identified one compound with higher activity against 8226/Dox40 cells compared to its parental counterpart RPMI 8226. This compound, chem Inhibitors,Modulators,Libraries ically a quinoline alkaloid, was designated VLX40, and subjected for detailed studies. Measurement of cancer Inhibitors,Modulators,Libraries drug activity The Fluorometric Microculture Cytotoxicity Assay, FMCA, described in detail previously, was used for measure ment of the cytotoxic effect of library compounds and the established standard drugs. The FMCA is based on measurement of fluorescence generated from hydrolysis of fluorescein diacetate to fluorescein by cells with intact plasma membranes. Cells were seeded in the drug prepared 384 well plates using the pipetting robot Pre cision 2000.

The number of cells per well was 2,500 5,000 for solid tumor samples and 10,000 20,000 for leukemic samples. In each plate, two columns without drugs served as controls and Inhibitors,Modulators,Libraries one column with medium only served as blank. The plates were incubated for 72 h and then transferred to an integrated HTS SAGIAN Inhibitors,Modulators,Libraries Core System consisting of an ORCA robot with CO2 incubator, dispenser module, washer module, de lidding station, plate hotels, barcode reader, liquid handler and a multipurpose reader for automated FMCA. Quality criteria for a successful assay included a mean coefficient of variation of less than 30% in the control wells and a fluorescence signal in control wells of more than 5 times the blank. Survival index is defined as the fluorescence of test wells in percentage of controls with blank values subtracted.

Multiparametric or high content evaluation of apoptosis and cell cycle arrest The fluorescence microscope ArrayScan High Content Screening system was used to study apoptosis and cell cycle arrest. For these assays, cells were seeded into 96 well plates, left to attach over night, before test compounds were added. Cell death characteristics were studied using a multi parametric HCS assay described in detail previously. Apoptosis was evaluated after 6, 24 and 48 h exposure to VLX40 in MCF 7 cells.