High throughput sonication: evaluation for compound solubilization.

Dissolution of organic compounds in DMSO in HTS plate or tube format is a difficult problem as users move to higher compression plate formats. Precipitation of compounds from DMSO screening stocks is a recognized problem in the HTS materials management process. The adverse effect of freeze thaw cycles on DMSO stock solutions stored in plate format as a result of cherry picking operations has led to the gradual replacement of plate-based storage with tube-based storage so as to minimize the number of freeze thaw cycles. Compound solubility in DMSO is markedly decreased by uptake of small quantities of water. We attribute this effect to the non ideal properties of DMSO water mixtures such that cavity formation in solvent, a necessary step in dissolution, is more difficult in wet DMSO than in dry DMSO or in pure water. We report here that efficient compound dissolution is possible even in 384 well format by the use of in-well plate-based sonication. Surprisingly, compounds precipitated from DMSO stocks either by water uptake or repeated freeze thaw cycles can be re-dissolved by low energy sonication. Finally, we demonstrate that precipitation of compound from DMSO stock solutions is synergistically enhanced by water uptake into DMSO compound stock solutions as well as by increasing the number of freeze thaw cycles.

Identification of Akt pathway inhibitors using redistribution screening on the FLIPR and the IN Cell 3000 analyzer.

The PI3-kinase/Akt pathway is an important cell survival pathway that is deregulated in the majority of human cancers. Despite the apparent druggability of several kinases in the pathway, no specific catalytic inhibitors have been reported in the literature. The authors describe the development of a fluorometric imaging plate reader (FLIPR)-based Akt1 translocation assay to discover inhibitors of Akt1 activation. Screening of a diverse chemical library of 45,000 compounds resulted in identification of several classes of Akt1 translocation inhibitors. Using a combination of classical in vitro assays and translocation assays directed at different steps of the Akt pathway, the mechanisms of action of 2 selected chemical classes were further defined. Protein translocation assays emerge as powerful tools for hit identification and characterization.

Changes in intracellular cAMP reported by a Redistribution assay using a cAMP-dependent protein kinase-green fluorescent protein chimera.

We report on a novel method to monitor changes in intracellular cAMP concentration ([cAMP]i) within intact living cells using a chimeric fusion of the catalytic subunit of cAMP-dependent protein kinase to green fluorescent protein (PKAcat-GFP). In stably transfected unstimulated fibroblasts, fusion protein fluorescence is highly concentrated in aggregates throughout the cytoplasm and absent in the nucleus. Elevation of [cAMP]i disperses GFP fluorescence from the cytoplasmic aggregates within minutes. Spot-photobleach measurements show that the rate of exchange of GFP-labeled catalytic subunits at these aggregates increases in proportion to [cAMP]i. For any given stimulus, the response curve for dispersal of GFP fluorescence from aggregates agrees closely with the increase in total [cAMP]i as measured by standard in vitro methods (SPA). The redistribution of fluorescence is completely reversible: reduction of [cAMP]i results in return of fluorescence to the cytoplasmic aggregates. Consistent behaviour of PKAcat-GFP is seen in different cell backgrounds. We demonstrate that PKA Redistribution assays are suitable for measurement of changes in [cAMP]i brought about by both Gs- and Gi-protein-coupled receptor stimulation as well as by inhibition of cAMP phosphodiesterases.

A simple technique for reducing edge effect in cell-based assays.

Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO(2) incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37 degrees C CO(2) incubator yields a significant reduction in edge effect.