As we enter a post-genomic era, it has become apparent that a significant number of the genes identified in bacterial genomes encode proteins of unknown function. The development and refinement of tools and techniques to elucidate the function of these poorly characterized proteins will be required. <P>One of the goals of this research is the development of peptide-based tools to determine the role of various proteins of unknown function in bacterial metabolism and virulence. In addition, many of the genes encoding proteins of unknown function are required for cell viability. These essential genes represent valid targets for antibacterial drug discovery but are typically not accessible using standard biochemical assays. The peptide-based tools developed to ascribe function to these essential gene products can be readily converted into high throughput screening (HTS) assays for drug discovery. Another goal of this research will be to develop assays and screen compound collections for new antibacterials.<P> Specific Objectives: <OL> <LI> Build 5-10 phage display peptide libraries and establish techniques for screening the libraries <LI> Identify peptides that bind to and inhibit the function of 5 essential bacterial genes <LI> Demonstrate that through in vivo expression of target-specific peptides that the peptides inhibit bacterial viability <LI> <P>Build peptide-based, HTS-compatible assays for 3 essential bacterial targets and use those assays to screen a compound collection
Non-Technical Summary: Infectious diseases are still a leading cause of death worldwide. With increasing antibiotic resistance among bacterial pathogens and the emergence of hard-to-treat opportunistic infections, the need for new antibiotics continues. This research is focused towards developing tools to increase our understanding of the functions required for bacterial viability and the development of assays to discover new antibiotics to treat bacterial infections. <P> Approach: Phage display is a powerful technology that can used to identify peptides that bind to a wide range of targets from proteins to biomaterials. Combinatorial peptide libraries are constructed by inserting peptide-encoding oligonucleotides into gene III of bacteriophage M13. The resulting peptide fusions are displayed on the surface of the bacteriophage and can be screened for binding to a target of interest. When applied to protein targets, the peptides isolated are directed to functional sites on the protein and don't bind randomly to the protein. For an enzyme, this includes targeting the active site and the peptide will function as an inhibitor of the enzymatic activity, even if the enzymatic substrates are none peptidic in nature. For example, peptides have been isolated by phage display that bind to alcohol dehydrogenase and inhibit the conversion of ethanol to acetaldehyde with a Ki of 80 nM. In addition to their use as inhibitors in biochemical reactions, peptides can be expressed inside cells to bind the target protein and disrupt its function. When applied to proteins that are essential for cell viability, this "protein knockout" will result in inhibition of cell growth. Peptide-based protein knockouts can be generated for proteins of unknown function that are indentified by genomics and used to characterize and dissect the proteins function. Monitoring cell viability, metabolism and gene expression profiles during expression of the peptide inhibitor will lead to an understanding of the role of various proteins of unknown function. The peptides identified by phage display are high affinity binding agents. The affinity of the peptide for a protein target is typically in the range of low nM to low ?M. Also, the peptides are generally very specific for their protein target with the ability to selectively bind to the target in the presence of high concentrations of other proteins. As such, these affinity reagents can be used in a variety of biochemical techniques including affinity-based protein purification, co-precipitation reactions and Western Blots to isolate and characterize a protein. In addition, the peptides can be labeled with a detector molecule and used to develop screening assays. With increasing antibiotic resistance among bacterial pathogens and the emergence of hard-to-treat opportunistic infections, the need for new antibiotics continues. Bacterial genomics has provided a large number of new targets for antibacterial drug discovery. As described above, a significant number of the essential genes identified by genomics encode proteins of unknown function. In previous work, we have shown that peptides isolated by phage display can be used as surrogate ligands to develop HTS-compatible assays to screen chemical compound collections for the discovery of new antibacterial agents.