Molecular therapeutics targeting the underlying defects in signaling pathways that contribute to cancer initiation and progression is a rapidly emerging avenue for patient therapy. The success of Imatinib Mesylate (Gleevec) has galvanized the field to identify and implement additional targeted therapeutics. A large number of inhibitors against specific kinases are currently in development. Most screening approaches analyze one or at most two criteria usually with purified enzymes or with a phenotypic assay. Many of these inhibitors demonstrated unexpected effects in intact cells, which reflect off target responses and the robust characteristics of signaling networks. There is an urgent need for function-based assays to prioritize and validate candidate targeting reagents. We propose the study of """"""""Tissue lysate array as a high throughput assay for molecular screening"""""""". The assay can rapidly assess over 100 different characteristics of functional proteomics, pathways and networks in intact cells, which greatly assists in selection of molecules for chemical genomics or lead compounds for pharmaceutical development. The assay is based on lysis of drug treated cells under stringent conditions followed by arraying on a solid matrix. The matrix can then be probed with pairs of antibodies identifying activation state and total amount of the protein. Based on this technology, we are able to gather multiple information from each compound and integrated into a """"""""fingerprint"""""""" database to allow rapid assessment of on and off target activity. We propose:
Specific Aim 1) To develop feasible and robust approaches to adapt tissue lysate arrays to a 96-well format, which will be applied to screen cellular responses to pharmacological standard.
Aim 2) To develop multiplex technology to improve the cost, efficacy and robustness of the tissue lysate array.
Aim 3) To develop and validate cell lines appropriate for high throughput screening by tissue lysate array.
Aim 4) To validate the technology in a synthetic lethality analysis by combinations of screening molecule with drug of known function. ? ?
