? The studies outlined in this proposal are the first steps towards the establishment of a high throughput in vitro toxicology screen using mammalian cells. Toxicological screens are a critical component of the drug discovery process but are often employed late in the process due to the constraints of cost and throughput. The enablement of valid toxicology screens capable of implementation early in the drug discovery process would have tremendous value for the pharmaceutical industry as well as the public in terms of lowered costs and better medicines. One of the major causes of adverse drug-drug interactions is caused by inhibitory interactions of drugs with metabolizing enzymes. Cytochromes P450 (CYP) are the principal enzymes for metabolizing drugs and other xenoboitics and the inhibition of these enzymes is often the mechanism for drug-drug interactions. In order to provide a tool for better toxicology screens, we propose to establish high-content, multiplexed CYP assays using cellular microarrays coupled with molecular based readouts. Cellular microarrays are produced using a technology termed reverse transfection whereby distinct cell clusters over-expressing or under-expressing specific cDNAs are created. In this case, each cell cluster will be expressing a different CYP allowing for the parallel examination of the enzymatic activity of each of these CYPs in context of exposure to a small molecule drug lead. The ability to perform these assays in 96-well plates, in which each well can contain up to 64 different cellular genotypes, provides a platform for the direct comparison of potency and selectivity of a given drug lead within a single experiment across many targets. These arrays, in combination with a variety of different molecular and cellular assay readouts, will allow for the high-throughput screening of 1000's of different molecules against 100's of different toxicologic targets across many different assay systems in parallel. Such a tool should greatly enhance to the discovery of new, and better, drugs regardless of the disease indication. ? ?
