In response to RFA-RM-04-020, """"""""Molecular Libraries Screening Instrumentation"""""""", we propose to develop a highly integrated hardware and software platform that performs highly multiplexed high throughput assays for signaling protein activation in extracts from cells treated with individual compounds. Current high throughput screening methods offer powerful tools to discover novel modulators of cell signaling in large libraries of small molecules by detecting the chemical modification of substrates by kinases, phosphatase, proteases and other signaling enzymes. This approach has proven its value in discovering inhibitors of oncogenic tyrosine kinases, leading to clinically important drugs such as Imatinib to target Bcr-Abl in CML and c-kit in GIST, Gefitinib to target EGFR in small cell lung cancer and a number of investigational kinase antagonists such as SU11248 to target Flt3 in AML. However, the state-of-the-art in molecular library screening falls short in identifying molecules that may be agonists or antagonists of specific signaling pathways but that are not necessarily targeted at a particular signaling protein. In prior work, we have developed sensitive assays using immunologic and MALDI-TOF detection of the phosphorylation of immobilized peptides and proteins that accurately report Bcr-Abl activity and inhibition in whole cell extracts from Ph+ leukemia cell lines. Here, we will extend this work and use our established copolymerization methodology to immobilize multiple tyrosine kinase substrate and isotope-coded control peptides in an acrylamide copolymer via photo-cleavable linkers. Peptides that display high specificity for individual tyrosine kinases or specific classes will be selected and validated in this format. Cell extracts will be applied to these surfaces in an array format where each spot corresponds to cells treated with a single library compound. After incubation, the surface will be washed, and the peptides released by UV photo-cleavage. MALDI-TOF analysis of each spot will lead to a spectrum of test and control peptides associated with each compound. These spectra will be normalized and compared with reference spectra to detect statistically significant changes in one or more signaling pathways. Compounds that appear to activate or inhibit one or more tyrosine kinases present in the cell extracts will be flagged for further analysis.
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