This project proposes to investigate the potential use of Display Cloning to rapidly identify and isolate the genes of protein-based therapeutic targets from biological warfare agents. This application will utilize the genome of Yersinia pestis, a Category A biological agent of potential use in biological warfare, to develop a panel of small-molecule probes that can be used in combination with gDNA phage display to identify genes of basic biological function and potential therapeutic importance. Once established, these probe panels can be used to provide a rapid-response approach for the target-directed drug development of therapeutic agents for novel and/or engineered biological warfare agents of known or unknown origin. Display Cloning is a procedure that combines small-molecule affinity chromatography with cDNA phage display. By utilizing a gDNA library, whole genomes of bacterial organisms can be displayed simultaneously, resulting in the ability to isolate genes from biological organisms, based on the functional ability of their resulting protein product to bind a small molecule. If the small molecule probe is a known transition-state analogue, putative protein function can be assigned. If the small molecule is a known enzyme inhibitor or drug, a therapeutic value can be ascribed to the target. An important benefit of this procedure is that not only the identity of the gene, but the gene itself is isolated. Additionally, on-phage binding analysis has been demonstrated, providing an immediate in vitro binding assay that can be used for target-based drug development. This method has been successfully utilized in mammalian systems to identify and isolate the genes of drug targets, signal transduction proteins, as well as RNA and carbohydrate binding proteins. Creating a gDNA library and performing a phage selection can be completed in less than one week. This is advantageous compared to the months or years that it may take to sequence the entire genome of an unknown or engineered organism. While microarray technology is a powerful method of looking at whole genomes, it cannot provide the functional information that can be obtained from a transition-state analogue affinity experiment. In addition to providing a starting point for drug development, analysis of biowarfare organisms with our probe panels will provide a functional taxonomic analysis of these organisms, well before their genomes can be sequenced or gene-chips constructed.
