The exponential growth in our knowledgebase of cancer genetics has led to the identification of a large array of genes, proteins and pathways that potentially play a central role in the carcinogenesis and may be potential targets for therapeutic intervention. The challenge now is to delve into how different compounds and compound classes' influence these genes. An enormous number of different cellular activities can be tracked at the gene expression level, including the ability to track in detail the response of cells and tissues to the introduction of a pharmacological agent. For this reason, gene expression analysis can be used to provide detailed and broad insight into the on and off-target mechanistic activities of existing and potential therapeutics. Proposed here is the demonstration and development of a new production-oriented, high throughput, PCR-based approach to gene expression analysis. The approach builds off of the fundamental strengths of PCR and adds a high level of multiplexing, 15-35 genes per PCR reaction. The principal goal of this proposal is to develop and demonstrate the value of a multiplexed PCR approach for use as a compound library gene expression screen. In order to achieve this goal we propose to (a) develop and validate 3 multiplex assays (genotoxicity, nephrotoxicity and HDAC-targeting antitumor activity), (b) develop protocols for high throughput screening, and (c) demonstrate the utility by screening the Gen- Plus and NCI chemical diversity set and performing a lead optimization study on HDAC-targeting candidates. The protocols and assays developed and the data from these demonstration screening studies will be made publicly available.
