) The long-term objective of my research is to apply combinatorial chemistry for basic research and drug development. In combinatorial chemistry, millions of compounds can be generated and screened for their ability to bind to a specific target macromolecule or to elicit a specific biological response. Over the same period, several molecular biology tools have emerged that rapidly analyze changes in gene expression. Differential display, serial analysis of gene expression or SAGE and more recently, cDNA microarrays are very powerful technologies enabling one to identify unique genes expressed in a disease state. Although 2- dimensional polyacrylamide gel electrophoresis was first described in 1975, it is only in the last few years that this technique resurfaced as a potential method for the identification of altered protein expressed in disease-tissues. Our laboratory has been awarded a two-year NIH R21 exploratory research grant (CA789909-01, 7/1/98-6/30/00) entitled """"""""Peptide Ligands for Altered Protein in Disease-tissues."""""""" The project involves the application of the """"""""one-bead one- compound combinatorial library method to differential genomic or protein display. Briefly, millions of peptide-beads are mixed with tagged-cell extracts from both cancer and normal cells. We are developing methods to identify individual peptide-beads which interact with altered proteins of cancer cell. These peptide-beads can then be isolated to determine their chemical structures. In principle, this new approach will enable us to screen millions of ligands against thousands of proteins simultaneously (over a billion interactions) in """"""""one-pot""""""""; altered proteins that bind to specific ligands from the library are identified. This last year, we have reached two technical milestones essential for the development and application of this novel and potentially very powerful technology. The first technique enable us to effectively examine billion of phosphoprotein/ligand interactions simultaneously, and to identify the ligands that interact with unique phosphoproteins derived from cancer cells. The second technique involves the development of a chemical microarray system in which thousands of peptides or small molecules are chemically immobilized on a glass slide in a microarray format, and the use of these chemical microarrays to profile functional properties of whole cell extract of tumor tissue. Further development and application of this novel technology on human cancer cell lines and fresh biopsy specimens will be the subject of this grant proposal. Potential applications of this technology include (1) tumor profiling for unique phosphoproteins, (2) identification of cancer drug targets, (3) and identification of new leads for drug development.
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